Process definitions

ATAC-Seq

data:workflow:atacseqworkflow-atac-seq (data:reads:fastq  reads, data:genome:fasta  genome, data:bed  promoter, basic:string  mode, basic:string  speed, basic:boolean  use_se, basic:boolean  discordantly, basic:boolean  rep_se, basic:integer  minins, basic:integer  maxins, basic:integer  trim_5, basic:integer  trim_3, basic:integer  trim_iter, basic:integer  trim_nucl, basic:string  rep_mode, basic:integer  k_reports, basic:integer  q_threshold, basic:integer  n_sub, basic:boolean  tn5, basic:integer  shift, basic:boolean  tagalign, basic:string  duplicates, basic:string  duplicates_prepeak, basic:decimal  qvalue, basic:decimal  pvalue, basic:decimal  pvalue_prepeak, basic:integer  cap_num, basic:integer  mfold_lower, basic:integer  mfold_upper, basic:integer  slocal, basic:integer  llocal, basic:integer  extsize, basic:integer  shift, basic:integer  band_width, basic:boolean  nolambda, basic:boolean  fix_bimodal, basic:boolean  nomodel, basic:boolean  nomodel_prepeak, basic:boolean  down_sample, basic:boolean  bedgraph, basic:boolean  spmr, basic:boolean  call_summits, basic:boolean  broad, basic:decimal  broad_cutoff)[Source: v2.0.2]

This ATAC-seq pipeline closely follows the official ENCODE DCC pipeline. It is comprised of three steps; alignment, pre-peakcall QC, and calling peaks (with post-peakcall QC). First, reads are aligned to a genome using [Bowtie2](http://bowtie-bio.sourceforge.net/index.shtml) aligner. Next, pre-peakcall QC metrics are calculated. QC report contains ENCODE 3 proposed QC metrics – [NRF](https://www.encodeproject.org/data-standards/terms/), [PBC bottlenecking coefficients, NSC, and RSC](https://genome.ucsc.edu/ENCODE/qualityMetrics.html#chipSeq). Finally, the peaks are called using [MACS2](https://github.com/taoliu/MACS/). The post-peakcall QC report includes additional QC metrics – number of peaks, fraction of reads in peaks (FRiP), number of reads in peaks, and if promoter regions BED file is provided, number of reads in promoter regions, fraction of reads in promoter regions, number of peaks in promoter regions, and fraction of reads in promoter regions.

Input arguments

reads
label:Select sample(s)
type:data:reads:fastq
genome
label:Genome
type:data:genome:fasta
promoter
label:Promoter regions BED file
type:data:bed
description:BED file containing promoter regions (TSS+-1000bp for example). Needed to get the number of peaks and reads mapped to promoter regions.
required:False
alignment.mode
label:Alignment mode
type:basic:string
description:End to end: Bowtie 2 requires that the entire read align from one end to the other, without any trimming (or “soft clipping”) of characters from either end. Local: Bowtie 2 does not require that the entire read align from one end to the other. Rather, some characters may be omitted (“soft clipped”) from the ends in order to achieve the greatest possible alignment score.
default:--local
choices:
  • end to end mode: --end-to-end
  • local: --local
alignment.speed
label:Speed vs. Sensitivity
type:basic:string
default:--sensitive
choices:
  • Very fast: --very-fast
  • Fast: --fast
  • Sensitive: --sensitive
  • Very sensitive: --very-sensitive
alignment.PE_options.use_se
label:Map as single-ended (for paired-end reads only)
type:basic:boolean
description:If this option is selected paired-end reads will be mapped as single-ended and other paired-end options are ignored.
default:False
alignment.PE_options.discordantly
label:Report discordantly matched read
type:basic:boolean
description:If both mates have unique alignments, but the alignments do not match paired-end expectations (orientation and relative distance) then alignment will be reported. Useful for detecting structural variations.
default:True
alignment.PE_options.rep_se
label:Report single ended
type:basic:boolean
description:If paired alignment can not be found Bowtie2 tries to find alignments for the individual mates.
default:True
alignment.PE_options.minins
label:Minimal distance
type:basic:integer
description:The minimum fragment length for valid paired-end alignments. 0 imposes no minimum.
default:0
alignment.PE_options.maxins
label:Maximal distance
type:basic:integer
description:The maximum fragment length for valid paired-end alignments.
default:2000
alignment.start_trimming.trim_5
label:Bases to trim from 5’
type:basic:integer
description:Number of bases to trim from from 5’ (left) end of each read before alignment.
default:0
alignment.start_trimming.trim_3
label:Bases to trim from 3’
type:basic:integer
description:Number of bases to trim from from 3’ (right) end of each read before alignment
default:0
alignment.trimming.trim_iter
label:Iterations
type:basic:integer
description:Number of iterations.
default:0
alignment.trimming.trim_nucl
label:Bases to trim
type:basic:integer
description:Number of bases to trim from 3’ end in each iteration.
default:2
alignment.reporting.rep_mode
label:Report mode
type:basic:string
description:Default mode: search for multiple alignments, report the best one; -k mode: search for one or more alignments, report each; -a mode: search for and report all alignments
default:def
choices:
  • Default mode: def
  • -k mode: k
  • -a mode (very slow): a
alignment.reporting.k_reports
label:Number of reports (for -k mode only)
type:basic:integer
description:Searches for at most X distinct, valid alignments for each read. The search terminates when it can’t find more distinct valid alignments, or when it finds X, whichever happens first.
default:5
prepeakqc_settings.q_threshold
label:Quality filtering threshold
type:basic:integer
default:30
prepeakqc_settings.n_sub
label:Number of reads to subsample
type:basic:integer
default:25000000
prepeakqc_settings.tn5
label:TN5 shifting
type:basic:boolean
description:Tn5 transposon shifting. Shift reads on “+” strand by 4bp and reads on “-” strand by 5bp.
default:True
prepeakqc_settings.shift
label:User-defined cross-correlation peak strandshift
type:basic:integer
description:If defined, SPP tool will not try to estimate fragment length but will use the given value as fragment length.
default:0
settings.tagalign
label:Use tagAlign files
type:basic:boolean
description:Use filtered tagAlign files as case (treatment) and control (background) samples. If extsize parameter is not set, run MACS using input’s estimated fragment length.
default:True
settings.duplicates
label:Number of duplicates
type:basic:string
description:It controls the MACS behavior towards duplicate tags at the exact same location – the same coordination and the same strand. The ‘auto’ option makes MACS calculate the maximum tags at the exact same location based on binomal distribution using 1e-5 as pvalue cutoff and the ‘all’ option keeps all the tags. If an integer is given, at most this number of tags will be kept at the same location. The default is to keep one tag at the same location.
required:False
hidden:settings.tagalign
choices:
  • 1: 1
  • auto: auto
  • all: all
settings.duplicates_prepeak
label:Number of duplicates
type:basic:string
description:It controls the MACS behavior towards duplicate tags at the exact same location – the same coordination and the same strand. The ‘auto’ option makes MACS calculate the maximum tags at the exact same location based on binomal distribution using 1e-5 as pvalue cutoff and the ‘all’ option keeps all the tags. If an integer is given, at most this number of tags will be kept at the same location. The default is to keep one tag at the same location.
required:False
hidden:!settings.tagalign
default:all
choices:
  • 1: 1
  • auto: auto
  • all: all
settings.qvalue
label:Q-value cutoff
type:basic:decimal
description:The q-value (minimum FDR) cutoff to call significant regions. Q-values are calculated from p-values using Benjamini-Hochberg procedure.
required:False
disabled:settings.pvalue && settings.pvalue_prepeak
settings.pvalue
label:P-value cutoff
type:basic:decimal
description:The p-value cutoff. If specified, MACS2 will use p-value instead of q-value cutoff.
required:False
disabled:settings.qvalue
hidden:settings.tagalign
settings.pvalue_prepeak
label:P-value cutoff
type:basic:decimal
description:The p-value cutoff. If specified, MACS2 will use p-value instead of q-value cutoff.
disabled:settings.qvalue
hidden:!settings.tagalign || settings.qvalue
default:0.01
settings.cap_num
label:Cap number of peaks by taking top N peaks
type:basic:integer
description:To keep all peaks set value to 0.
disabled:settings.broad
default:300000
settings.mfold_lower
label:MFOLD range (lower limit)
type:basic:integer
description:This parameter is used to select the regions within MFOLD range of high-confidence enrichment ratio against background to build model. The regions must be lower than upper limit, and higher than the lower limit of fold enrichment. DEFAULT:10,30 means using all regions not too low (>10) and not too high (<30) to build paired-peaks model. If MACS can not find more than 100 regions to build model, it will use the –extsize parameter to continue the peak detection ONLY if –fix-bimodal is set.
required:False
settings.mfold_upper
label:MFOLD range (upper limit)
type:basic:integer
description:This parameter is used to select the regions within MFOLD range of high-confidence enrichment ratio against background to build model. The regions must be lower than upper limit, and higher than the lower limit of fold enrichment. DEFAULT:10,30 means using all regions not too low (>10) and not too high (<30) to build paired-peaks model. If MACS can not find more than 100 regions to build model, it will use the –extsize parameter to continue the peak detection ONLY if –fix-bimodal is set.
required:False
settings.slocal
label:Small local region
type:basic:integer
description:Slocal and llocal parameters control which two levels of regions will be checked around the peak regions to calculate the maximum lambda as local lambda. By default, MACS considers 1000bp for small local region (–slocal), and 10000bps for large local region (–llocal) which captures the bias from a long range effect like an open chromatin domain. You can tweak these according to your project. Remember that if the region is set too small, a sharp spike in the input data may kill the significant peak.
required:False
settings.llocal
label:Large local region
type:basic:integer
description:Slocal and llocal parameters control which two levels of regions will be checked around the peak regions to calculate the maximum lambda as local lambda. By default, MACS considers 1000bp for small local region (–slocal), and 10000bps for large local region (–llocal) which captures the bias from a long range effect like an open chromatin domain. You can tweak these according to your project. Remember that if the region is set too small, a sharp spike in the input data may kill the significant peak.
required:False
settings.extsize
label:extsize
type:basic:integer
description:While ‘–nomodel’ is set, MACS uses this parameter to extend reads in 5’->3’ direction to fix-sized fragments. For example, if the size of binding region for your transcription factor is 200 bp, and you want to bypass the model building by MACS, this parameter can be set as 200. This option is only valid when –nomodel is set or when MACS fails to build model and –fix-bimodal is on.
default:150
settings.shift
label:Shift
type:basic:integer
description:Note, this is NOT the legacy –shiftsize option which is replaced by –extsize! You can set an arbitrary shift in bp here. Please Use discretion while setting it other than default value (0). When –nomodel is set, MACS will use this value to move cutting ends (5’) then apply –extsize from 5’ to 3’ direction to extend them to fragments. When this value is negative, ends will be moved toward 3’->5’ direction, otherwise 5’->3’ direction. Recommended to keep it as default 0 for ChIP-Seq datasets, or -1 * half of EXTSIZE together with –extsize option for detecting enriched cutting loci such as certain DNAseI-Seq datasets. Note, you can’t set values other than 0 if format is BAMPE for paired-end data. Default is 0.
default:-75
settings.band_width
label:Band width
type:basic:integer
description:The band width which is used to scan the genome ONLY for model building. You can set this parameter as the sonication fragment size expected from wet experiment. The previous side effect on the peak detection process has been removed. So this parameter only affects the model building.
required:False
settings.nolambda
label:Use backgroud lambda as local lambda
type:basic:boolean
description:With this flag on, MACS will use the background lambda as local lambda. This means MACS will not consider the local bias at peak candidate regions.
default:False
settings.fix_bimodal
label:Turn on the auto paired-peak model process
type:basic:boolean
description:Whether turn on the auto paired-peak model process. If it’s set, when MACS failed to build paired model, it will use the nomodel settings, the ‘–extsize’ parameter to extend each tags. If set, MACS will be terminated if paired-peak model is failed.
default:False
settings.nomodel
label:Bypass building the shifting model
type:basic:boolean
description:While on, MACS will bypass building the shifting model.
hidden:settings.tagalign
default:False
settings.nomodel_prepeak
label:Bypass building the shifting model
type:basic:boolean
description:While on, MACS will bypass building the shifting model.
hidden:!settings.tagalign
default:True
settings.down_sample
label:Down-sample
type:basic:boolean
description:When set, random sampling method will scale down the bigger sample. By default, MACS uses linear scaling. This option will make the results unstable and irreproducible since each time, random reads would be selected, especially the numbers (pileup, pvalue, qvalue) would change. Consider to use ‘randsample’ script before MACS2 runs instead.
default:False
settings.bedgraph
label:Save fragment pileup and control lambda
type:basic:boolean
description:If this flag is on, MACS will store the fragment pileup, control lambda, -log10pvalue and -log10qvalue scores in bedGraph files. The bedGraph files will be stored in current directory named NAME+’_treat_pileup.bdg’ for treatment data, NAME+’_control_lambda.bdg’ for local lambda values from control, NAME+’_treat_pvalue.bdg’ for Poisson pvalue scores (in -log10(pvalue) form), and NAME+’_treat_qvalue.bdg’ for q-value scores from Benjamini-Hochberg-Yekutieli procedure.
default:True
settings.spmr
label:Save signal per million reads for fragment pileup profiles
type:basic:boolean
disabled:settings.bedgraph === false
default:True
settings.call_summits
label:Call summits
type:basic:boolean
description:MACS will now reanalyze the shape of signal profile (p or q-score depending on cutoff setting) to deconvolve subpeaks within each peak called from general procedure. It’s highly recommended to detect adjacent binding events. While used, the output subpeaks of a big peak region will have the same peak boundaries, and different scores and peak summit positions.
default:True
settings.broad
label:Composite broad regions
type:basic:boolean
description:When this flag is on, MACS will try to composite broad regions in BED12 (a gene-model-like format) by putting nearby highly enriched regions into a broad region with loose cutoff. The broad region is controlled by another cutoff through –broad-cutoff. The maximum length of broad region length is 4 times of d from MACS.
disabled:settings.call_summits === true
default:False
settings.broad_cutoff
label:Broad cutoff
type:basic:decimal
description:Cutoff for broad region. This option is not available unless –broad is set. If -p is set, this is a p-value cutoff, otherwise, it’s a q-value cutoff. DEFAULT = 0.1
required:False
disabled:settings.call_summits === true || settings.broad !== true

Output results

Abstract alignment process

data:alignmentabstract-alignment ()[Source: v1.0.0]

Input arguments

Output results

bam
label:Alignment file
type:basic:file
bai
label:Alignment index BAI
type:basic:file
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Abstract annotation process

data:annotationabstract-annotation ()[Source: v1.0.0]

Input arguments

Output results

annot
label:Uploaded file
type:basic:file
source
label:Gene ID source
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Abstract bed process

data:bedabstract-bed ()[Source: v1.0.0]

Input arguments

Output results

bed
label:BED
type:basic:file
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Abstract differential expression process

data:differentialexpressionabstract-differentialexpression ()[Source: v1.0.0]

Input arguments

Output results

raw
label:Differential expression (gene level)
type:basic:file
de_json
label:Results table (JSON)
type:basic:json
de_file
label:Results table (file)
type:basic:file
source
label:Gene ID source
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string

Abstract expression process

data:expressionabstract-expression ()[Source: v1.0.0]

Input arguments

Output results

exp
label:Normalized expression
type:basic:file
rc
label:Read counts
type:basic:file
required:False
exp_json
label:Expression (json)
type:basic:json
exp_type
label:Expression type
type:basic:string
source
label:Gene ID source
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string

Accel Amplicon Pipeline

data:workflow:ampliconworkflow-accel (data:reads:fastq:paired  reads, data:genome:fasta  genome, data:masterfile:amplicon  master_file, data:seq:nucleotide  adapters, list:data:variants:vcf  known_indels, list:data:variants:vcf  known_vars, data:variants:vcf  dbsnp, basic:integer  mbq, basic:integer  stand_call_conf, basic:integer  min_bq, basic:integer  min_alt_bq, list:data:variants:vcf  known_vars_db, basic:decimal  af_threshold)[Source: v4.0.1]

Processing pipeline to analyse the Accel-Amplicon NGS panel data. The raw amplicon sequencing reads are quality trimmed using Trimmomatic. The quality of the raw and trimmed data is assesed using the FASTQC tool. Quality trimmed reads are aligned to a reference genome using BWA mem. Sequencing primers are removed from the aligned reads using Primerclip. Amplicon performance stats are calculated using Bedtools coveragebed and Picard CollectTargetedPcrMetrics programs. Prior to variant calling, the alignment file is preprocessed using the GATK IndelRealigner and BaseRecalibrator tools. GATK HaplotypeCaller and Lofreq tools are used to call germline variants. Called variants are annotated using the SnpEff tool. Finally, the amplicon performance metrics and identified variants data are used to generate the PDF analysis report.

Input arguments

reads
label:Input reads
type:data:reads:fastq:paired
genome
label:Genome
type:data:genome:fasta
master_file
label:Experiment Master file
type:data:masterfile:amplicon
adapters
label:Adapters
type:data:seq:nucleotide
description:Provide an Illumina sequencing adapters file (.fasta) with adapters to be removed by Trimmomatic.
preprocess_bam.known_indels
label:Known indels
type:list:data:variants:vcf
preprocess_bam.known_vars
label:Known variants
type:list:data:variants:vcf
gatk.dbsnp
label:dbSNP
type:data:variants:vcf
gatk.mbq
label:Min Base Quality
type:basic:integer
description:Minimum base quality required to consider a base for calling.
default:20
gatk.stand_call_conf
label:Min call confidence threshold
type:basic:integer
description:The minimum phred-scaled confidence threshold at which variants should be called.
default:20
lofreq.min_bq
label:Min baseQ
type:basic:integer
description:Skip any base with baseQ smaller than the default value.
default:20
lofreq.min_alt_bq
label:Min alternate baseQ
type:basic:integer
description:Skip alternate bases with baseQ smaller than the default value.
default:20
var_annot.known_vars_db
label:Known variants
type:list:data:variants:vcf
report.af_threshold
label:Allele frequency threshold
type:basic:decimal
default:0.01

Output results

Align (BWA) and trim adapters

data:alignment:bam:bwatrimalign-bwa-trim (data:masterfile:amplicon  master_file, data:genome:fasta  genome, data:reads:fastq  reads, basic:integer  seed_l, basic:integer  band_w, basic:decimal  re_seeding, basic:boolean  m, basic:integer  match, basic:integer  missmatch, basic:integer  gap_o, basic:integer  gap_e, basic:integer  clipping, basic:integer  unpaired_p, basic:boolean  report_all, basic:integer  report_tr)[Source: v1.2.2]

Align with BWA mem and trim the sam output. The process uses the memory-optimized Primertrim tool.

Input arguments

master_file
label:Master file
type:data:masterfile:amplicon
description:Amplicon experiment design file that holds the information about the primers to be removed.
genome
label:Reference genome
type:data:genome:fasta
reads
label:Reads
type:data:reads:fastq
seed_l
label:Minimum seed length
type:basic:integer
description:Minimum seed length. Matches shorter than minimum seed length will be missed. The alignment speed is usually insensitive to this value unless it significantly deviates 20.
default:19
band_w
label:Band width
type:basic:integer
description:Gaps longer than this will not be found.
default:100
re_seeding
label:Re-seeding factor
type:basic:decimal
description:Trigger re-seeding for a MEM longer than minSeedLen*FACTOR. This is a key heuristic parameter for tuning the performance. Larger value yields fewer seeds, which leads to faster alignment speed but lower accuracy.
default:1.5
m
label:Mark shorter split hits as secondary
type:basic:boolean
description:Mark shorter split hits as secondary (for Picard compatibility)
default:False
scoring.match
label:Score of a match
type:basic:integer
default:1
scoring.missmatch
label:Mismatch penalty
type:basic:integer
default:4
scoring.gap_o
label:Gap open penalty
type:basic:integer
default:6
scoring.gap_e
label:Gap extension penalty
type:basic:integer
default:1
scoring.clipping
label:Clipping penalty
type:basic:integer
description:Clipping is applied if final alignment score is smaller than (best score reaching the end of query) - (Clipping penalty)
default:5
scoring.unpaired_p
label:Penalty for an unpaired read pair
type:basic:integer
description:Affinity to force pair. Score: scoreRead1+scoreRead2-Penalty
default:9
reporting.report_all
label:Report all found alignments
type:basic:boolean
description:Output all found alignments for single-end or unpaired paired-end reads. These alignments will be flagged as secondary alignments.
default:False
reporting.report_tr
label:Report threshold score
type:basic:integer
description:Don’t output alignment with score lower than defined number. This option only affects output.
default:30

Output results

bam
label:Alignment file
type:basic:file
description:Position sorted alignment
bai
label:Index BAI
type:basic:file
stats
label:Statistics
type:basic:file
bigwig
label:BigWig file
type:basic:file
required:False
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Amplicon report

data:report:ampliconamplicon-report (data:picard:coverage  pcr_metrics, data:coverage  coverage, data:masterfile:amplicon  master_file, list:data:snpeff  annot_vars, basic:decimal  af_threshold)[Source: v1.0.4]

Create amplicon report.

Input arguments

pcr_metrics
label:Picard TargetedPcrMetrics
type:data:picard:coverage
coverage
label:Coverage
type:data:coverage
master_file
label:Amplicon master file
type:data:masterfile:amplicon
annot_vars
label:Annotated variants (snpEff)
type:list:data:snpeff
af_threshold
label:Allele frequency threshold
type:basic:decimal
default:0.01

Output results

report
label:Report
type:basic:file
panel_name
label:Panel name
type:basic:string
stats
label:File with sample statistics
type:basic:file
amplicon_cov
label:Amplicon coverage file (nomergebed)
type:basic:file
variant_tables
label:Variant tabels (snpEff)
type:list:basic:file

Amplicon table

data:varianttable:ampliconamplicon-table (data:masterfile:amplicon  master_file, data:coverage  coverage, list:data:snpeff  annot_vars, basic:boolean  all_amplicons, basic:string  table_name)[Source: v1.0.1]

Create variant table for use together with the genome browser.

Input arguments

master_file
label:Master file
type:data:masterfile:amplicon
coverage
label:Amplicon coverage
type:data:coverage
annot_vars
label:Annotated variants
type:list:data:snpeff
all_amplicons
label:Report all amplicons
type:basic:boolean
default:False
table_name
label:Amplicon table name
type:basic:string
default:Amplicons containing variants

Output results

variant_table
label:Variant table
type:basic:json

Annotate novel splice junctions (regtools)

data:junctions:regtoolsregtools-junctions-annotate (data:genome:fasta  genome, data:annotation:gtf  annotation, data:alignment:bam:star  alignment_star, data:alignment:bam  alignment, data:bed  input_bed_junctions)[Source: v0.2.1]

Identify novel splice junctions by using regtools to annotate against a reference. The process accepts reference genome, reference genome annotation (GTF), and input with reads information (STAR aligment or reads aligned by any other aligner or junctions in BED12 format). If STAR aligner data is given as input, the process calculates BED12 file from STAR ‘SJ.out.tab’ file, and annotates all junctions with ‘regtools junctions annotate’ command. When reads are aligned by other aligner, junctions are extracted with ‘regtools junctions extract’ tool and then annotated with ‘junction annotate’ command. Third option allows user to provide directly BED12 file with junctions, which are then annotated. Finnally, annotated novel junctions are filtered in a separate output file. More information can be found in the [regtools manual](https://regtools.readthedocs.io/en/latest/).

Input arguments

genome
label:Reference genome
type:data:genome:fasta
annotation
label:Reference genome annotation (GTF)
type:data:annotation:gtf
alignment_star
label:STAR alignment
type:data:alignment:bam:star
description:Splice junctions detected by STAR aligner (SJ.out.tab STAR output file). Please provide one input ‘STAR alignment’ or ‘Alignment’ by any aligner or directly ‘Junctions in BED12 format’.
required:False
alignment
label:Alignment
type:data:alignment:bam
description:Aligned reads from which splice junctions are going to be extracted. Please provide one input ‘STAR alignment’ or ‘Alignment’ by any aligner or directly ‘Junctions in BED12 format’.
required:False
input_bed_junctions
label:Junctions in BED12 format
type:data:bed
description:Splice junctions in BED12 format. Please provide one input ‘STAR alignment’ or ‘Alignment’ by any aligner or directly ‘Junctions in BED12 format’.
required:False

Output results

novel_splice_junctions
label:Table of annotated novel splice junctions
type:basic:file
splice_junctions
label:Table of annotated splice junctions
type:basic:file
novel_sj_bed
label:Novel splice junctions in BED format
type:basic:file
bed
label:Splice junctions in BED format
type:basic:file
novel_sj_bigbed_igv_ucsc
label:Novel splice junctions in BigBed format
type:basic:file
required:False
bigbed_igv_ucsc
label:Splice junctions in BigBed format
type:basic:file
required:False
novel_sj_tbi_jbrowse
label:Novel splice junctions bed tbi index for JBrowse
type:basic:file
tbi_jbrowse
label:Bed tbi index for JBrowse
type:basic:file
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Archive and make multi-sample report for amplicon data

data:archive:samples:ampliconamplicon-archive-multi-report (list:data  data, list:basic:string  fields, basic:boolean  j)[Source: v0.2.5]

Create an archive of output files. The ouput folder structure is organized by sample slug and data object’s output-field names. Additionally, create multi-sample report for selected samples.

Input arguments

data
label:Data list
type:list:data
fields
label:Output file fields
type:list:basic:string
j
label:Junk paths
type:basic:boolean
description:Store just names of saved files (junk the path)
default:False

Output results

archive
label:Archive of selected samples and a heatmap comparing them
type:basic:file

Archive samples

data:archive:samplesarchive-samples (list:data  data, list:basic:string  fields, basic:boolean  j)[Source: v0.2.3]

Create an archive of output files. The ouput folder structure is organized by sample slug and data object’s output-field names.

Input arguments

data
label:Data list
type:list:data
fields
label:Output file fields
type:list:basic:string
j
label:Junk paths
type:basic:boolean
description:Store just names of saved files (junk the path)
default:False

Output results

archive
label:Archive
type:basic:file

BAM file

data:alignment:bam:uploadupload-bam (basic:file  src, basic:string  species, basic:string  build)[Source: v1.4.1]

Import a BAM file (.bam), which is the binary format for storing sequence alignment data. This format is described on the [SAM Tools web site](http://samtools.github.io/hts-specs/).

Input arguments

src
label:Mapping (BAM)
type:basic:file
description:A mapping file in BAM format. The file will be indexed on upload, so additional BAI files are not required.
validate_regex:\.(bam)$
species
label:Species
type:basic:string
description:Species latin name.
choices:
  • Homo sapiens: Homo sapiens
  • Mus musculus: Mus musculus
  • Rattus norvegicus: Rattus norvegicus
  • Dictyostelium discoideum: Dictyostelium discoideum
  • Odocoileus virginianus texanus: Odocoileus virginianus texanus
  • Solanum tuberosum: Solanum tuberosum
build
label:Build
type:basic:string

Output results

bam
label:Uploaded file
type:basic:file
bai
label:Index BAI
type:basic:file
stats
label:Alignment statistics
type:basic:file
bigwig
label:BigWig file
type:basic:file
required:False
species
label:Species
type:basic:string
build
label:Build
type:basic:string

BAM file and index

data:alignment:bam:uploadupload-bam-indexed (basic:file  src, basic:file  src2, basic:string  species, basic:string  build)[Source: v1.4.1]

Import a BAM file (.bam) and BAM index (.bam.bai). BAM file is the binary format for storing sequence alignment data. This format is described on the [SAM Tools web site](http://samtools.github.io/hts-specs/).

Input arguments

src
label:Mapping (BAM)
type:basic:file
description:A mapping file in BAM format.
validate_regex:\.(bam)$
src2
label:bam index (*.bam.bai file)
type:basic:file
description:An index file of a BAM mapping file (ending with bam.bai).
validate_regex:\.(bam.bai)$
species
label:Species
type:basic:string
description:Species latin name.
choices:
  • Homo sapiens: Homo sapiens
  • Mus musculus: Mus musculus
  • Rattus norvegicus: Rattus norvegicus
  • Dictyostelium discoideum: Dictyostelium discoideum
  • Odocoileus virginianus texanus: Odocoileus virginianus texanus
  • Solanum tuberosum: Solanum tuberosum
build
label:Build
type:basic:string

Output results

bam
label:Uploaded file
type:basic:file
bai
label:Index BAI
type:basic:file
stats
label:Alignment statistics
type:basic:file
bigwig
label:BigWig file
type:basic:file
required:False
species
label:Species
type:basic:string
build
label:Build
type:basic:string

BBDuk (paired-end)

data:reads:fastq:paired:bbdukbbduk-paired (data:reads:fastq:paired  reads, basic:integer  min_length, basic:boolean  show_advanced, list:data:seq:nucleotide  sequences, list:basic:string  literal_sequences, basic:integer  kmer_length, basic:boolean  check_reverse_complements, basic:boolean  mask_middle_base, basic:integer  min_kmer_hits, basic:decimal  min_kmer_fraction, basic:decimal  min_coverage_fraction, basic:integer  hamming_distance, basic:integer  query_hamming_distance, basic:integer  edit_distance, basic:integer  hamming_distance2, basic:integer  query_hamming_distance2, basic:integer  edit_distance2, basic:boolean  forbid_N, basic:boolean  remove_if_either_bad, basic:boolean  find_best_match, basic:boolean  perform_error_correction, basic:string  k_trim, basic:string  k_mask, basic:boolean  mask_fully_covered, basic:integer  min_k, basic:string  quality_trim, basic:integer  trim_quality, basic:integer  trim_poly_A, basic:decimal  min_length_fraction, basic:integer  max_length, basic:integer  min_average_quality, basic:integer  min_average_quality_bases, basic:integer  min_base_quality, basic:integer  min_consecutive_bases, basic:integer  trim_pad, basic:boolean  trim_by_overlap, basic:boolean  strict_overlap, basic:integer  min_overlap, basic:integer  min_insert, basic:boolean  trim_pairs_evenly, basic:integer  force_trim_left, basic:integer  force_trim_right, basic:integer  force_trim_right2, basic:integer  force_trim_mod, basic:integer  restrict_left, basic:integer  restrict_right, basic:decimal  min_GC, basic:decimal  max_GC, basic:integer  maxns, basic:boolean  toss_junk, basic:boolean  chastity_filter, basic:boolean  barcode_filter, list:data:seq:nucleotide  barcode_files, list:basic:string  barcode_sequences, basic:integer  x_min, basic:integer  y_min, basic:integer  x_max, basic:integer  y_max, basic:decimal  entropy, basic:integer  entropy_window, basic:integer  entropy_k, basic:boolean  entropy_mask, basic:integer  min_base_frequency, basic:boolean  nogroup)[Source: v2.2.2]

BBDuk combines the most common data-quality-related trimming, filtering, and masking operations into a single high-performance tool. It is capable of quality-trimming and filtering, adapter-trimming, contaminant-filtering via kmer matching, sequence masking, GC-filtering, length filtering, entropy-filtering, format conversion, histogram generation, subsampling, quality-score recalibration, kmer cardinality estimation, and various other operations in a single pass. See [here](https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/) for more information.

Input arguments

reads
label:Reads
type:data:reads:fastq:paired
min_length
label:Minimum length [minlength=10]
type:basic:integer
description:Reads shorter than the minimum length will be discarded after trimming.
default:10
show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
reference.sequences
label:Sequences [ref]
type:list:data:seq:nucleotide
description:Reference sequences include adapters, contaminants, and degenerate sequences. They can be provided in a multi-sequence FASTA file or as a set of literal sequences below.
required:False
reference.literal_sequences
label:Literal sequences [literal]
type:list:basic:string
description:Literal sequences can be specified by inputting them one by one and pressing Enter after each sequence.
required:False
default:[]
processing.kmer_length
label:Kmer length [k=27]
type:basic:integer
description:Kmer length used for finding contaminants. Contaminants shorter than kmer length will not be found. Kmer length must be at least 1.
default:27
processing.check_reverse_complements
label:Look for reverse complements of kmers in addition to forward kmers [rcomp=t]
type:basic:boolean
default:True
processing.mask_middle_base
label:Treat the middle base of a kmer as a wildcard to increase sensitivity in the presence of errors [maskmiddle=t]
type:basic:boolean
default:True
processing.min_kmer_hits
label:Minimum number of kmer hits [minkmerhits=1]
type:basic:integer
description:Reads need at least this many matching kmers to be considered as matching the reference.
default:1
processing.min_kmer_fraction
label:Minimum kmer fraction [minkmerfraction=0.0]
type:basic:decimal
description:A read needs at least this fraction of its total kmers to hit a reference in order to be considered a match. If this and ‘Minimum number of kmer hits’ are set, the greater is used.
default:0.0
processing.min_coverage_fraction
label:Minimum coverage fraction [mincovfraction=0.0]
type:basic:decimal
description:A read needs at least this fraction of its total bases to be covered by reference kmers to be considered a match. If specified, ‘Minimum coverage fraction’ overrides ‘Minimum number of kmer hits’ and ‘Minimum kmer fraction’.
default:0.0
processing.hamming_distance
label:Maximum Hamming distance for kmers (substitutions only) [hammingdistance=0]
type:basic:integer
default:0
processing.query_hamming_distance
label:Hamming distance for query kmers [qhdist=0]
type:basic:integer
default:0
processing.edit_distance
label:Maximum edit distance from reference kmers (substitutions and indels) [editdistance=0]
type:basic:integer
default:0
processing.hamming_distance2
label:Hamming distance for short kmers when looking for shorter kmers [hammingdistance2=0]
type:basic:integer
default:0
processing.query_hamming_distance2
label:Hamming distance for short query kmers when looking for shorter kmers [qhdist2=0]
type:basic:integer
default:0
processing.edit_distance2
label:Maximum edit distance from short reference kmers (substitutions and indels) when looking for shorter kmers [editdistance2=0]
type:basic:integer
default:0
processing.forbid_N
label:Forbid matching of read kmers containing N [forbidn=f]
type:basic:boolean
description:By default, these will match a reference ‘A’ if ‘Maximum Hamming distance for kmers’ > 0 or ‘Maximum edit distance from reference kmers’ > 0, to increase sensitivity.
default:False
processing.remove_if_either_bad
label:Remove both sequences of a paired-end read, if either of them is to be removed [removeifeitherbad=t]
type:basic:boolean
default:True
processing.find_best_match
label:If multiple matches, associate read with sequence sharing most kmers [findbestmatch=t]
type:basic:boolean
default:True
processing.perform_error_correction
label:Perform error correction with BBMerge prior to kmer operations [ecco=f]
type:basic:boolean
default:False
operations.k_trim
label:Trimming protocol to remove bases matching reference kmers from reads [ktrim=f]
type:basic:string
default:f
choices:
  • Don’t trim: f
  • Trim to the right: r
  • Trim to the left: l
operations.k_mask
label:Symbol to replace bases matching reference kmers [kmask=f]
type:basic:string
description:Allows any non-whitespace character other than t or f. Processes short kmers on both ends.
default:f
operations.mask_fully_covered
label:Only mask bases that are fully covered by kmers [maskfullycovered=f]
type:basic:boolean
default:False
operations.min_k
label:Look for shorter kmers at read tips down to this length when k-trimming or masking [mink=0]
type:basic:integer
description:-1 means disabled. Enabling this will disable treating the middle base of a kmer as a wildcard to increase sensitivity in the presence of errors.
default:-1
operations.quality_trim
label:Trimming protocol to remove bases with quality below the minimum average region quality from read ends [qtrim=f]
type:basic:string
description:Performed after looking for kmers. If enabled, set also ‘Average quality below which to trim region’.
default:f
choices:
  • Trim neither end: f
  • Trim both ends: rl
  • Trim only right end: r
  • Trim only left end: l
  • Use sliding window: w
operations.trim_quality
label:Average quality below which to trim region [trimq=6]
type:basic:integer
description:Set trimming protocol to enable this parameter.
disabled:operations.quality_trim == ‘f’
default:6
operations.trim_poly_A
label:Minimum length of poly-A or poly-T tails to trim on either end of reads [trimpolya=0]
type:basic:integer
default:0
operations.min_length_fraction
label:Minimum length fraction [mlf=0.0]
type:basic:decimal
description:Reads shorter than this fraction of original length after trimming will be discarded.
default:0.0
operations.max_length
label:Maximum length [maxlength]
type:basic:integer
description:Reads longer than this after trimming will be discarded.
required:False
operations.min_average_quality
label:Minimum average quality [minavgquality=0]
type:basic:integer
description:Reads with average quality (after trimming) below this will be discarded.
default:0
operations.min_average_quality_bases
label:Number of initial bases to calculate minimum average quality from [maqb=0]
type:basic:integer
description:Used only if positive.
default:0
operations.min_base_quality
label:Minimum base quality below which reads are discarded after trimming [minbasequality=0]
type:basic:integer
default:0
operations.min_consecutive_bases
label:Minimum number of consecutive called bases [mcb=0]
type:basic:integer
default:0
operations.trim_pad
label:Number of bases to trim around matching kmers [tp=0]
type:basic:integer
default:0
operations.trim_by_overlap
label:Trim adapters based on where paired-end reads overlap [tbo=f]
type:basic:boolean
default:False
operations.strict_overlap
label:Adjust sensitivity in ‘Trim adapters based on where paired-end reads overlap’ mode [strictoverlap=t]
type:basic:boolean
default:True
operations.min_overlap
label:Minimum number of overlapping bases [minoverlap=14]
type:basic:integer
description:Require this many bases of overlap for detection.
default:14
operations.min_insert
label:Minimum insert size [mininsert=40]
type:basic:integer
description:Require insert size of at least this for overlap. Should be reduced to 16 for small RNA sequencing.
default:40
operations.trim_pairs_evenly
label:Trim both sequences of paired-end reads to the minimum length of either sequence [tpe=f]
type:basic:boolean
default:False
operations.force_trim_left
label:Position from which to trim bases to the left [forcetrimleft=0]
type:basic:integer
default:0
operations.force_trim_right
label:Position from which to trim bases to the right [forcetrimright=0]
type:basic:integer
default:0
operations.force_trim_right2
label:Number of bases to trim from the right end [forcetrimright2=0]
type:basic:integer
default:0
operations.force_trim_mod
label:Modulo to right-trim reads [forcetrimmod=0]
type:basic:integer
description:Trim reads to the largest multiple of modulo.
default:0
operations.restrict_left
label:Number of leftmost bases to look in for kmer matches [restrictleft=0]
type:basic:integer
default:0
operations.restrict_right
label:Number of rightmosot bases to look in for kmer matches [restrictright=0]
type:basic:integer
default:0
operations.min_GC
label:Minimum GC content [mingc=0.0]
type:basic:decimal
description:Discard reads with lower GC content.
default:0.0
operations.max_GC
label:Maximum GC content [maxgc=1.0]
type:basic:decimal
description:Discard reads with higher GC content.
default:1.0
operations.maxns
label:Max Ns after trimming [maxns=-1]
type:basic:integer
description:If non-negative, reads with more Ns than this (after trimming) will be discarded.
default:-1
operations.toss_junk
label:Discard reads with invalid characters as bases [tossjunk=f]
type:basic:boolean
default:False
header_parsing.chastity_filter
label:Discard reads that fail Illumina chastity filtering [chastityfilter=f]
type:basic:boolean
description:Discard reads with id containing ‘ 1:Y:’ or ‘ 2:Y:’.
default:False
header_parsing.barcode_filter
label:Remove reads with unexpected barcodes if barcodes are set, or barcodes containing ‘N’ otherwise [barcodefilter=f]
type:basic:boolean
description:A barcode must be the last part of the read header.
default:False
header_parsing.barcode_files
label:Barcode sequences [barcodes]
type:list:data:seq:nucleotide
required:False
header_parsing.barcode_sequences
label:Literal barcode sequences [barcodes]
type:list:basic:string
description:Literal barcode sequences can be specified by inputting them one by one and pressing Enter after each sequence.
required:False
default:[]
header_parsing.x_min
label:Minimum X coordinate [xmin=-1]
type:basic:integer
description:If positive, discard reads with a smaller X coordinate.
default:-1
header_parsing.y_min
label:Minimum Y coordinate [ymin=-1]
type:basic:integer
description:If positive, discard reads with a smaller Y coordinate.
default:-1
header_parsing.x_max
label:Maximum X coordinate [xmax=-1]
type:basic:integer
description:If positive, discard reads with a larger X coordinate.
default:-1
header_parsing.y_max
label:Maximum Y coordinate [ymax=-1]
type:basic:integer
description:If positive, discard reads with a larger Y coordinate.
default:-1
complexity.entropy
label:Minimum entropy [entropy=-1.0]
type:basic:decimal
description:Set between 0 and 1 to filter reads with entropy below that value. Higher is more stringent.
default:-1.0
complexity.entropy_window
label:Length of sliding window used to calculate entropy [entropywindow=50]
type:basic:integer
description:To use the sliding window set minimum entropy in range between 0.0 and 1.0.
default:50
complexity.entropy_k
label:Length of kmers used to calcuate entropy [entropyk=5]
type:basic:integer
default:5
complexity.entropy_mask
label:Mask low-entropy parts of sequences with N instead of discarding [entropymask=f]
type:basic:boolean
default:False
complexity.min_base_frequency
label:Minimum base frequency [minbasefrequency=0]
type:basic:integer
default:0
fastqc.nogroup
label:Disable grouping of bases for reads >50bp [nogroup]
type:basic:boolean
description:All reports will show data for every base in the read. Using this option will cause fastqc to crash and burn if you use it on really long reads.
default:False

Output results

fastq
label:Remaining upstream reads
type:list:basic:file
fastq2
label:Remaining downstream reads
type:list:basic:file
statistics
label:Statistics
type:list:basic:file
fastqc_url
label:Upstream quality control with FastQC
type:list:basic:file:html
fastqc_url2
label:Downstream quality control with FastQC
type:list:basic:file:html
fastqc_archive
label:Download upstream FastQC archive
type:list:basic:file
fastqc_archive2
label:Download downstream FastQC archive
type:list:basic:file

BBDuk (single-end)

data:reads:fastq:single:bbdukbbduk-single (data:reads:fastq:single  reads, basic:integer  min_length, basic:boolean  show_advanced, list:data:seq:nucleotide  sequences, list:basic:string  literal_sequences, basic:integer  kmer_length, basic:boolean  check_reverse_complements, basic:boolean  mask_middle_base, basic:integer  min_kmer_hits, basic:decimal  min_kmer_fraction, basic:decimal  min_coverage_fraction, basic:integer  hamming_distance, basic:integer  query_hamming_distance, basic:integer  edit_distance, basic:integer  hamming_distance2, basic:integer  query_hamming_distance2, basic:integer  edit_distance2, basic:boolean  forbid_N, basic:boolean  find_best_match, basic:string  k_trim, basic:string  k_mask, basic:boolean  mask_fully_covered, basic:integer  min_k, basic:string  quality_trim, basic:integer  trim_quality, basic:integer  trim_poly_A, basic:decimal  min_length_fraction, basic:integer  max_length, basic:integer  min_average_quality, basic:integer  min_average_quality_bases, basic:integer  min_base_quality, basic:integer  min_consecutive_bases, basic:integer  trim_pad, basic:integer  min_overlap, basic:integer  min_insert, basic:integer  force_trim_left, basic:integer  force_trim_right, basic:integer  force_trim_right2, basic:integer  force_trim_mod, basic:integer  restrict_left, basic:integer  restrict_right, basic:decimal  min_GC, basic:decimal  max_GC, basic:integer  maxns, basic:boolean  toss_junk, basic:boolean  chastity_filter, basic:boolean  barcode_filter, list:data:seq:nucleotide  barcode_files, list:basic:string  barcode_sequences, basic:integer  x_min, basic:integer  y_min, basic:integer  x_max, basic:integer  y_max, basic:decimal  entropy, basic:integer  entropy_window, basic:integer  entropy_k, basic:boolean  entropy_mask, basic:integer  min_base_frequency, basic:boolean  nogroup)[Source: v2.2.2]

BBDuk combines the most common data-quality-related trimming, filtering, and masking operations into a single high-performance tool. It is capable of quality-trimming and filtering, adapter-trimming, contaminant-filtering via kmer matching, sequence masking, GC-filtering, length filtering, entropy-filtering, format conversion, histogram generation, subsampling, quality-score recalibration, kmer cardinality estimation, and various other operations in a single pass. See [here](https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/) for more information.

Input arguments

reads
label:Reads
type:data:reads:fastq:single
min_length
label:Minimum length [minlength=10]
type:basic:integer
description:Reads shorter than the minimum length will be discarded after trimming.
default:10
show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
reference.sequences
label:Sequences [ref]
type:list:data:seq:nucleotide
description:Reference sequences include adapters, contaminants, and degenerate sequences. They can be provided in a multi-sequence FASTA file or as a set of literal sequences below.
required:False
reference.literal_sequences
label:Literal sequences [literal]
type:list:basic:string
description:Literal sequences can be specified by inputting them one by one and pressing Enter after each sequence.
required:False
default:[]
processing.kmer_length
label:Kmer length [k=27]
type:basic:integer
description:Kmer length used for finding contaminants. Contaminants shorter than Kmer length will not be found. Kmer length must be at least 1.
default:27
processing.check_reverse_complements
label:Look for reverse complements of kmers in addition to forward kmers [rcomp=t]
type:basic:boolean
default:True
processing.mask_middle_base
label:Treat the middle base of a kmer as a wildcard to increase sensitivity in the presence of errors [maskmiddle=t]
type:basic:boolean
default:True
processing.min_kmer_hits
label:Minimum number of kmer hits [minkmerhits=1]
type:basic:integer
description:Reads need at least this many matching kmers to be considered matching the reference.
default:1
processing.min_kmer_fraction
label:Minimum kmer fraction [minkmerfraction=0.0]
type:basic:decimal
description:A read needs at least this fraction of its total kmers to hit a reference in order to be considered a match. If this and ‘Minimum number of kmer hits’ are set, the greater is used.
default:0.0
processing.min_coverage_fraction
label:Minimum coverage fraction [mincovfraction=0.0]
type:basic:decimal
description:A read needs at least this fraction of its total bases to be covered by reference kmers to be considered a match. If specified, ‘Minimum coverage fraction’ overrides ‘Minimum number of kmer hits’ and ‘Minimum kmer fraction’.
default:0.0
processing.hamming_distance
label:Maximum Hamming distance for kmers (substitutions only) [hammingdistance=0]
type:basic:integer
default:0
processing.query_hamming_distance
label:Hamming distance for query kmers [qhdist=0]
type:basic:integer
default:0
processing.edit_distance
label:Maximum edit distance from reference kmers (substitutions and indels) [editdistance=0]
type:basic:integer
default:0
processing.hamming_distance2
label:Hamming distance for short kmers when looking for shorter kmers [hammingdistance2=0]
type:basic:integer
default:0
processing.query_hamming_distance2
label:Hamming distance for short query kmers when looking for shorter kmers [qhdist2=0]
type:basic:integer
default:0
processing.edit_distance2
label:Maximum edit distance from short reference kmers (substitutions and indels) when looking for shorter kmers [editdistance2=0]
type:basic:integer
default:0
processing.forbid_N
label:Forbid matching of read kmers containing N [forbidn=f]
type:basic:boolean
description:By default, these will match a reference ‘A’ if ‘Maximum Hamming distance for kmers’ > 0 or ‘Maximum edit distance from reference kmers’ > 0, to increase sensitivity.
default:False
processing.find_best_match
label:If multiple matches, associate read with sequence sharing most kmers [findbestmatch=f]
type:basic:boolean
default:True
operations.k_trim
label:Trimming protocol to remove bases matching reference kmers from reads [ktrim=f]
type:basic:string
default:f
choices:
  • Don’t trim: f
  • Trim to the right: r
  • Trim to the left: l
operations.k_mask
label:Symbol to replace bases matching reference kmers [kmask=f]
type:basic:string
description:Allows any non-whitespace character other than t or f. Processes short kmers on both ends.
default:f
operations.mask_fully_covered
label:Only mask bases that are fully covered by kmers [maskfullycovered=f]
type:basic:boolean
default:False
operations.min_k
label:Look for shorter kmers at read tips down to this length when k-trimming or masking [mink=0]
type:basic:integer
description:-1 means disabled. Enabling this will disable treating the middle base of a kmer as a wildcard to increase sensitivity in the presence of errors.
default:-1
operations.quality_trim
label:Trimming protocol to remove bases with quality below the minimum average region quality from read ends [qtrim=f]
type:basic:string
description:Performed after looking for kmers. If enabled, set also ‘Average quality below which to trim region’.
default:f
choices:
  • Trim neither end: f
  • Trim both ends: rl
  • Trim only right end: r
  • Trim only left end: l
  • Use sliding window: w
operations.trim_quality
label:Average quality below which to trim region [trimq=6]
type:basic:integer
description:Set trimming protocol to enable this parameter.
disabled:operations.quality_trim == ‘f’
default:6
operations.trim_poly_A
label:Minimum length of poly-A or poly-T tails to trim on either end of reads [trimpolya=0]
type:basic:integer
default:0
operations.min_length_fraction
label:Minimum length fraction [mlf=0]
type:basic:decimal
description:Reads shorter than this fraction of original length after trimming will be discarded.
default:0.0
operations.max_length
label:Maximum length [maxlength]
type:basic:integer
description:Reads longer than this after trimming will be discarded.
required:False
operations.min_average_quality
label:Minimum average quality [minavgquality=0]
type:basic:integer
description:Reads with average quality (after trimming) below this will be discarded.
default:0
operations.min_average_quality_bases
label:Number of initial bases to calculate minimum average quality from [maqb=0]
type:basic:integer
description:Used only if positive.
default:0
operations.min_base_quality
label:Minimum base quality below which reads are discarded after trimming [minbasequality=0]
type:basic:integer
default:0
operations.min_consecutive_bases
label:Minimum number of consecutive called bases [mcb=0]
type:basic:integer
default:0
operations.trim_pad
label:Number of bases to trim around matching kmers [tp=0]
type:basic:integer
default:0
operations.min_overlap
label:Minimum number of overlapping bases [minoverlap=14]
type:basic:integer
description:Require this many bases of overlap for detection.
default:14
operations.min_insert
label:Minimum insert size [mininsert=40]
type:basic:integer
description:Require insert size of at least this for overlap. Should be reduced to 16 for small RNA sequencing.
default:40
operations.force_trim_left
label:Position from which to trim bases to the left [forcetrimleft=0]
type:basic:integer
default:0
operations.force_trim_right
label:Position from which to trim bases to the right [forcetrimright=0]
type:basic:integer
default:0
operations.force_trim_right2
label:Number of bases to trim from the right end [forcetrimright2=0]
type:basic:integer
default:0
operations.force_trim_mod
label:Modulo to right-trim reads [forcetrimmod=0]
type:basic:integer
description:Trim reads to the largest multiple of modulo.
default:0
operations.restrict_left
label:Number of leftmost bases to look in for kmer matches [restrictleft=0]
type:basic:integer
default:0
operations.restrict_right
label:Number of rightmosot bases to look in for kmer matches [restricright=0]
type:basic:integer
default:0
operations.min_GC
label:Minimum GC content [mingc=0.0]
type:basic:decimal
description:Discard reads with lower GC content.
default:0.0
operations.max_GC
label:Maximum GC content [maxgc=1.0]
type:basic:decimal
description:Discard reads with higher GC content.
default:1.0
operations.maxns
label:Max Ns after trimming [maxns=-1]
type:basic:integer
description:If non-negative, reads with more Ns than this (after trimming) will be discarded.
default:-1
operations.toss_junk
label:Discard reads with invalid characters as bases [tossjunk=f]
type:basic:boolean
default:False
header_parsing.chastity_filter
label:Discard reads that fail Illumina chastity filtering [chastityfilter=f]
type:basic:boolean
description:Discard reads with id containing ‘ 1:Y:’ or ‘ 2:Y:’.
default:False
header_parsing.barcode_filter
label:Remove reads with unexpected barcodes if barcodes are set, or barcodes containing ‘N’ otherwise [barcodefilter=f]
type:basic:boolean
description:A barcode must be the last part of the read header.
default:False
header_parsing.barcode_files
label:Barcode sequences [barcodes]
type:list:data:seq:nucleotide
required:False
header_parsing.barcode_sequences
label:Literal barcode sequences [barcodes]
type:list:basic:string
description:Literal barcode sequences can be specified by inputting them one by one and pressing Enter after each sequence.
required:False
default:[]
header_parsing.x_min
label:Minimum X coordinate [xmin=-1]
type:basic:integer
description:If positive, discard reads with a smaller X coordinate.
default:-1
header_parsing.y_min
label:Minimum Y coordinate [ymin=-1]
type:basic:integer
description:If positive, discard reads with a smaller Y coordinate.
default:-1
header_parsing.x_max
label:Maximum X coordinate [xmax=-1]
type:basic:integer
description:If positive, discard reads with a larger X coordinate.
default:-1
header_parsing.y_max
label:Maximum Y coordinate [ymax=-1]
type:basic:integer
description:If positive, discard reads with a larger Y coordinate.
default:-1
complexity.entropy
label:Minimum entropy [entropy=-1]
type:basic:decimal
description:Set between 0 and 1 to filter reads with entropy below that value. Higher is more stringent.
default:-1.0
complexity.entropy_window
label:Length of sliding window used to calculate entropy [entropywindow=50]
type:basic:integer
description:To use the sliding window set minimum entropy in range between 0.0 and 1.0.
default:50
complexity.entropy_k
label:Length of kmers used to calcuate entropy [entropyk=5]
type:basic:integer
default:5
complexity.entropy_mask
label:Mask low-entropy parts of sequences with N instead of discarding [entropymask=f]
type:basic:boolean
default:False
complexity.min_base_frequency
label:Minimum base frequency [minbasefrequency=0]
type:basic:integer
default:0
fastqc.nogroup
label:Disable grouping of bases for reads >50bp [nogroup]
type:basic:boolean
description:All reports will show data for every base in the read. Using this option will cause fastqc to crash and burn if you use it on really long reads.
default:False

Output results

fastq
label:Remaining reads
type:list:basic:file
statistics
label:Statistics
type:list:basic:file
fastqc_url
label:Quality control with FastQC
type:list:basic:file:html
fastqc_archive
label:Download FastQC archive
type:list:basic:file

BBDuk - STAR - FeatureCounts (3’ mRNA-Seq, paired-end)

data:workflow:quant:featurecounts:pairedworkflow-bbduk-star-fc-quant-paired (data:reads:fastq:paired  reads, data:genomeindex:star  star_index, list:data:seq:nucleotide  adapters, data:annotation  annotation, basic:string  stranded, basic:integer  n_reads, basic:integer  seed, basic:decimal  fraction, basic:boolean  two_pass, data:genomeindex:star  rrna_reference, data:genomeindex:star  globin_reference)[Source: v1.1.0]

This 3’ mRNA-Seq pipeline is comprised of QC, preprocessing, alignment and quantification steps. Reads are preprocessed by __BBDuk__ which removes adapters, trims reads for quality from the 3’-end, and discards reads that are too short after trimming. Preprocessed reads are aligned by __STAR__ aligner. For read-count quantification, the __FeatureCounts__ tool is used. QC steps include downsampling, QoRTs QC analysis and alignment of input reads to the rRNA/globin reference sequences. The reported alignment rate is used to asses the rRNA/globin sequence depletion rate.

Input arguments

reads
label:Paired-end reads
type:data:reads:fastq:paired
star_index
label:Star index
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
adapters
label:Adapters
type:list:data:seq:nucleotide
description:Provide a list of sequencing adapters files (.fasta) to be removed by BBDuk.
required:False
annotation
label:Annotation
type:data:annotation
stranded
label:Select the type of kit used for library preparation.
type:basic:string
choices:
  • Strand-specific forward: forward
  • Strand-specific reverse: reverse
downsampling.n_reads
label:Number of reads
type:basic:integer
default:1000000
downsampling.advanced.seed
label:Seed
type:basic:integer
default:11
downsampling.advanced.fraction
label:Fraction
type:basic:decimal
description:Use the fraction of reads in range [0.0, 1.0] from the orignal input file instead of the absolute number of reads. If set, this will override the “Number of reads” input parameter.
required:False
downsampling.advanced.two_pass
label:2-pass mode
type:basic:boolean
description:Enable two-pass mode when down-sampling. Two-pass mode is twice as slow but with much reduced memory.
default:False
qc.rrna_reference
label:Indexed rRNA reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
qc.globin_reference
label:Indexed Globin reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.

Output results

BBDuk - STAR - FeatureCounts (3’ mRNA-Seq, single-end)

data:workflow:quant:featurecounts:singleworkflow-bbduk-star-fc-quant-single (data:reads:fastq:single  reads, data:genomeindex:star  star_index, list:data:seq:nucleotide  adapters, data:annotation  annotation, basic:string  stranded, basic:integer  n_reads, basic:integer  seed, basic:decimal  fraction, basic:boolean  two_pass, data:genomeindex:star  rrna_reference, data:genomeindex:star  globin_reference)[Source: v1.1.0]

This 3’ mRNA-Seq pipeline is comprised of QC, preprocessing, alignment and quantification steps. Reads are preprocessed by __BBDuk__ which removes adapters, trims reads for quality from the 3’-end, and discards reads that are too short after trimming. Preprocessed reads are aligned by __STAR__ aligner. For read-count quantification, the __FeatureCounts__ tool is used. QC steps include downsampling, QoRTs QC analysis and alignment of input reads to the rRNA/globin reference sequences. The reported alignment rate is used to asses the rRNA/globin sequence depletion rate.

Input arguments

reads
label:Input single-end reads
type:data:reads:fastq:single
star_index
label:Star index
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
adapters
label:Adapters
type:list:data:seq:nucleotide
description:Provide a list of sequencing adapters files (.fasta) to be removed by BBDuk.
required:False
annotation
label:Annotation
type:data:annotation
stranded
label:Select the type of kit used for library preparation.
type:basic:string
choices:
  • Strand-specific forward: forward
  • Strand-specific reverse: reverse
downsampling.n_reads
label:Number of reads
type:basic:integer
default:1000000
downsampling.advanced.seed
label:Seed
type:basic:integer
default:11
downsampling.advanced.fraction
label:Fraction
type:basic:decimal
description:Use the fraction of reads in range [0.0, 1.0] from the orignal input file instead of the absolute number of reads. If set, this will override the “Number of reads” input parameter.
required:False
downsampling.advanced.two_pass
label:2-pass mode
type:basic:boolean
description:Enable two-pass mode when down-sampling. Two-pass mode is twice as slow but with much reduced memory.
default:False
qc.rrna_reference
label:Indexed rRNA reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
qc.globin_reference
label:Indexed Globin reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.

Output results

BBDuk - STAR - HTSeq-count (paired-end)

data:workflow:rnaseq:htseq:pairedworkflow-bbduk-star-htseq-paired (data:reads:fastq:paired  reads, data:genomeindex:star  star_index, list:data:seq:nucleotide  adapters, data:annotation  annotation, basic:string  stranded)[Source: v1.0.1]

This RNA-seq pipeline is comprised of three steps, preprocessing, alignment, and quantification. First, reads are preprocessed by __BBDuk__ which removes adapters, trims reads for quality from the 3’-end, and discards reads that are too short after trimming. Compared to similar tools, BBDuk is regarded for its computational efficiency. Next, preprocessed reads are aligned by __STAR__ aligner. At the time of implementation, STAR is considered a state-of-the-art tool that consistently produces accurate results from diverse sets of reads, and performs well even with default settings. For more information see [this comparison of RNA-seq aligners](https://www.nature.com/articles/nmeth.4106). Finally, aligned reads are summarized to genes by __HTSeq-count__. Compared to featureCounts, HTSeq-count is not as computationally efficient. All three tools in this workflow support parallelization to accelerate the analysis.

Input arguments

reads
label:Paired-end reads
type:data:reads:fastq:paired
star_index
label:Star index
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
adapters
label:Adapters
type:list:data:seq:nucleotide
description:Provide a list of sequencing adapters files (.fasta) to be removed by BBDuk.
required:False
annotation
label:Annotation
type:data:annotation
stranded
label:Select the QuantSeq kit used for library preparation.
type:basic:string
choices:
  • QuantSeq FWD: yes
  • QuantSeq REV: reverse

Output results

BBDuk - STAR - HTSeq-count (single-end)

data:workflow:rnaseq:htseq:singleworkflow-bbduk-star-htseq (data:reads:fastq:single  reads, data:genomeindex:star  star_index, list:data:seq:nucleotide  adapters, data:annotation  annotation, basic:string  stranded)[Source: v1.0.1]

This RNA-seq pipeline is comprised of three steps, preprocessing, alignment, and quantification. First, reads are preprocessed by __BBDuk__ which removes adapters, trims reads for quality from the 3’-end, and discards reads that are too short after trimming. Compared to similar tools, BBDuk is regarded for its computational efficiency. Next, preprocessed reads are aligned by __STAR__ aligner. At the time of implementation, STAR is considered a state-of-the-art tool that consistently produces accurate results from diverse sets of reads, and performs well even with default settings. For more information see [this comparison of RNA-seq aligners](https://www.nature.com/articles/nmeth.4106). Finally, aligned reads are summarized to genes by __HTSeq-count__. Compared to featureCounts, HTSeq-count is not as computationally efficient. All three tools in this workflow support parallelization to accelerate the analysis.

Input arguments

reads
label:Input single-end reads
type:data:reads:fastq:single
star_index
label:Star index
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
adapters
label:Adapters
type:list:data:seq:nucleotide
description:Provide a list of sequencing adapters files (.fasta) to be removed by BBDuk.
required:False
annotation
label:annotation
type:data:annotation
stranded
label:Select the QuantSeq kit used for library preparation.
type:basic:string
choices:
  • QuantSeq FWD: yes
  • QuantSeq REV: reverse

Output results

BBDuk - STAR - featureCounts - QC (paired-end)

data:workflow:rnaseq:featurecounts:qcworkflow-bbduk-star-featurecounts-qc-paired (data:reads:fastq:paired  reads, list:data:seq:nucleotide  adapters, basic:boolean  show_advanced, list:basic:string  custom_adapter_sequences, basic:integer  kmer_length, basic:integer  min_k, basic:integer  hamming_distance, basic:integer  maxns, basic:integer  trim_quality, basic:integer  min_length, data:genomeindex:star  genome, basic:boolean  show_advanced, basic:boolean  unstranded, basic:boolean  noncannonical, basic:boolean  chimeric, basic:integer  chimSegmentMin, basic:boolean  quantmode, basic:boolean  singleend, basic:boolean  gene_counts, basic:string  outFilterType, basic:integer  outFilterMultimapNmax, basic:integer  outFilterMismatchNmax, basic:decimal  outFilterMismatchNoverLmax, basic:integer  outFilterScoreMin, basic:integer  alignSJoverhangMin, basic:integer  alignSJDBoverhangMin, basic:integer  alignIntronMin, basic:integer  alignIntronMax, basic:integer  alignMatesGapMax, basic:string  alignEndsType, basic:string  outSAMunmapped, basic:string  outSAMattributes, basic:string  outSAMattrRGline, data:annotation  annotation, basic:boolean  show_advanced, basic:string  assay_type, data:index:salmon  cdna_index, basic:integer  n_reads, basic:string  feature_class, basic:string  feature_type, basic:string  id_attribute, basic:integer  n_reads, basic:integer  seed, basic:decimal  fraction, basic:boolean  two_pass, data:genomeindex:star  rrna_reference, data:genomeindex:star  globin_reference)[Source: v1.4.0]

This RNA-seq pipeline is comprised of three steps preprocessing, alignment, and quantification. First, reads are preprocessed by __BBDuk__ which removes adapters, trims reads for quality from the 3’-end, and discards reads that are too short after trimming. Compared to similar tools, BBDuk is regarded for its computational efficiency. Next, preprocessed reads are aligned by __STAR__ aligner. At the time of implementation, STAR is considered a state-of-the-art tool that consistently produces accurate results from diverse sets of reads, and performs well even with default settings. For more information see [this comparison of RNA-seq aligners](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792058/). Finally, aligned reads are summarized to genes by __featureCounts__. Gaining wide adoption among the bioinformatics community, featureCounts yields expressions in a computationally efficient manner. All three tools in this workflow support parallelization to accelerate the analysis. rRNA contamination rate in the sample is determined using the STAR aligner. Quality-trimmed reads are down-sampled (using Seqtk tool) and aligned to the rRNA reference sequences. The alignment rate indicates the percentage of the reads in the sample that are derived from the rRNA sequences.

Input arguments

preprocessing.reads
label:Reads
type:data:reads:fastq:paired
preprocessing.adapters
label:Adapters
type:list:data:seq:nucleotide
required:False
preprocessing.show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
preprocessing.custom_adapter_sequences
label:Custom adapter sequences [literal]
type:list:basic:string
description:Custom adapter sequences can be specified by inputting them one by one and pressing Enter after each sequence.
required:False
hidden:!preprocessing.show_advanced
default:[]
preprocessing.kmer_length
label:K-mer length
type:basic:integer
description:K-mer length must be smaller or equal to the length of adapters.
hidden:!preprocessing.show_advanced
default:23
preprocessing.min_k
label:Minimum k-mer length at right end of reads used for trimming
type:basic:integer
disabled:preprocessing.adapters.length === 0 && preprocessing.custom_adapter_sequences.length === 0
hidden:!preprocessing.show_advanced
default:11
preprocessing.hamming_distance
label:Maximum Hamming distance for k-mers
type:basic:integer
hidden:!preprocessing.show_advanced
default:1
preprocessing.maxns
label:Max Ns after trimming [maxns=-1]
type:basic:integer
description:If non-negative, reads with more Ns than this (after trimming) will be discarded.
hidden:!preprocessing.show_advanced
default:-1
preprocessing.trim_quality
label:Quality below which to trim reads from the right end
type:basic:integer
description:Phred algorithm is used, which is more accurate than naive trimming.
hidden:!preprocessing.show_advanced
default:10
preprocessing.min_length
label:Minimum read length
type:basic:integer
description:Reads shorter than minimum read length after trimming are discarded.
hidden:!preprocessing.show_advanced
default:20
alignment.genome
label:Indexed reference genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
alignment.show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
alignment.unstranded
label:The data is unstranded
type:basic:boolean
description:For unstranded RNA-seq data, Cufflinks/Cuffdiff require spliced alignments with XS strand attribute, which STAR will generate with –outSAMstrandField intronMotif option. As required, the XS strand attribute will be generated for all alignments that contain splice junctions. The spliced alignments that have undefined strand (i.e. containing only non-canonical unannotated junctions) will be suppressed. If you have stranded RNA-seq data, you do not need to use any specific STAR options. Instead, you need to run Cufflinks with the library option –library-type options. For example, c ufflinks –library-type fr-firststrand should be used for the standard dUTP protocol, including Illumina’s stranded Tru-Seq. This option has to be used only for Cufflinks runs and not for STAR runs.
hidden:!alignment.show_advanced
default:False
alignment.noncannonical
label:Remove non-cannonical junctions (Cufflinks compatibility)
type:basic:boolean
description:It is recommended to remove the non-canonical junctions for Cufflinks runs using –outFilterIntronMotifs RemoveNoncanonical.
hidden:!alignment.show_advanced
default:False
alignment.detect_chimeric.chimeric
label:Detect chimeric and circular alignments
type:basic:boolean
description:To switch on detection of chimeric (fusion) alignments (in addition to normal mapping), –chimSegmentMin should be set to a positive value. Each chimeric alignment consists of two “segments”. Each segment is non-chimeric on its own, but the segments are chimeric to each other (i.e. the segments belong to different chromosomes, or different strands, or are far from each other). Both segments may contain splice junctions, and one of the segments may contain portions of both mates. –chimSegmentMin parameter controls the minimum mapped length of the two segments that is allowed. For example, if you have 2x75 reads and used –chimSegmentMin 20, a chimeric alignment with 130b on one chromosome and 20b on the other will be output, while 135 + 15 won’t be.
default:False
alignment.detect_chimeric.chimSegmentMin
label:–chimSegmentMin
type:basic:integer
disabled:detect_chimeric.chimeric != true
default:20
alignment.t_coordinates.quantmode
label:Output in transcript coordinates
type:basic:boolean
description:With –quantMode TranscriptomeSAM option STAR will output alignments translated into transcript coordinates in the Aligned.toTranscriptome.out.bam file (in addition to alignments in genomic coordinates in Aligned.*.sam/bam files). These transcriptomic alignments can be used with various transcript quantification software that require reads to be mapped to transcriptome, such as RSEM or eXpress.
default:False
alignment.t_coordinates.singleend
label:Allow soft-clipping and indels
type:basic:boolean
description:By default, the output satisfies RSEM requirements: soft-clipping or indels are not allowed. Use –quantTranscriptomeBan Singleend to allow insertions, deletions ans soft-clips in the transcriptomic alignments, which can be used by some expression quantification software (e.g. eXpress).
disabled:t_coordinates.quantmode != true
default:False
alignment.t_coordinates.gene_counts
label:Count reads
type:basic:boolean
description:With –quantMode GeneCounts option STAR will count number reads per gene while mapping. A read is counted if it overlaps (1nt or more) one and only one gene. Both ends of the paired-end read are checked for overlaps. The counts coincide with those produced by htseq-count with default parameters. ReadsPerGene.out.tab file with 4 columns which correspond to different strandedness options: column 1: gene ID; column 2: counts for unstranded RNA-seq; column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes); column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse).
disabled:t_coordinates.quantmode != true
default:False
alignment.filtering.outFilterType
label:Type of filtering
type:basic:string
description:Normal: standard filtering using only current alignment; BySJout: keep only those reads that contain junctions that passed filtering into SJ.out.tab
default:Normal
choices:
  • Normal: Normal
  • BySJout: BySJout
alignment.filtering.outFilterMultimapNmax
label:–outFilterMultimapNmax
type:basic:integer
description:Read alignments will be output only if the read maps fewer than this value, otherwise no alignments will be output (default: 10).
required:False
alignment.filtering.outFilterMismatchNmax
label:–outFilterMismatchNmax
type:basic:integer
description:Alignment will be output only if it has fewer mismatches than this value (default: 10).
required:False
alignment.filtering.outFilterMismatchNoverLmax
label:–outFilterMismatchNoverLmax
type:basic:decimal
description:Max number of mismatches per pair relative to read length: for 2x100b, max number of mismatches is 0.06*200=8 for the paired read.
required:False
alignment.filtering.outFilterScoreMin
label:–outFilterScoreMin
type:basic:integer
description:Alignment will be output only if its score is higher than or equal to this value (default: 0).
required:False
alignment.alignment.alignSJoverhangMin
label:–alignSJoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for spliced alignments (default: 5).
required:False
alignment.alignment.alignSJDBoverhangMin
label:–alignSJDBoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for annotated (sjdb) spliced alignments (default: 3).
required:False
alignment.alignment.alignIntronMin
label:–alignIntronMin
type:basic:integer
description:Minimum intron size: genomic gap is considered intron if its length >= alignIntronMin, otherwise it is considered Deletion (default: 21).
required:False
alignment.alignment.alignIntronMax
label:–alignIntronMax
type:basic:integer
description:Maximum intron size, if 0, max intron size will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
alignment.alignment.alignMatesGapMax
label:–alignMatesGapMax
type:basic:integer
description:Maximum gap between two mates, if 0, max intron gap will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
alignment.alignment.alignEndsType
label:–alignEndsType
type:basic:string
description:Type of read ends alignment (default: Local).
required:False
default:Local
choices:
  • Local: Local
  • EndToEnd: EndToEnd
  • Extend5pOfRead1: Extend5pOfRead1
  • Extend5pOfReads12: Extend5pOfReads12
alignment.output_sam_bam.outSAMunmapped
label:–outSAMunmapped
type:basic:string
description:Output of unmapped reads in the SAM format.
required:False
default:None
choices:
  • None: None
  • Within: Within
alignment.output_sam_bam.outSAMattributes
label:–outSAMattributes
type:basic:string
description:a string of desired SAM attributes, in the order desired for the output SAM.
required:False
default:Standard
choices:
  • None: None
  • Standard: Standard
  • All: All
alignment.output_sam_bam.outSAMattrRGline
label:–outSAMattrRGline
type:basic:string
description:SAM/BAM read group line. The first word contains the read group identifier and must start with “ID:”, e.g. –outSAMattrRGline ID:xxx CN:yy “DS:z z z”
required:False
quantification.annotation
label:Annotation
type:data:annotation
quantification.show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
quantification.assay_type
label:Assay type
type:basic:string
description:In strand non-specific assay a read is considered overlapping with a feature regardless of whether it is mapped to the same or the opposite strand as the feature. In strand-specific forward assay and single reads, the read has to be mapped to the same strand as the feature. For paired-end reads, the first read has to be on the same strand and the second read on the opposite strand. In strand-specific reverse assay these rules are reversed.
hidden:!quantification.show_advanced
default:non_specific
choices:
  • Strand non-specific: non_specific
  • Strand-specific forward: forward
  • Strand-specific reverse: reverse
  • Detect automatically: auto
quantification.cdna_index
label:cDNA index file
type:data:index:salmon
description:Transcriptome index file created using the Salmon indexing tool. cDNA (transcriptome) sequences used for index file creation must be derived from the same species as the input sequencing reads to obtain the reliable analysis results.
required:False
hidden:quantification.assay_type != ‘auto’
quantification.n_reads
label:Number of reads in subsampled alignment file
type:basic:integer
description:Alignment (.bam) file subsample size. Increase the number of reads to make automatic detection more reliable. Decrease the number of reads to make automatic detection run faster.
hidden:quantification.assay_type != ‘auto’
default:5000000
quantification.feature_class
label:Feature class
type:basic:string
description:Feature class (3rd column in GTF/GFF3 file) to be used. All other features will be ignored.
hidden:!quantification.show_advanced
default:exon
quantification.feature_type
label:Feature type
type:basic:string
description:The type of feature the quantification program summarizes over (e.g. gene or transcript-level analysis). The value of this parameter needs to be chosen in line with ‘ID attribute’ below.
hidden:!quantification.show_advanced
default:gene
choices:
  • gene: gene
  • transcript: transcript
quantification.id_attribute
label:ID attribute
type:basic:string
description:GTF/GFF3 attribute to be used as feature ID. Several GTF/GFF3 lines with the same feature ID are considered as parts of the same feature. The feature ID is used to identify the counts in the output table. In GTF files this is usually ‘gene_id’, in GFF3 files this is often ‘ID’, and ‘transcript_id’ is frequently a valid choice for both annotation formats.
hidden:!quantification.show_advanced
default:gene_id
choices:
  • gene_id: gene_id
  • transcript_id: transcript_id
  • ID: ID
  • geneid: geneid
downsampling.n_reads
label:Number of reads
type:basic:integer
default:1000000
downsampling.advanced.seed
label:Seed
type:basic:integer
default:11
downsampling.advanced.fraction
label:Fraction
type:basic:decimal
description:Use the fraction of reads [0 - 1.0] from the orignal input file instead of the absolute number of reads. If set, this will override the “Number of reads” input parameter.
required:False
downsampling.advanced.two_pass
label:2-pass mode
type:basic:boolean
description:Enable two-pass mode when down-sampling. Two-pass mode is twice as slow but with much reduced memory.
default:False
qc.rrna_reference
label:Indexed rRNA reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
qc.globin_reference
label:Indexed Globin reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.

Output results

BBDuk - STAR - featureCounts - QC (single-end)

data:workflow:rnaseq:featurecounts:qcworkflow-bbduk-star-featurecounts-qc-single (data:reads:fastq:single  reads, list:data:seq:nucleotide  adapters, basic:boolean  show_advanced, list:basic:string  custom_adapter_sequences, basic:integer  kmer_length, basic:integer  min_k, basic:integer  hamming_distance, basic:integer  maxns, basic:integer  trim_quality, basic:integer  min_length, data:genomeindex:star  genome, basic:boolean  show_advanced, basic:boolean  unstranded, basic:boolean  noncannonical, basic:boolean  chimeric, basic:integer  chimSegmentMin, basic:boolean  quantmode, basic:boolean  singleend, basic:boolean  gene_counts, basic:string  outFilterType, basic:integer  outFilterMultimapNmax, basic:integer  outFilterMismatchNmax, basic:decimal  outFilterMismatchNoverLmax, basic:integer  outFilterScoreMin, basic:integer  alignSJoverhangMin, basic:integer  alignSJDBoverhangMin, basic:integer  alignIntronMin, basic:integer  alignIntronMax, basic:integer  alignMatesGapMax, basic:string  alignEndsType, basic:string  outSAMunmapped, basic:string  outSAMattributes, basic:string  outSAMattrRGline, data:annotation  annotation, basic:boolean  show_advanced, basic:string  assay_type, data:index:salmon  cdna_index, basic:integer  n_reads, basic:string  feature_class, basic:string  feature_type, basic:string  id_attribute, basic:integer  n_reads, basic:integer  seed, basic:decimal  fraction, basic:boolean  two_pass, data:genomeindex:star  rrna_reference, data:genomeindex:star  globin_reference)[Source: v1.4.0]

This RNA-seq pipeline is comprised of three steps preprocessing, alignment, and quantification. First, reads are preprocessed by __BBDuk__ which removes adapters, trims reads for quality from the 3’-end, and discards reads that are too short after trimming. Compared to similar tools, BBDuk is regarded for its computational efficiency. Next, preprocessed reads are aligned by __STAR__ aligner. At the time of implementation, STAR is considered a state-of-the-art tool that consistently produces accurate results from diverse sets of reads, and performs well even with default settings. For more information see [this comparison of RNA-seq aligners](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792058/). Finally, aligned reads are summarized to genes by __featureCounts__. Gaining wide adoption among the bioinformatics community, featureCounts yields expressions in a computationally efficient manner. All three tools in this workflow support parallelization to accelerate the analysis. rRNA contamination rate in the sample is determined using the STAR aligner. Quality-trimmed reads are down-sampled (using Seqtk tool) and aligned to the rRNA reference sequences. The alignment rate indicates the percentage of the reads in the sample that are derived from the rRNA sequences.

Input arguments

preprocessing.reads
label:Reads
type:data:reads:fastq:single
preprocessing.adapters
label:Adapters
type:list:data:seq:nucleotide
required:False
preprocessing.show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
preprocessing.custom_adapter_sequences
label:Custom adapter sequences [literal]
type:list:basic:string
description:Custom adapter sequences can be specified by inputting them one by one and pressing Enter after each sequence.
required:False
hidden:!preprocessing.show_advanced
default:[]
preprocessing.kmer_length
label:K-mer length
type:basic:integer
description:K-mer length must be smaller or equal to the length of adapters.
hidden:!preprocessing.show_advanced
default:23
preprocessing.min_k
label:Minimum k-mer length at right end of reads used for trimming
type:basic:integer
disabled:preprocessing.adapters.length === 0 && preprocessing.custom_adapter_sequences.length === 0
hidden:!preprocessing.show_advanced
default:11
preprocessing.hamming_distance
label:Maximum Hamming distance for k-mers
type:basic:integer
hidden:!preprocessing.show_advanced
default:1
preprocessing.maxns
label:Max Ns after trimming [maxns=-1]
type:basic:integer
description:If non-negative, reads with more Ns than this (after trimming) will be discarded.
hidden:!preprocessing.show_advanced
default:-1
preprocessing.trim_quality
label:Quality below which to trim reads from the right end
type:basic:integer
description:Phred algorithm is used, which is more accurate than naive trimming.
hidden:!preprocessing.show_advanced
default:10
preprocessing.min_length
label:Minimum read length
type:basic:integer
description:Reads shorter than minimum read length after trimming are discarded.
hidden:!preprocessing.show_advanced
default:20
alignment.genome
label:Indexed reference genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
alignment.show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
alignment.unstranded
label:The data is unstranded
type:basic:boolean
description:For unstranded RNA-seq data, Cufflinks/Cuffdiff require spliced alignments with XS strand attribute, which STAR will generate with –outSAMstrandField intronMotif option. As required, the XS strand attribute will be generated for all alignments that contain splice junctions. The spliced alignments that have undefined strand (i.e. containing only non-canonical unannotated junctions) will be suppressed. If you have stranded RNA-seq data, you do not need to use any specific STAR options. Instead, you need to run Cufflinks with the library option –library-type options. For example, c ufflinks –library-type fr-firststrand should be used for the standard dUTP protocol, including Illumina’s stranded Tru-Seq. This option has to be used only for Cufflinks runs and not for STAR runs.
hidden:!alignment.show_advanced
default:False
alignment.noncannonical
label:Remove non-cannonical junctions (Cufflinks compatibility)
type:basic:boolean
description:It is recommended to remove the non-canonical junctions for Cufflinks runs using –outFilterIntronMotifs RemoveNoncanonical.
hidden:!alignment.show_advanced
default:False
alignment.detect_chimeric.chimeric
label:Detect chimeric and circular alignments
type:basic:boolean
description:To switch on detection of chimeric (fusion) alignments (in addition to normal mapping), –chimSegmentMin should be set to a positive value. Each chimeric alignment consists of two “segments”. Each segment is non-chimeric on its own, but the segments are chimeric to each other (i.e. the segments belong to different chromosomes, or different strands, or are far from each other). Both segments may contain splice junctions, and one of the segments may contain portions of both mates. –chimSegmentMin parameter controls the minimum mapped length of the two segments that is allowed. For example, if you have 2x75 reads and used –chimSegmentMin 20, a chimeric alignment with 130b on one chromosome and 20b on the other will be output, while 135 + 15 won’t be.
default:False
alignment.detect_chimeric.chimSegmentMin
label:–chimSegmentMin
type:basic:integer
disabled:detect_chimeric.chimeric != true
default:20
alignment.t_coordinates.quantmode
label:Output in transcript coordinates
type:basic:boolean
description:With –quantMode TranscriptomeSAM option STAR will output alignments translated into transcript coordinates in the Aligned.toTranscriptome.out.bam file (in addition to alignments in genomic coordinates in Aligned.*.sam/bam files). These transcriptomic alignments can be used with various transcript quantification software that require reads to be mapped to transcriptome, such as RSEM or eXpress.
default:False
alignment.t_coordinates.singleend
label:Allow soft-clipping and indels
type:basic:boolean
description:By default, the output satisfies RSEM requirements: soft-clipping or indels are not allowed. Use –quantTranscriptomeBan Singleend to allow insertions, deletions ans soft-clips in the transcriptomic alignments, which can be used by some expression quantification software (e.g. eXpress).
disabled:t_coordinates.quantmode != true
default:False
alignment.t_coordinates.gene_counts
label:Count reads
type:basic:boolean
description:With –quantMode GeneCounts option STAR will count number reads per gene while mapping. A read is counted if it overlaps (1nt or more) one and only one gene. Both ends of the paired-end read are checked for overlaps. The counts coincide with those produced by htseq-count with default parameters. ReadsPerGene.out.tab file with 4 columns which correspond to different strandedness options: column 1: gene ID; column 2: counts for unstranded RNA-seq; column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes); column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse).
disabled:t_coordinates.quantmode != true
default:False
alignment.filtering.outFilterType
label:Type of filtering
type:basic:string
description:Normal: standard filtering using only current alignment; BySJout: keep only those reads that contain junctions that passed filtering into SJ.out.tab
default:Normal
choices:
  • Normal: Normal
  • BySJout: BySJout
alignment.filtering.outFilterMultimapNmax
label:–outFilterMultimapNmax
type:basic:integer
description:Read alignments will be output only if the read maps fewer than this value, otherwise no alignments will be output (default: 10).
required:False
alignment.filtering.outFilterMismatchNmax
label:–outFilterMismatchNmax
type:basic:integer
description:Alignment will be output only if it has fewer mismatches than this value (default: 10).
required:False
alignment.filtering.outFilterMismatchNoverLmax
label:–outFilterMismatchNoverLmax
type:basic:decimal
description:Max number of mismatches per pair relative to read length: for 2x100b, max number of mismatches is 0.06*200=8 for the paired read.
required:False
alignment.filtering.outFilterScoreMin
label:–outFilterScoreMin
type:basic:integer
description:Alignment will be output only if its score is higher than or equal to this value (default: 0).
required:False
alignment.alignment.alignSJoverhangMin
label:–alignSJoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for spliced alignments (default: 5).
required:False
alignment.alignment.alignSJDBoverhangMin
label:–alignSJDBoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for annotated (sjdb) spliced alignments (default: 3).
required:False
alignment.alignment.alignIntronMin
label:–alignIntronMin
type:basic:integer
description:Minimum intron size: genomic gap is considered intron if its length >= alignIntronMin, otherwise it is considered Deletion (default: 21).
required:False
alignment.alignment.alignIntronMax
label:–alignIntronMax
type:basic:integer
description:Maximum intron size, if 0, max intron size will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
alignment.alignment.alignMatesGapMax
label:–alignMatesGapMax
type:basic:integer
description:Maximum gap between two mates, if 0, max intron gap will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
alignment.alignment.alignEndsType
label:–alignEndsType
type:basic:string
description:Type of read ends alignment (default: Local).
required:False
default:Local
choices:
  • Local: Local
  • EndToEnd: EndToEnd
  • Extend5pOfRead1: Extend5pOfRead1
  • Extend5pOfReads12: Extend5pOfReads12
alignment.output_sam_bam.outSAMunmapped
label:–outSAMunmapped
type:basic:string
description:Output of unmapped reads in the SAM format.
required:False
default:None
choices:
  • None: None
  • Within: Within
alignment.output_sam_bam.outSAMattributes
label:–outSAMattributes
type:basic:string
description:a string of desired SAM attributes, in the order desired for the output SAM.
required:False
default:Standard
choices:
  • None: None
  • Standard: Standard
  • All: All
alignment.output_sam_bam.outSAMattrRGline
label:–outSAMattrRGline
type:basic:string
description:SAM/BAM read group line. The first word contains the read group identifier and must start with “ID:”, e.g. –outSAMattrRGline ID:xxx CN:yy “DS:z z z”
required:False
quantification.annotation
label:Annotation
type:data:annotation
quantification.show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
quantification.assay_type
label:Assay type
type:basic:string
description:In strand non-specific assay a read is considered overlapping with a feature regardless of whether it is mapped to the same or the opposite strand as the feature. In strand-specific forward assay and single reads, the read has to be mapped to the same strand as the feature. For paired-end reads, the first read has to be on the same strand and the second read on the opposite strand. In strand-specific reverse assay these rules are reversed.
hidden:!quantification.show_advanced
default:non_specific
choices:
  • Strand non-specific: non_specific
  • Strand-specific forward: forward
  • Strand-specific reverse: reverse
  • Detect automatically: auto
quantification.cdna_index
label:cDNA index file
type:data:index:salmon
description:Transcriptome index file created using the Salmon indexing tool. cDNA (transcriptome) sequences used for index file creation must be derived from the same species as the input sequencing reads to obtain the reliable analysis results.
required:False
hidden:quantification.assay_type != ‘auto’
quantification.n_reads
label:Number of reads in subsampled alignment file
type:basic:integer
description:Alignment (.bam) file subsample size. Increase the number of reads to make automatic detection more reliable. Decrease the number of reads to make automatic detection run faster.
hidden:quantification.assay_type != ‘auto’
default:5000000
quantification.feature_class
label:Feature class
type:basic:string
description:Feature class (3rd column in GTF/GFF3 file) to be used. All other features will be ignored.
hidden:!quantification.show_advanced
default:exon
quantification.feature_type
label:Feature type
type:basic:string
description:The type of feature the quantification program summarizes over (e.g. gene or transcript-level analysis). The value of this parameter needs to be chosen in line with ‘ID attribute’ below.
hidden:!quantification.show_advanced
default:gene
choices:
  • gene: gene
  • transcript: transcript
quantification.id_attribute
label:ID attribute
type:basic:string
description:GTF/GFF3 attribute to be used as feature ID. Several GTF/GFF3 lines with the same feature ID will be considered as parts of the same feature. The feature ID is used to identify the counts in the output table. In GTF files this is usually ‘gene_id’, in GFF3 files this is often ‘ID’, and ‘transcript_id’ is frequently a valid choice for both annotation formats.
hidden:!quantification.show_advanced
default:gene_id
choices:
  • gene_id: gene_id
  • transcript_id: transcript_id
  • ID: ID
  • geneid: geneid
downsampling.n_reads
label:Number of reads
type:basic:integer
default:1000000
downsampling.advanced.seed
label:Seed
type:basic:integer
default:11
downsampling.advanced.fraction
label:Fraction
type:basic:decimal
description:Use the fraction of reads [0 - 1.0] from the orignal input file instead of the absolute number of reads. If set, this will override the “Number of reads” input parameter.
required:False
downsampling.advanced.two_pass
label:2-pass mode
type:basic:boolean
description:Enable two-pass mode when down-sampling. Two-pass mode is twice as slow but with much reduced memory.
default:False
qc.rrna_reference
label:Indexed rRNA reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
qc.globin_reference
label:Indexed Globin reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.

Output results

BBDuk - Salmon - QC (paired-end)

data:workflow:rnaseq:salmonworkflow-bbduk-salmon-qc-paired (data:reads:fastq:paired  reads, data:index:salmon  salmon_index, data:genomeindex:star  genome, data:annotation  annotation, data:genomeindex:star  rrna_reference, data:genomeindex:star  globin_reference, basic:boolean  show_advanced, list:data:seq:nucleotide  adapters, list:basic:string  custom_adapter_sequences, basic:integer  kmer_length, basic:integer  min_k, basic:integer  hamming_distance, basic:integer  maxns, basic:integer  trim_quality, basic:integer  min_length, basic:boolean  seq_bias, basic:boolean  gc_bias, basic:boolean  validate_mappings, basic:decimal  consensus_slack, basic:decimal  min_score_fraction, basic:integer  range_factorization_bins, basic:integer  min_assigned_frag, basic:integer  n_reads, basic:integer  seed, basic:decimal  fraction, basic:boolean  two_pass)[Source: v1.0.1]

Alignment-free RNA-seq pipeline. Salmon tool and tximport package are used in quantification step to produce gene-level abundance estimates. rRNA and globin-sequence contamination rate in the sample is determined using STAR aligner. Quality-trimmed reads are down-sampled (using Seqtk tool) and aligned to the genome, rRNA and globin reference sequences. The rRNA and globin-sequence alignment rates indicate the percentage of the reads in the sample that are of rRNA and globin origin, respectively. Alignment of down-sampled data to a whole genome reference sequence is used to produce an alignment file suitable for Samtools and QoRTs QC analysis. Per-sample analysis results and QC data is summarized by the MultiQC tool.

Input arguments

reads
label:Select sample(s)
type:data:reads:fastq:paired
salmon_index
label:Salmon index
type:data:index:salmon
genome
label:Indexed reference genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
annotation
label:Annotation
type:data:annotation
rrna_reference
label:Indexed rRNA reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
globin_reference
label:Indexed Globin reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
preprocessing.adapters
label:Adapters
type:list:data:seq:nucleotide
required:False
preprocessing.custom_adapter_sequences
label:Custom adapter sequences [literal]
type:list:basic:string
description:Custom adapter sequences can be specified by inputting them one by one and pressing Enter after each sequence.
required:False
default:[]
preprocessing.kmer_length
label:K-mer length
type:basic:integer
description:K-mer length must be smaller or equal to the length of adapters.
default:23
preprocessing.min_k
label:Minimum k-mer length at right end of reads used for trimming
type:basic:integer
disabled:preprocessing.adapters.length === 0 && preprocessing.custom_adapter_sequences.length === 0
default:11
preprocessing.hamming_distance
label:Maximum Hamming distance for k-mers
type:basic:integer
default:1
preprocessing.maxns
label:Max Ns after trimming [maxns=-1]
type:basic:integer
description:If non-negative, reads with more Ns than this (after trimming) will be discarded.
default:-1
preprocessing.trim_quality
label:Quality below which to trim reads from the right end
type:basic:integer
description:Phred algorithm is used, which is more accurate than naive trimming.
default:10
preprocessing.min_length
label:Minimum read length
type:basic:integer
description:Reads shorter than minimum read length after trimming are discarded.
default:20
quantification.seq_bias
label:Perform sequence-specific bias correction
type:basic:boolean
default:True
quantification.gc_bias
label:Perform fragment GC bias correction.
type:basic:boolean
default:True
quantification.validate_mappings
label:Validate mappings using alignment-based verification.
type:basic:boolean
default:True
quantification.consensus_slack
label:Consensus slack
type:basic:decimal
description:The amount of slack allowed in the quasi-mapping consensus mechanism. Normally, a transcript must cover all hits to be considered for mapping. If this is set to a fraction, X, greater than 0 (and in [0,1)), then a transcript can fail to cover up to (100 * X)% of the hits before it is discounted as a mapping candidate. The default value of this option is 0.2 if –validateMappings is given and 0 otherwise”.
required:False
hidden:!quantification.validate_mappings
quantification.min_score_fraction
label:Minimum alignment score fraction
type:basic:decimal
description:The fraction of the optimal possible alignment score that a mapping must achieve in order to be considered valid - should be in (0,1].
hidden:!quantification.validate_mappings
default:0.65
quantification.range_factorization_bins
label:Range factorization bins
type:basic:integer
description:Factorizes the likelihood used in quantification by adopting a new notion of equivalence classes based on the conditional probabilities with which fragments are generated from different transcripts. This is a more fine-grained factorization than the normal rich equivalence classes. The default value (0) corresponds to the standard rich equivalence classes, and larger values imply a more fine-grained factorization. If range factorization is enabled, a common value to select for this parameter is 4.
default:4
quantification.min_assigned_frag
label:Minimum number of assigned fragments
type:basic:integer
description:The minimum number of fragments that must be assigned to the transcriptome for quantification to proceed.
default:10
downsampling.n_reads
label:Number of reads
type:basic:integer
default:10000000
downsampling.seed
label:Seed
type:basic:integer
default:11
downsampling.fraction
label:Fraction
type:basic:decimal
description:Use the fraction of reads [0 - 1.0] from the original input file instead of the absolute number of reads. If set, this will override the “Number of reads” input parameter.
required:False
downsampling.two_pass
label:2-pass mode
type:basic:boolean
description:Enable two-pass mode when down-sampling. Two-pass mode is twice as slow but with much reduced memory.
default:False

Output results

BBDuk - Salmon - QC (single-end)

data:workflow:rnaseq:salmonworkflow-bbduk-salmon-qc-single (data:reads:fastq:single  reads, data:index:salmon  salmon_index, data:genomeindex:star  genome, data:annotation  annotation, data:genomeindex:star  rrna_reference, data:genomeindex:star  globin_reference, basic:boolean  show_advanced, list:data:seq:nucleotide  adapters, list:basic:string  custom_adapter_sequences, basic:integer  kmer_length, basic:integer  min_k, basic:integer  hamming_distance, basic:integer  maxns, basic:integer  trim_quality, basic:integer  min_length, basic:boolean  seq_bias, basic:boolean  gc_bias, basic:boolean  validate_mappings, basic:decimal  consensus_slack, basic:decimal  min_score_fraction, basic:integer  range_factorization_bins, basic:integer  min_assigned_frag, basic:integer  n_reads, basic:integer  seed, basic:decimal  fraction, basic:boolean  two_pass)[Source: v1.0.1]

Alignment-free RNA-seq pipeline. Salmon tool and tximport package are used in quantification step to produce gene-level abundance estimates. rRNA and globin-sequence contamination rate in the sample is determined using STAR aligner. Quality-trimmed reads are down-sampled (using Seqtk tool) and aligned to the genome, rRNA and globin reference sequences. The rRNA and globin-sequence alignment rates indicate the percentage of the reads in the sample that are of rRNA and globin origin, respectively. Alignment of down-sampled data to a whole genome reference sequence is used to produce an alignment file suitable for Samtools and QoRTs QC analysis. Per-sample analysis results and QC data is summarized by the MultiQC tool.

Input arguments

reads
label:Select sample(s)
type:data:reads:fastq:single
salmon_index
label:Salmon index
type:data:index:salmon
genome
label:Indexed reference genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
annotation
label:Annotation
type:data:annotation
rrna_reference
label:Indexed rRNA reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
globin_reference
label:Indexed Globin reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
preprocessing.adapters
label:Adapters
type:list:data:seq:nucleotide
required:False
preprocessing.custom_adapter_sequences
label:Custom adapter sequences [literal]
type:list:basic:string
description:Custom adapter sequences can be specified by inputting them one by one and pressing Enter after each sequence.
required:False
default:[]
preprocessing.kmer_length
label:K-mer length
type:basic:integer
description:K-mer length must be smaller or equal to the length of adapters.
default:23
preprocessing.min_k
label:Minimum k-mer length at right end of reads used for trimming
type:basic:integer
disabled:preprocessing.adapters.length === 0 && preprocessing.custom_adapter_sequences.length === 0
default:11
preprocessing.hamming_distance
label:Maximum Hamming distance for k-mers
type:basic:integer
default:1
preprocessing.maxns
label:Max Ns after trimming [maxns=-1]
type:basic:integer
description:If non-negative, reads with more Ns than this (after trimming) will be discarded.
default:-1
preprocessing.trim_quality
label:Quality below which to trim reads from the right end
type:basic:integer
description:Phred algorithm is used, which is more accurate than naive trimming.
default:10
preprocessing.min_length
label:Minimum read length
type:basic:integer
description:Reads shorter than minimum read length after trimming are discarded.
default:20
quantification.seq_bias
label:Perform sequence-specific bias correction
type:basic:boolean
default:True
quantification.gc_bias
label:Perform fragment GC bias correction.
type:basic:boolean
default:False
quantification.validate_mappings
label:Validate mappings using alignment-based verification.
type:basic:boolean
default:True
quantification.consensus_slack
label:Consensus slack
type:basic:decimal
description:The amount of slack allowed in the quasi-mapping consensus mechanism. Normally, a transcript must cover all hits to be considered for mapping. If this is set to a fraction, X, greater than 0 (and in [0,1)), then a transcript can fail to cover up to (100 * X)% of the hits before it is discounted as a mapping candidate. The default value of this option is 0.2 if –validateMappings is given and 0 otherwise”.
required:False
hidden:!quantification.validate_mappings
quantification.min_score_fraction
label:Minimum alignment score fraction
type:basic:decimal
description:The fraction of the optimal possible alignment score that a mapping must achieve in order to be considered valid - should be in (0,1].
hidden:!quantification.validate_mappings
default:0.65
quantification.range_factorization_bins
label:Range factorization bins
type:basic:integer
description:Factorizes the likelihood used in quantification by adopting a new notion of equivalence classes based on the conditional probabilities with which fragments are generated from different transcripts. This is a more fine-grained factorization than the normal rich equivalence classes. The default value (0) corresponds to the standard rich equivalence classes, and larger values imply a more fine-grained factorization. If range factorization is enabled, a common value to select for this parameter is 4.
default:4
quantification.min_assigned_frag
label:Minimum number of assigned fragments
type:basic:integer
description:The minimum number of fragments that must be assigned to the transcriptome for quantification to proceed.
default:10
downsampling.n_reads
label:Number of reads
type:basic:integer
default:10000000
downsampling.seed
label:Seed
type:basic:integer
default:11
downsampling.fraction
label:Fraction
type:basic:decimal
description:Use the fraction of reads [0 - 1.0] from the original input file instead of the absolute number of reads. If set, this will override the “Number of reads” input parameter.
required:False
downsampling.two_pass
label:2-pass mode
type:basic:boolean
description:Enable two-pass mode when down-sampling. Two-pass mode is twice as slow but with much reduced memory.
default:False

Output results

BED file

data:bedupload-bed (basic:file  src, basic:string  species, basic:string  build)[Source: v1.3.1]

Import a BED file (.bed) which is a tab-delimited text file that defines a feature track. It can have any file extension, but .bed is recommended. The BED file format is described on the [UCSC Genome Bioinformatics web site](http://genome.ucsc.edu/FAQ/FAQformat#format1).

Input arguments

src
label:BED file
type:basic:file
description:Upload BED file annotation track. The first three required BED fields are chrom, chromStart and chromEnd.
required:True
validate_regex:\.(bed|narrowPeak)$
species
label:Species
type:basic:string
description:Species latin name.
choices:
  • Homo sapiens: Homo sapiens
  • Mus musculus: Mus musculus
  • Rattus norvegicus: Rattus norvegicus
  • Dictyostelium discoideum: Dictyostelium discoideum
  • Odocoileus virginianus texanus: Odocoileus virginianus texanus
  • Solanum tuberosum: Solanum tuberosum
build
label:Genome build
type:basic:string

Output results

bed
label:BED file
type:basic:file
bed_jbrowse
label:Bgzip bed file for JBrowse
type:basic:file
tbi_jbrowse
label:Bed file index for Jbrowse
type:basic:file
species
label:Species
type:basic:string
build
label:Build
type:basic:string

BWA ALN

data:alignment:bam:bwaalnalignment-bwa-aln (data:genome:fasta  genome, data:reads:fastq  reads, basic:integer  q, basic:boolean  use_edit, basic:integer  edit_value, basic:decimal  fraction, basic:boolean  seeds, basic:integer  seed_length, basic:integer  seed_dist)[Source: v1.5.0]

Read aligner for mapping low-divergent sequences against a large reference genome. Designed for Illumina sequence reads up to 100bp.

Input arguments

genome
label:Reference genome
type:data:genome:fasta
reads
label:Reads
type:data:reads:fastq
q
label:Quality threshold
type:basic:integer
description:Parameter for dynamic read trimming.
default:0
use_edit
label:Use maximum edit distance (excludes fraction of missing alignments)
type:basic:boolean
default:False
edit_value
label:Maximum edit distance
type:basic:integer
hidden:!use_edit
default:5
fraction
label:Fraction of missing alignments
type:basic:decimal
description:The fraction of missing alignments given 2% uniform base error rate. The maximum edit distance is automatically chosen for different read lengths.
hidden:use_edit
default:0.04
seeds
label:Use seeds
type:basic:boolean
default:False
seed_length
label:Seed length
type:basic:integer
description:Take the first X subsequence as seed. If X is larger than the query sequence, seeding will be disabled. For long reads, this option is typically ranged from 25 to 35 for value 2 in seed maximum edit distance.
hidden:!seeds
default:35
seed_dist
label:Seed maximum edit distance
type:basic:integer
hidden:!seeds
default:2

Output results

bam
label:Alignment file
type:basic:file
description:Position sorted alignment
bai
label:Index BAI
type:basic:file
unmapped
label:Unmapped reads
type:basic:file
required:False
stats
label:Statistics
type:basic:file
bigwig
label:BigWig file
type:basic:file
required:False
species
label:Species
type:basic:string
build
label:Build
type:basic:string

BWA MEM

data:alignment:bam:bwamemalignment-bwa-mem (data:genome:fasta  genome, data:reads:fastq  reads, basic:integer  seed_l, basic:integer  band_w, basic:decimal  re_seeding, basic:boolean  m, basic:integer  match, basic:integer  missmatch, basic:integer  gap_o, basic:integer  gap_e, basic:integer  clipping, basic:integer  unpaired_p, basic:boolean  report_all, basic:integer  report_tr)[Source: v2.3.0]

BWA MEM is a read aligner for mapping low-divergent sequences against a large reference genome. Designed for longer sequences ranged from 70bp to 1Mbp. The algorithm works by seeding alignments with maximal exact matches (MEMs) and then extending seeds with the affine-gap Smith-Waterman algorithm (SW). See [here](http://bio-bwa.sourceforge.net/) for more information.

Input arguments

genome
label:Reference genome
type:data:genome:fasta
reads
label:Reads
type:data:reads:fastq
seed_l
label:Minimum seed length
type:basic:integer
description:Minimum seed length. Matches shorter than minimum seed length will be missed. The alignment speed is usually insensitive to this value unless it significantly deviates from 20.
default:19
band_w
label:Band width
type:basic:integer
description:Gaps longer than this will not be found.
default:100
re_seeding
label:Re-seeding factor
type:basic:decimal
description:Trigger re-seeding for a MEM longer than minSeedLen*FACTOR. This is a key heuristic parameter for tuning the performance. Larger value yields fewer seeds, which leads to faster alignment speed but lower accuracy.
default:1.5
m
label:Mark shorter split hits as secondary
type:basic:boolean
description:Mark shorter split hits as secondary (for Picard compatibility)
default:False
scoring.match
label:Score of a match
type:basic:integer
default:1
scoring.missmatch
label:Mismatch penalty
type:basic:integer
default:4
scoring.gap_o
label:Gap open penalty
type:basic:integer
default:6
scoring.gap_e
label:Gap extension penalty
type:basic:integer
default:1
scoring.clipping
label:Clipping penalty
type:basic:integer
description:Clipping is applied if final alignment score is smaller than (best score reaching the end of query) - (Clipping penalty)
default:5
scoring.unpaired_p
label:Penalty for an unpaired read pair
type:basic:integer
description:Affinity to force pair. Score: scoreRead1+scoreRead2-Penalty
default:9
reporting.report_all
label:Report all found alignments
type:basic:boolean
description:Output all found alignments for single-end or unpaired paired-end reads. These alignments will be flagged as secondary alignments.
default:False
reporting.report_tr
label:Report threshold score
type:basic:integer
description:Don’t output alignment with score lower than defined number. This option only affects output.
default:30

Output results

bam
label:Alignment file
type:basic:file
description:Position sorted alignment
bai
label:Index BAI
type:basic:file
unmapped
label:Unmapped reads
type:basic:file
required:False
stats
label:Statistics
type:basic:file
bigwig
label:BigWig file
type:basic:file
required:False
species
label:Species
type:basic:string
build
label:Build
type:basic:string

BWA SW

data:alignment:bam:bwaswalignment-bwa-sw (data:genome:fasta  genome, data:reads:fastq  reads, basic:integer  match, basic:integer  missmatch, basic:integer  gap_o, basic:integer  gap_e)[Source: v1.4.0]

Read aligner for mapping low-divergent sequences against a large reference genome. Designed for longer sequences ranged from 70bp to 1Mbp. The paired-end mode only works for reads Illumina short-insert libraries.

Input arguments

genome
label:Reference genome
type:data:genome:fasta
reads
label:Reads
type:data:reads:fastq
match
label:Score of a match
type:basic:integer
default:1
missmatch
label:Mismatch penalty
type:basic:integer
default:3
gap_o
label:Gap open penalty
type:basic:integer
default:5
gap_e
label:Gap extension penalty
type:basic:integer
default:2

Output results

bam
label:Alignment file
type:basic:file
description:Position sorted alignment
bai
label:Index BAI
type:basic:file
unmapped
label:Unmapped reads
type:basic:file
required:False
stats
label:Statistics
type:basic:file
bigwig
label:BigWig file
type:basic:file
required:False
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Bam split

data:alignment:bam:primarybam-split (data:alignment:bam  bam, data:sam:header  header, data:sam:header  header2)[Source: v0.4.0]

Split hybrid bam file into two bam files.

Input arguments

bam
label:Hybrid alignment bam
type:data:alignment:bam
header
label:Primary header sam file (optional)
type:data:sam:header
description:If no header file is provided, the headers will be extracted from the hybrid alignment bam file.
required:False
header2
label:Secondary header sam file (optional)
type:data:sam:header
description:If no header file is provided, the headers will be extracted from the hybrid alignment bam file.
required:False

Output results

bam
label:Uploaded file
type:basic:file
bai
label:Index BAI
type:basic:file
bigwig
label:BigWig file
type:basic:file
required:False
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Bamliquidator

data:bam:plot:bamliquidatorbamliquidator (basic:string  analysis_type, list:data:alignment:bam  bam, basic:string  cell_type, basic:integer  bin_size, data:annotation:gtf  regions_gtf, data:bed  regions_bed, basic:integer  extension, basic:string  sense, basic:boolean  skip_plot, list:basic:string  black_list, basic:integer  threads)[Source: v0.2.1]

Set of tools for analyzing the density of short DNA sequence read alignments in the BAM file format.

Input arguments

analysis_type
label:Analysis type
type:basic:string
default:bin
choices:
  • Bin mode: bin
  • Region mode: region
  • BED mode: bed
bam
label:BAM File
type:list:data:alignment:bam
cell_type
label:Cell type
type:basic:string
default:cell_type
bin_size
label:Bin size
type:basic:integer
description:Number of base pairs in each bin. The smaller the bin size the longer the runtime and the larger the data files. Default is 100000.
required:False
hidden:analysis_type != ‘bin’
regions_gtf
label:Region gff file / Annotation file (.gff|.gtf)
type:data:annotation:gtf
required:False
hidden:analysis_type != ‘region’
regions_bed
label:Region bed file / Annotation file (.bed)
type:data:bed
required:False
hidden:analysis_type != ‘bed’
extension
label:Extension
type:basic:integer
description:Extends reads by number of bp
default:200
sense
label:Mapping strand to gff file
type:basic:string
default:.
choices:
  • Forward: +
  • Reverse: -
  • Both: .
skip_plot
label:Skip plot
type:basic:boolean
required:False
black_list
label:Black list
type:list:basic:string
description:One or more chromosome patterns to skip during bin liquidation. Default is to skip any chromosomes that contain any of the following substrings chrUn _random Zv9_ _hap.
required:False
threads
label:Threads
type:basic:integer
description:Number of threads to run concurrently during liquidation.
default:1

Output results

analysis_type
label:Analysis type
type:basic:string
hidden:True
output_dir
label:Output directory
type:basic:file
counts
label:Counts HDF5 file
type:basic:file
matrix
label:Matrix file
type:basic:file
required:False
hidden:analysis_type != ‘region’
summary
label:Summary file
type:basic:file:html
required:False
hidden:analysis_type != ‘bin’

Bamplot

data:bam:plot:bamplotbamplot (basic:string  genome, data:annotation:gtf  input_gff, basic:string  input_region, list:data:alignment:bam  bam, basic:integer  stretch_input, basic:string  color, basic:string  sense, basic:integer  extension, basic:boolean  rpm, basic:string  yscale, list:basic:string  names, basic:string  plot, basic:string  title, basic:string  scale, list:data:bed  bed, basic:boolean  multi_page)[Source: v1.3.1]

Plot a single locus from a bam.

Input arguments

genome
label:Genome
type:basic:string
choices:
  • HG19: HG19
  • HG18: HG18
  • MM8: MM8
  • MM9: MM9
  • MM10: MM10
  • RN6: RN6
  • RN4: RN4
input_gff
label:Region string
type:data:annotation:gtf
description:Enter .gff file.
required:False
input_region
label:Region string
type:basic:string
description:Enter genomic region e.g. chr1:+:1-1000.
required:False
bam
label:Bam
type:list:data:alignment:bam
description:bam to plot from
required:False
stretch_input
label:Stretch-input
type:basic:integer
description:Stretch the input regions to a minimum length in bp, e.g. 10000 (for 10kb).
required:False
color
label:Color
type:basic:string
description:Enter a colon separated list of colors e.g. 255,0,0:255,125,0, default samples the rainbow.
default:255,0,0:255,125,0
sense
label:Sense
type:basic:string
description:Map to forward, reverse or’both strands. Default maps to both.
default:both
choices:
  • Forward: forward
  • Reverse: reverse
  • Both: both
extension
label:Extension
type:basic:integer
description:Extends reads by n bp. Default value is 200bp.
default:200
rpm
label:rpm
type:basic:boolean
description:Normalizes density to reads per million (rpm) Default is False.
required:False
yscale
label:y scale
type:basic:string
description:Choose either relative or uniform y axis scaling. Default is relative scaling.
default:relative
choices:
  • relative: relative
  • uniform: uniform
names
label:Names
type:list:basic:string
description:Enter a comma separated list of names for your bams.
required:False
plot
label:Single or multiple polt
type:basic:string
description:Choose either all lines on a single plot or multiple plots.
default:merge
choices:
  • single: single
  • multiple: multiple
  • merge: merge
title
label:Title
type:basic:string
description:Specify a title for the output plot(s), default will be the coordinate region.
default:output
scale
label:Scale
type:basic:string
description:Enter a comma separated list of multiplicative scaling factors for your bams. Default is none.
required:False
bed
label:Bed
type:list:data:bed
description:Add a space-delimited list of bed files to plot.
required:False
multi_page
label:Multi page
type:basic:boolean
description:If flagged will create a new pdf for each region.
default:False

Output results

plot
label:region plot
type:basic:file

BaseSpace file

data:filebasespace-file-import (basic:string  file_id, basic:secret  access_token_secret)[Source: v1.0.3]

Import a file from Illumina BaseSpace.

Input arguments

file_id
label:BaseSpace file ID
type:basic:string
access_token_secret
label:BaseSpace access token
type:basic:secret
description:BaseSpace access token secret handle needed to download the file.

Output results

file
label:File
type:basic:file

Bowtie (Dicty)

data:alignment:bam:bowtie1alignment-bowtie (data:genome:fasta  genome, data:reads:fastq  reads, basic:string  mode, basic:integer  m, basic:integer  l, basic:boolean  use_se, basic:integer  trim_5, basic:integer  trim_3, basic:integer  trim_nucl, basic:integer  trim_iter, basic:string  r)[Source: v1.4.1]

An ultrafast memory-efficient short read aligner.

Input arguments

genome
label:Reference genome
type:data:genome:fasta
reads
label:Reads
type:data:reads:fastq
mode
label:Alignment mode
type:basic:string
description:When the -n option is specified (which is the default), bowtie determines which alignments are valid according to the following policy, which is similar to Maq’s default policy. 1. Alignments may have no more than N mismatches (where N is a number 0-3, set with -n) in the first L bases (where L is a number 5 or greater, set with -l) on the high-quality (left) end of the read. The first L bases are called the “seed”. 2. The sum of the Phred quality values at all mismatched positions (not just in the seed) may not exceed E (set with -e). Where qualities are unavailable (e.g. if the reads are from a FASTA file), the Phred quality defaults to 40. In -v mode, alignments may have no more than V mismatches, where V may be a number from 0 through 3 set using the -v option. Quality values are ignored. The -v option is mutually exclusive with the -n option.
default:-n
choices:
  • Use qualities (-n): -n
  • Use mismatches (-v): -v
m
label:Allowed mismatches
type:basic:integer
description:When used with “Use qualities (-n)” it is the maximum number of mismatches permitted in the “seed”, i.e. the first L base pairs of the read (where L is set with -l/–seedlen). This may be 0, 1, 2 or 3 and the default is 2 When used with “Use mismatches (-v)” report alignments with at most <int> mismatches.
default:2
l
label:Seed length (for -n only)
type:basic:integer
description:Only for “Use qualities (-n)”. Seed length (-l) is the number of bases on the high-quality end of the read to which the -n ceiling applies. The lowest permitted setting is 5 and the default is 28. bowtie is faster for larger values of -l.
default:28
use_se
label:Map as single-ended (for paired end reads only)
type:basic:boolean
description:If this option is selected paired-end reads will be mapped as single-ended.
default:False
start_trimming.trim_5
label:Bases to trim from 5’
type:basic:integer
description:Number of bases to trim from from 5’ (left) end of each read before alignment
default:0
start_trimming.trim_3
label:Bases to trim from 3’
type:basic:integer
description:Number of bases to trim from from 3’ (right) end of each read before alignment
default:0
trimming.trim_nucl
label:Bases to trim
type:basic:integer
description:Number of bases to trim from 3’ end in each iteration.
default:2
trimming.trim_iter
label:Iterations
type:basic:integer
description:Number of iterations.
default:0
reporting.r
label:Reporting mode
type:basic:string
description:Report up to <int> valid alignments per read or pair (-k) (default: 1). Validity of alignments is determined by the alignment policy (combined effects of -n, -v, -l, and -e). If more than one valid alignment exists and the –best and –strata options are specified, then only those alignments belonging to the best alignment “stratum” will be reported. Bowtie is designed to be very fast for small -k but bowtie can become significantly slower as -k increases. If you would like to use Bowtie for larger values of -k, consider building an index with a denser suffix-array sample, i.e. specify a smaller -o/–offrate when invoking bowtie-build for the relevant index (see the Performance tuning section for details).
default:-a -m 1 --best --strata
choices:
  • Report unique alignments: -a -m 1 --best --strata
  • Report all alignments: -a --best
  • Report all alignments in the best stratum: -a --best --strata

Output results

bam
label:Alignment file
type:basic:file
description:Position sorted alignment
bai
label:Index BAI
type:basic:file
unmapped
label:Unmapped reads
type:basic:file
required:False
stats
label:Statistics
type:basic:file
bigwig
label:BigWig file
type:basic:file
required:False
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Bowtie2

data:alignment:bam:bowtie2alignment-bowtie2 (data:genome:fasta  genome, data:reads:fastq  reads, basic:string  mode, basic:string  speed, basic:boolean  use_se, basic:boolean  discordantly, basic:boolean  rep_se, basic:integer  minins, basic:integer  maxins, basic:integer  N, basic:integer  L, basic:integer  gbar, basic:string  mp, basic:string  rdg, basic:string  rfg, basic:string  score_min, basic:integer  trim_5, basic:integer  trim_3, basic:integer  trim_iter, basic:integer  trim_nucl, basic:string  rep_mode, basic:integer  k_reports)[Source: v1.5.0]

Bowtie is an ultrafast, memory-efficient short read aligner. It aligns short DNA sequences (reads) to the human genome at a rate of over 25 million 35-bp reads per hour. Bowtie indexes the genome with a Burrows-Wheeler index to keep its memory footprint small–typically about 2.2 GB for the human genome (2.9 GB for paired-end). See [here](http://bowtie-bio.sourceforge.net/index.shtml) for more information.

Input arguments

genome
label:Reference genome
type:data:genome:fasta
reads
label:Reads
type:data:reads:fastq
mode
label:Alignment mode
type:basic:string
description:End to end: Bowtie 2 requires that the entire read align from one end to the other, without any trimming (or “soft clipping”) of characters from either end. local: Bowtie 2 does not require that the entire read align from one end to the other. Rather, some characters may be omitted (“soft clipped”) from the ends in order to achieve the greatest possible alignment score.
default:--end-to-end
choices:
  • end to end mode: --end-to-end
  • local: --local
speed
label:Speed vs. Sensitivity
type:basic:string
description:A quick setting for aligning fast or accurately. This option is a shortcut for parameters as follows: For –end-to-end: –very-fast -D 5 -R 1 -N 0 -L 22 -i S,0,2.50 –fast -D 10 -R 2 -N 0 -L 22 -i S,0,2.50 –sensitive -D 15 -R 2 -N 0 -L 22 -i S,1,1.15 (default) –very-sensitive -D 20 -R 3 -N 0 -L 20 -i S,1,0.50 For –local: –very-fast-local -D 5 -R 1 -N 0 -L 25 -i S,1,2.00 –fast-local -D 10 -R 2 -N 0 -L 22 -i S,1,1.75 –sensitive-local -D 15 -R 2 -N 0 -L 20 -i S,1,0.75 (default) –very-sensitive-local -D 20 -R 3 -N 0 -L 20 -i S,1,0.50
required:False
choices:
  • Very fast: --very-fast
  • Fast: --fast
  • Sensitive: --sensitive
  • Very sensitive: --very-sensitive
PE_options.use_se
label:Map as single-ended (for paired-end reads only)
type:basic:boolean
description:If this option is selected paired-end reads will be mapped as single-ended and other paired-end options are ignored.
default:False
PE_options.discordantly
label:Report discordantly matched read
type:basic:boolean
description:If both mates have unique alignments, but the alignments do not match paired-end expectations (orientation and relative distance) then alignment will be reported. Useful for detecting structural variations.
default:True
PE_options.rep_se
label:Report single ended
type:basic:boolean
description:If paired alignment can not be found Bowtie2 tries to find alignments for the individual mates.
default:True
PE_options.minins
label:Minimal distance
type:basic:integer
description:The minimum fragment length for valid paired-end alignments. 0 imposes no minimum.
default:0
PE_options.maxins
label:Maximal distance
type:basic:integer
description:The maximum fragment length for valid paired-end alignments.
default:500
alignment_options.N
label:Number of mismatches allowed in seed alignment (N)
type:basic:integer
description:Sets the number of mismatches to allowed in a seed alignment during multiseed alignment. Can be set to 0 or 1. Setting this higher makes alignment slower (often much slower) but increases sensitivity. Default: 0.
required:False
alignment_options.L
label:Length of seed substrings (L)
type:basic:integer
description:Sets the length of the seed substrings to align during multiseed alignment. Smaller values make alignment slower but more sensitive. Default: the –sensitive preset is used by default for end-to-end alignment and –sensitive-local for local alignment. See documentation for details.
required:False
alignment_options.gbar
label:Disallow gaps within positions (gbar)
type:basic:integer
description:Disallow gaps within <int> positions of the beginning or end of the read. Default: 4.
required:False
alignment_options.mp
label:Maximal and minimal mismatch penalty (mp)
type:basic:string
description:Sets the maximum (MX) and minimum (MN) mismatch penalties, both integers. A number less than or equal to MX and greater than or equal to MN is subtracted from the alignment score for each position where a read character aligns to a reference character, the characters do not match, and neither is an N. If –ignore-quals is specified, the number subtracted quals MX. Otherwise, the number subtracted is MN + floor((MX-MN)(MIN(Q, 40.0)/40.0)) where Q is the Phred quality value. Default for MX, MN: 6,2.
required:False
alignment_options.rdg
label:Set read gap open and extend penalties (rdg)
type:basic:string
description:Sets the read gap open (<int1>) and extend (<int2>) penalties. A read gap of length N gets a penalty of <int1> + N * <int2>. Default: 5,3.
required:False
alignment_options.rfg
label:Set reference gap open and close penalties (rfg)
type:basic:string
description:Sets the reference gap open (<int1>) and extend (<int2>) penalties. A reference gap of length N gets a penalty of <int1> + N * <int2>. Default: 5,3.
required:False
alignment_options.score_min
label:Minimum alignment score needed for “valid” alignment (score_min)
type:basic:string
description:Sets a function governing the minimum alignment score needed for an alignment to be considered “valid” (i.e. good enough to report). This is a function of read length. For instance, specifying L,0,-0.6 sets the minimum-score function to f(x) = 0 + -0.6 * x, where x is the read length. The default in –end-to-end mode is L,-0.6,-0.6 and the default in –local mode is G,20,8.
required:False
start_trimming.trim_5
label:Bases to trim from 5’
type:basic:integer
description:Number of bases to trim from from 5’ (left) end of each read before alignment
default:0
start_trimming.trim_3
label:Bases to trim from 3’
type:basic:integer
description:Number of bases to trim from from 3’ (right) end of each read before alignment
default:0
trimming.trim_iter
label:Iterations
type:basic:integer
description:Number of iterations.
default:0
trimming.trim_nucl
label:Bases to trim
type:basic:integer
description:Number of bases to trim from 3’ end in each iteration.
default:2
reporting.rep_mode
label:Report mode
type:basic:string
description:Default mode: search for multiple alignments, report the best one; -k mode: search for one or more alignments, report each; -a mode: search for and report all alignments
default:def
choices:
  • Default mode: def
  • -k mode: k
  • -a mode (very slow): a
reporting.k_reports
label:Number of reports (for -k mode only)
type:basic:integer
description:Searches for at most X distinct, valid alignments for each read. The search terminates when it can’t find more distinct valid alignments, or when it finds X, whichever happens first. default: 5
default:5

Output results

bam
label:Alignment file
type:basic:file
description:Position sorted alignment
bai
label:Index BAI
type:basic:file
unmapped
label:Unmapped reads
type:basic:file
required:False
stats
label:Statistics
type:basic:file
bigwig
label:BigWig file
type:basic:file
required:False
species
label:Species
type:basic:string
build
label:Build
type:basic:string

ChIP-Seq (Gene Score)

data:chipseq:genescorechipseq-genescore (data:chipseq:peakscore  peakscore, basic:decimal  fdr, basic:decimal  pval, basic:decimal  logratio)[Source: v1.1.1]

Chip-Seq analysis - Gene Score (BCM)

Input arguments

peakscore
label:PeakScore file
type:data:chipseq:peakscore
description:PeakScore file
fdr
label:FDR threshold
type:basic:decimal
description:FDR threshold value (default = 0.00005).
default:5e-05
pval
label:Pval threshold
type:basic:decimal
description:Pval threshold value (default = 0.00005).
default:5e-05
logratio
label:Log-ratio threshold
type:basic:decimal
description:Log-ratio threshold value (default = 2).
default:2.0

Output results

genescore
label:Gene Score
type:basic:file

ChIP-Seq (Peak Score)

data:chipseq:peakscorechipseq-peakscore (data:chipseq:callpeak:macs2  peaks, data:bed  bed)[Source: v2.1.0]

Chip-Seq analysis - Peak Score (BCM)

Input arguments

peaks
label:MACS2 results
type:data:chipseq:callpeak:macs2
description:MACS2 results file (NarrowPeak)
bed
label:BED file
type:data:bed

Output results

peak_score
label:Peak Score
type:basic:file

ChIP-seq (MACS2)

data:chipseq:batch:macs2macs2-batch (list:data:alignment:bam  alignments, basic:boolean  advanced, data:bed  promoter, basic:boolean  tagalign, basic:integer  q_threshold, basic:integer  n_sub, basic:boolean  tn5, basic:integer  shift, basic:string  duplicates, basic:string  duplicates_prepeak, basic:decimal  qvalue, basic:decimal  pvalue, basic:decimal  pvalue_prepeak, basic:integer  cap_num, basic:integer  mfold_lower, basic:integer  mfold_upper, basic:integer  slocal, basic:integer  llocal, basic:integer  extsize, basic:integer  shift, basic:integer  band_width, basic:boolean  nolambda, basic:boolean  fix_bimodal, basic:boolean  nomodel, basic:boolean  nomodel_prepeak, basic:boolean  down_sample, basic:boolean  bedgraph, basic:boolean  spmr, basic:boolean  call_summits, basic:boolean  broad, basic:decimal  broad_cutoff)[Source: v1.0.3]

This process runs MACS2 in batch mode. MACS2 analysis is triggered for pairs of samples as defined using treatment-background sample relations. If there are no sample relations defined, each sample is treated individually for the MACS analysis. Model-based Analysis of ChIP-Seq (MACS 2.0), is used to identify transcript factor binding sites. MACS 2.0 captures the influence of genome complexity to evaluate the significance of enriched ChIP regions, and MACS improves the spatial resolution of binding sites through combining the information of both sequencing tag position and orientation. It has also an option to link nearby peaks together in order to call broad peaks. See [here](https://github.com/taoliu/MACS/) for more information. In addition to peak-calling, this process computes ChIP-Seq and ATAC-Seq QC metrics. Process returns a QC metrics report, fragment length estimation, and a deduplicated tagAlign file. QC report contains ENCODE 3 proposed QC metrics – [NRF](https://www.encodeproject.org/data-standards/terms/), [PBC bottlenecking coefficients, NSC, and RSC](https://genome.ucsc.edu/ENCODE/qualityMetrics.html#chipSeq).

Input arguments

alignments
label:Aligned reads
type:list:data:alignment:bam
description:Select multiple treatment/background samples.
advanced
label:Show advanced options
type:basic:boolean
description:Inspect and modify parameters.
default:False
promoter
label:Promoter regions BED file
type:data:bed
description:BED file containing promoter regions (TSS+-1000bp for example). Needed to get the number of peaks and reads mapped to promoter regions.
required:False
hidden:!advanced
tagalign
label:Use tagAlign files
type:basic:boolean
description:Use filtered tagAlign files as case (treatment) and control (background) samples. If extsize parameter is not set, run MACS using input’s estimated fragment length.
hidden:!advanced
default:False
prepeakqc_settings.q_threshold
label:Quality filtering threshold
type:basic:integer
default:30
prepeakqc_settings.n_sub
label:Number of reads to subsample
type:basic:integer
default:15000000
prepeakqc_settings.tn5
label:TN5 shifting
type:basic:boolean
description:Tn5 transposon shifting. Shift reads on “+” strand by 4bp and reads on “-” strand by 5bp.
default:False
prepeakqc_settings.shift
label:User-defined cross-correlation peak strandshift
type:basic:integer
description:If defined, SPP tool will not try to estimate fragment length but will use the given value as fragment length.
required:False
settings.duplicates
label:Number of duplicates
type:basic:string
description:It controls the MACS behavior towards duplicate tags at the exact same location – the same coordination and the same strand. The ‘auto’ option makes MACS calculate the maximum tags at the exact same location based on binomal distribution using 1e-5 as pvalue cutoff and the ‘all’ option keeps all the tags. If an integer is given, at most this number of tags will be kept at the same location. The default is to keep one tag at the same location.
required:False
hidden:tagalign
choices:
  • 1: 1
  • auto: auto
  • all: all
settings.duplicates_prepeak
label:Number of duplicates
type:basic:string
description:It controls the MACS behavior towards duplicate tags at the exact same location – the same coordination and the same strand. The ‘auto’ option makes MACS calculate the maximum tags at the exact same location based on binomal distribution using 1e-5 as pvalue cutoff and the ‘all’ option keeps all the tags. If an integer is given, at most this number of tags will be kept at the same location. The default is to keep one tag at the same location.
required:False
hidden:!tagalign
default:all
choices:
  • 1: 1
  • auto: auto
  • all: all
settings.qvalue
label:Q-value cutoff
type:basic:decimal
description:The q-value (minimum FDR) cutoff to call significant regions. Q-values are calculated from p-values using Benjamini-Hochberg procedure.
required:False
disabled:settings.pvalue && settings.pvalue_prepeak
settings.pvalue
label:P-value cutoff
type:basic:decimal
description:The p-value cutoff. If specified, MACS2 will use p-value instead of q-value cutoff.
required:False
disabled:settings.qvalue
hidden:tagalign
settings.pvalue_prepeak
label:P-value cutoff
type:basic:decimal
description:The p-value cutoff. If specified, MACS2 will use p-value instead of q-value cutoff.
disabled:settings.qvalue
hidden:!tagalign || settings.qvalue
default:1e-05
settings.cap_num
label:Cap number of peaks by taking top N peaks
type:basic:integer
description:To keep all peaks set value to 0.
disabled:settings.broad
default:500000
settings.mfold_lower
label:MFOLD range (lower limit)
type:basic:integer
description:This parameter is used to select the regions within MFOLD range of high-confidence enrichment ratio against background to build model. The regions must be lower than upper limit, and higher than the lower limit of fold enrichment. DEFAULT:10,30 means using all regions not too low (>10) and not too high (<30) to build paired-peaks model. If MACS can not find more than 100 regions to build model, it will use the –extsize parameter to continue the peak detection ONLY if –fix-bimodal is set.
required:False
settings.mfold_upper
label:MFOLD range (upper limit)
type:basic:integer
description:This parameter is used to select the regions within MFOLD range of high-confidence enrichment ratio against background to build model. The regions must be lower than upper limit, and higher than the lower limit of fold enrichment. DEFAULT:10,30 means using all regions not too low (>10) and not too high (<30) to build paired-peaks model. If MACS can not find more than 100 regions to build model, it will use the –extsize parameter to continue the peak detection ONLY if –fix-bimodal is set.
required:False
settings.slocal
label:Small local region
type:basic:integer
description:Slocal and llocal parameters control which two levels of regions will be checked around the peak regions to calculate the maximum lambda as local lambda. By default, MACS considers 1000bp for small local region (–slocal), and 10000bps for large local region (–llocal) which captures the bias from a long range effect like an open chromatin domain. You can tweak these according to your project. Remember that if the region is set too small, a sharp spike in the input data may kill the significant peak.
required:False
settings.llocal
label:Large local region
type:basic:integer
description:Slocal and llocal parameters control which two levels of regions will be checked around the peak regions to calculate the maximum lambda as local lambda. By default, MACS considers 1000bp for small local region (–slocal), and 10000bps for large local region (–llocal) which captures the bias from a long range effect like an open chromatin domain. You can tweak these according to your project. Remember that if the region is set too small, a sharp spike in the input data may kill the significant peak.
required:False
settings.extsize
label:extsize
type:basic:integer
description:While ‘–nomodel’ is set, MACS uses this parameter to extend reads in 5’->3’ direction to fix-sized fragments. For example, if the size of binding region for your transcription factor is 200 bp, and you want to bypass the model building by MACS, this parameter can be set as 200. This option is only valid when –nomodel is set or when MACS fails to build model and –fix-bimodal is on.
required:False
settings.shift
label:Shift
type:basic:integer
description:Note, this is NOT the legacy –shiftsize option which is replaced by –extsize! You can set an arbitrary shift in bp here. Please Use discretion while setting it other than default value (0). When –nomodel is set, MACS will use this value to move cutting ends (5’) then apply –extsize from 5’ to 3’ direction to extend them to fragments. When this value is negative, ends will be moved toward 3’->5’ direction, otherwise 5’->3’ direction. Recommended to keep it as default 0 for ChIP-Seq datasets, or -1 * half of EXTSIZE together with –extsize option for detecting enriched cutting loci such as certain DNAseI-Seq datasets. Note, you can’t set values other than 0 if format is BAMPE for paired-end data. Default is 0.
required:False
settings.band_width
label:Band width
type:basic:integer
description:The band width which is used to scan the genome ONLY for model building. You can set this parameter as the sonication fragment size expected from wet experiment. The previous side effect on the peak detection process has been removed. So this parameter only affects the model building.
required:False
settings.nolambda
label:Use backgroud lambda as local lambda
type:basic:boolean
description:With this flag on, MACS will use the background lambda as local lambda. This means MACS will not consider the local bias at peak candidate regions.
default:False
settings.fix_bimodal
label:Turn on the auto paired-peak model process
type:basic:boolean
description:Whether turn on the auto paired-peak model process. If it’s set, when MACS failed to build paired model, it will use the nomodel settings, the ‘–extsize’ parameter to extend each tags. If set, MACS will be terminated if paired-peak model is failed.
default:False
settings.nomodel
label:Bypass building the shifting model
type:basic:boolean
description:While on, MACS will bypass building the shifting model.
hidden:tagalign
default:False
settings.nomodel_prepeak
label:Bypass building the shifting model
type:basic:boolean
description:While on, MACS will bypass building the shifting model.
hidden:!tagalign
default:True
settings.down_sample
label:Down-sample
type:basic:boolean
description:When set, random sampling method will scale down the bigger sample. By default, MACS uses linear scaling. This option will make the results unstable and irreproducible since each time, random reads would be selected, especially the numbers (pileup, pvalue, qvalue) would change. Consider to use ‘randsample’ script before MACS2 runs instead.
default:False
settings.bedgraph
label:Save fragment pileup and control lambda
type:basic:boolean
description:If this flag is on, MACS will store the fragment pileup, control lambda, -log10pvalue and -log10qvalue scores in bedGraph files. The bedGraph files will be stored in current directory named NAME+’_treat_pileup.bdg’ for treatment data, NAME+’_control_lambda.bdg’ for local lambda values from control, NAME+’_treat_pvalue.bdg’ for Poisson pvalue scores (in -log10(pvalue) form), and NAME+’_treat_qvalue.bdg’ for q-value scores from Benjamini-Hochberg-Yekutieli procedure.
default:True
settings.spmr
label:Save signal per million reads for fragment pileup profiles
type:basic:boolean
disabled:settings.bedgraph === false
default:True
settings.call_summits
label:Call summits
type:basic:boolean
description:MACS will now reanalyze the shape of signal profile (p or q-score depending on cutoff setting) to deconvolve subpeaks within each peak called from general procedure. It’s highly recommended to detect adjacent binding events. While used, the output subpeaks of a big peak region will have the same peak boundaries, and different scores and peak summit positions.
default:False
settings.broad
label:Composite broad regions
type:basic:boolean
description:When this flag is on, MACS will try to composite broad regions in BED12 (a gene-model-like format) by putting nearby highly enriched regions into a broad region with loose cutoff. The broad region is controlled by another cutoff through –broad-cutoff. The maximum length of broad region length is 4 times of d from MACS.
disabled:settings.call_summits === true
default:False
settings.broad_cutoff
label:Broad cutoff
type:basic:decimal
description:Cutoff for broad region. This option is not available unless –broad is set. If -p is set, this is a p-value cutoff, otherwise, it’s a q-value cutoff. DEFAULT = 0.1
required:False
disabled:settings.call_summits === true || settings.broad !== true

Output results

ChIP-seq (MACS2-ROSE2)

data:chipseq:batch:macs2macs2-rose2-batch (list:data:alignment:bam  alignments, basic:boolean  advanced, data:bed  promoter, basic:boolean  tagalign, basic:integer  q_threshold, basic:integer  n_sub, basic:boolean  tn5, basic:integer  shift, basic:string  duplicates, basic:string  duplicates_prepeak, basic:decimal  qvalue, basic:decimal  pvalue, basic:decimal  pvalue_prepeak, basic:integer  cap_num, basic:integer  mfold_lower, basic:integer  mfold_upper, basic:integer  slocal, basic:integer  llocal, basic:integer  extsize, basic:integer  shift, basic:integer  band_width, basic:boolean  nolambda, basic:boolean  fix_bimodal, basic:boolean  nomodel, basic:boolean  nomodel_prepeak, basic:boolean  down_sample, basic:boolean  bedgraph, basic:boolean  spmr, basic:boolean  call_summits, basic:boolean  broad, basic:decimal  broad_cutoff, basic:integer  tss, basic:integer  stitch, data:bed  mask)[Source: v1.0.3]

This process runs MACS2 in batch mode. MACS2 analysis is triggered for pairs of samples as defined using treatment-background sample relations. If there are no sample relations defined, each sample is treated individually for the MACS analysis. Model-based Analysis of ChIP-Seq (MACS 2.0), is used to identify transcript factor binding sites. MACS 2.0 captures the influence of genome complexity to evaluate the significance of enriched ChIP regions, and MACS improves the spatial resolution of binding sites through combining the information of both sequencing tag position and orientation. It has also an option to link nearby peaks together in order to call broad peaks. See [here](https://github.com/taoliu/MACS/) for more information. In addition to peak-calling, this process computes ChIP-Seq and ATAC-Seq QC metrics. Process returns a QC metrics report, fragment length estimation, and a deduplicated tagAlign file. QC report contains ENCODE 3 proposed QC metrics – [NRF](https://www.encodeproject.org/data-standards/terms/), [PBC bottlenecking coefficients, NSC, and RSC](https://genome.ucsc.edu/ENCODE/qualityMetrics.html#chipSeq). For identification of super enhancers R2 uses the Rank Ordering of Super-Enhancers algorithm (ROSE2). This takes the peaks called by RSEG for acetylation and calculates the distances in-between to judge whether they can be considered super-enhancers. The ranked values can be plotted and by locating the inflection point in the resulting graph, super-enhancers can be assigned. It can also be used with the MACS calculated data. See [here](http://younglab.wi.mit.edu/super_enhancer_code.html) for more information.

Input arguments

alignments
label:Aligned reads
type:list:data:alignment:bam
description:Select multiple treatment/background samples.
advanced
label:Show advanced options
type:basic:boolean
description:Inspect and modify parameters.
default:False
promoter
label:Promoter regions BED file
type:data:bed
description:BED file containing promoter regions (TSS+-1000bp for example). Needed to get the number of peaks and reads mapped to promoter regions.
required:False
hidden:!advanced
tagalign
label:Use tagAlign files
type:basic:boolean
description:Use filtered tagAlign files as case (treatment) and control (background) samples. If extsize parameter is not set, run MACS using input’s estimated fragment length.
hidden:!advanced
default:False
prepeakqc_settings.q_threshold
label:Quality filtering threshold
type:basic:integer
default:30
prepeakqc_settings.n_sub
label:Number of reads to subsample
type:basic:integer
default:15000000
prepeakqc_settings.tn5
label:TN5 shifting
type:basic:boolean
description:Tn5 transposon shifting. Shift reads on “+” strand by 4bp and reads on “-” strand by 5bp.
default:False
prepeakqc_settings.shift
label:User-defined cross-correlation peak strandshift
type:basic:integer
description:If defined, SPP tool will not try to estimate fragment length but will use the given value as fragment length.
required:False
settings.duplicates
label:Number of duplicates
type:basic:string
description:It controls the MACS behavior towards duplicate tags at the exact same location – the same coordination and the same strand. The ‘auto’ option makes MACS calculate the maximum tags at the exact same location based on binomal distribution using 1e-5 as pvalue cutoff and the ‘all’ option keeps all the tags. If an integer is given, at most this number of tags will be kept at the same location. The default is to keep one tag at the same location.
required:False
hidden:tagalign
choices:
  • 1: 1
  • auto: auto
  • all: all
settings.duplicates_prepeak
label:Number of duplicates
type:basic:string
description:It controls the MACS behavior towards duplicate tags at the exact same location – the same coordination and the same strand. The ‘auto’ option makes MACS calculate the maximum tags at the exact same location based on binomal distribution using 1e-5 as pvalue cutoff and the ‘all’ option keeps all the tags. If an integer is given, at most this number of tags will be kept at the same location. The default is to keep one tag at the same location.
required:False
hidden:!tagalign
default:all
choices:
  • 1: 1
  • auto: auto
  • all: all
settings.qvalue
label:Q-value cutoff
type:basic:decimal
description:The q-value (minimum FDR) cutoff to call significant regions. Q-values are calculated from p-values using Benjamini-Hochberg procedure.
required:False
disabled:settings.pvalue && settings.pvalue_prepeak
settings.pvalue
label:P-value cutoff
type:basic:decimal
description:The p-value cutoff. If specified, MACS2 will use p-value instead of q-value cutoff.
required:False
disabled:settings.qvalue
hidden:tagalign
settings.pvalue_prepeak
label:P-value cutoff
type:basic:decimal
description:The p-value cutoff. If specified, MACS2 will use p-value instead of q-value cutoff.
disabled:settings.qvalue
hidden:!tagalign || settings.qvalue
default:1e-05
settings.cap_num
label:Cap number of peaks by taking top N peaks
type:basic:integer
description:To keep all peaks set value to 0.
disabled:settings.broad
default:500000
settings.mfold_lower
label:MFOLD range (lower limit)
type:basic:integer
description:This parameter is used to select the regions within MFOLD range of high-confidence enrichment ratio against background to build model. The regions must be lower than upper limit, and higher than the lower limit of fold enrichment. DEFAULT:10,30 means using all regions not too low (>10) and not too high (<30) to build paired-peaks model. If MACS can not find more than 100 regions to build model, it will use the –extsize parameter to continue the peak detection ONLY if –fix-bimodal is set.
required:False
settings.mfold_upper
label:MFOLD range (upper limit)
type:basic:integer
description:This parameter is used to select the regions within MFOLD range of high-confidence enrichment ratio against background to build model. The regions must be lower than upper limit, and higher than the lower limit of fold enrichment. DEFAULT:10,30 means using all regions not too low (>10) and not too high (<30) to build paired-peaks model. If MACS can not find more than 100 regions to build model, it will use the –extsize parameter to continue the peak detection ONLY if –fix-bimodal is set.
required:False
settings.slocal
label:Small local region
type:basic:integer
description:Slocal and llocal parameters control which two levels of regions will be checked around the peak regions to calculate the maximum lambda as local lambda. By default, MACS considers 1000bp for small local region (–slocal), and 10000bps for large local region (–llocal) which captures the bias from a long range effect like an open chromatin domain. You can tweak these according to your project. Remember that if the region is set too small, a sharp spike in the input data may kill the significant peak.
required:False
settings.llocal
label:Large local region
type:basic:integer
description:Slocal and llocal parameters control which two levels of regions will be checked around the peak regions to calculate the maximum lambda as local lambda. By default, MACS considers 1000bp for small local region (–slocal), and 10000bps for large local region (–llocal) which captures the bias from a long range effect like an open chromatin domain. You can tweak these according to your project. Remember that if the region is set too small, a sharp spike in the input data may kill the significant peak.
required:False
settings.extsize
label:extsize
type:basic:integer
description:While ‘–nomodel’ is set, MACS uses this parameter to extend reads in 5’->3’ direction to fix-sized fragments. For example, if the size of binding region for your transcription factor is 200 bp, and you want to bypass the model building by MACS, this parameter can be set as 200. This option is only valid when –nomodel is set or when MACS fails to build model and –fix-bimodal is on.
required:False
settings.shift
label:Shift
type:basic:integer
description:Note, this is NOT the legacy –shiftsize option which is replaced by –extsize! You can set an arbitrary shift in bp here. Please Use discretion while setting it other than default value (0). When –nomodel is set, MACS will use this value to move cutting ends (5’) then apply –extsize from 5’ to 3’ direction to extend them to fragments. When this value is negative, ends will be moved toward 3’->5’ direction, otherwise 5’->3’ direction. Recommended to keep it as default 0 for ChIP-Seq datasets, or -1 * half of EXTSIZE together with –extsize option for detecting enriched cutting loci such as certain DNAseI-Seq datasets. Note, you can’t set values other than 0 if format is BAMPE for paired-end data. Default is 0.
required:False
settings.band_width
label:Band width
type:basic:integer
description:The band width which is used to scan the genome ONLY for model building. You can set this parameter as the sonication fragment size expected from wet experiment. The previous side effect on the peak detection process has been removed. So this parameter only affects the model building.
required:False
settings.nolambda
label:Use backgroud lambda as local lambda
type:basic:boolean
description:With this flag on, MACS will use the background lambda as local lambda. This means MACS will not consider the local bias at peak candidate regions.
default:False
settings.fix_bimodal
label:Turn on the auto paired-peak model process
type:basic:boolean
description:Whether turn on the auto paired-peak model process. If it’s set, when MACS failed to build paired model, it will use the nomodel settings, the ‘–extsize’ parameter to extend each tags. If set, MACS will be terminated if paired-peak model is failed.
default:False
settings.nomodel
label:Bypass building the shifting model
type:basic:boolean
description:While on, MACS will bypass building the shifting model.
hidden:tagalign
default:False
settings.nomodel_prepeak
label:Bypass building the shifting model
type:basic:boolean
description:While on, MACS will bypass building the shifting model.
hidden:!tagalign
default:True
settings.down_sample
label:Down-sample
type:basic:boolean
description:When set, random sampling method will scale down the bigger sample. By default, MACS uses linear scaling. This option will make the results unstable and irreproducible since each time, random reads would be selected, especially the numbers (pileup, pvalue, qvalue) would change. Consider to use ‘randsample’ script before MACS2 runs instead.
default:False
settings.bedgraph
label:Save fragment pileup and control lambda
type:basic:boolean
description:If this flag is on, MACS will store the fragment pileup, control lambda, -log10pvalue and -log10qvalue scores in bedGraph files. The bedGraph files will be stored in current directory named NAME+’_treat_pileup.bdg’ for treatment data, NAME+’_control_lambda.bdg’ for local lambda values from control, NAME+’_treat_pvalue.bdg’ for Poisson pvalue scores (in -log10(pvalue) form), and NAME+’_treat_qvalue.bdg’ for q-value scores from Benjamini-Hochberg-Yekutieli procedure.
default:True
settings.spmr
label:Save signal per million reads for fragment pileup profiles
type:basic:boolean
disabled:settings.bedgraph === false
default:True
settings.call_summits
label:Call summits
type:basic:boolean
description:MACS will now reanalyze the shape of signal profile (p or q-score depending on cutoff setting) to deconvolve subpeaks within each peak called from general procedure. It’s highly recommended to detect adjacent binding events. While used, the output subpeaks of a big peak region will have the same peak boundaries, and different scores and peak summit positions.
default:False
settings.broad
label:Composite broad regions
type:basic:boolean
description:When this flag is on, MACS will try to composite broad regions in BED12 (a gene-model-like format) by putting nearby highly enriched regions into a broad region with loose cutoff. The broad region is controlled by another cutoff through –broad-cutoff. The maximum length of broad region length is 4 times of d from MACS.
disabled:settings.call_summits === true
default:False
settings.broad_cutoff
label:Broad cutoff
type:basic:decimal
description:Cutoff for broad region. This option is not available unless –broad is set. If -p is set, this is a p-value cutoff, otherwise, it’s a q-value cutoff. DEFAULT = 0.1
required:False
disabled:settings.call_summits === true || settings.broad !== true
rose_settings.tss
label:TSS exclusion
type:basic:integer
description:Enter a distance from TSS to exclude. 0 = no TSS exclusion
default:0
rose_settings.stitch
label:Stitch
type:basic:integer
description:Enter a max linking distance for stitching. If not given, optimal stitching parameter will be determined automatically.
required:False
rose_settings.mask
label:Masking BED file
type:data:bed
description:Mask a set of regions from analysis. Provide a BED of masking regions.
required:False

Output results

Chemical Mutagenesis

data:workflow:chemutworkflow-chemut (basic:string  analysis_type, data:genome:fasta  genome, list:data:alignment:bam  parental_strains, list:data:alignment:bam  mutant_strains, basic:boolean  advanced, basic:boolean  br_and_ind_ra, basic:boolean  dbsnp, data:variants:vcf  known_sites, list:data:variants:vcf  known_indels, basic:integer  stand_emit_conf, basic:integer  stand_call_conf, basic:boolean  rf, basic:boolean  advanced, basic:integer  read_depth)[Source: v0.0.6]

Input arguments

analysis_type
label:Analysis type
type:basic:string
description:Choice of the analysis type. Use “SNV” or “INDEL” options to run the GATK analysis only on the haploid portion of the dicty genome. Choose options SNV_CHR2 or INDEL_CHR2 to run the analysis only on the diploid portion of CHR2 (-ploidy 2 -L chr2:2263132-3015703).
default:snv
choices:
  • SNV: snv
  • INDEL: indel
  • SNV_CHR2: snv_chr2
  • INDEL_CHR2: indel_chr2
genome
label:Reference genome
type:data:genome:fasta
parental_strains
label:Parental strains
type:list:data:alignment:bam
mutant_strains
label:Mutant strains
type:list:data:alignment:bam
Vc.advanced
label:Advanced options
type:basic:boolean
required:False
default:False
Vc.br_and_ind_ra
label:Do variant base recalibration and indel realignment
type:basic:boolean
required:False
hidden:Vc.advanced === false
default:False
Vc.dbsnp
label:Use dbSNP file
type:basic:boolean
description:rsIDs from this file are used to populate the ID column of the output. Also, the DB INFO flag will be set when appropriate. dbSNP is not used in any way for the calculations themselves.
required:False
hidden:Vc.advanced === false
default:False
Vc.known_sites
label:Known sites (dbSNP)
type:data:variants:vcf
required:False
hidden:Vc.advanced === false || Vc.br_and_ind_ra === false && Vc.dbsnp === false
Vc.known_indels
label:Known indels
type:list:data:variants:vcf
required:False
hidden:Vc.advanced === false || Vc.br_and_ind_ra === false
default:[]
Vc.stand_emit_conf
label:Emission confidence threshold
type:basic:integer
description:The minimum confidence threshold (phred-scaled) at which the program should emit sites that appear to be possibly variant.
required:False
hidden:Vc.advanced === false
default:10
Vc.stand_call_conf
label:Calling confidence threshold
type:basic:integer
description:The minimum confidence threshold (phred-scaled) at which the program should emit variant sites as called. If a site’s associated genotype has a confidence score lower than the calling threshold, the program will emit the site as filtered and will annotate it as LowQual. This threshold separates high confidence calls from low confidence calls.
required:False
hidden:Vc.advanced === false
default:30
Vc.rf
label:ReasignOneMappingQuality Filter
type:basic:boolean
description:This read transformer will change a certain read mapping quality to a different value without affecting reads that have other mapping qualities. This is intended primarily for users of RNA-Seq data handling programs such as TopHat, which use MAPQ = 255 to designate uniquely aligned reads. According to convention, 255 normally designates “unknown” quality, and most GATK tools automatically ignore such reads. By reassigning a different mapping quality to those specific reads, users of TopHat and other tools can circumvent this problem without affecting the rest of their dataset.
required:False
hidden:Vc.advanced === false
default:False
Vf.advanced
label:Advanced options
type:basic:boolean
required:False
default:False
Vf.read_depth
label:Read depth cutoff
type:basic:integer
description:The minimum number of replicate reads required for a variant site to be included.
required:False
hidden:Vf.advanced === false
default:5

Output results

Convert GFF3 to GTF

data:annotation:gtfgff-to-gtf (data:annotation:gff3  annotation)[Source: v0.3.1]

Convert GFF3 file to GTF format.

Input arguments

annotation
label:Annotation (GFF3)
type:data:annotation:gff3
description:Annotation in GFF3 format.

Output results

annot
label:Converted GTF file
type:basic:file
annot_sorted
label:Sorted GTF file
type:basic:file
annot_sorted_idx_igv
label:Igv index for sorted GTF file
type:basic:file
annot_sorted_track_jbrowse
label:Jbrowse track for sorted GTF
type:basic:file
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Convert files to reads (paired-end)

data:reads:fastq:pairedfiles-to-fastq-paired (list:data:file  src1, list:data:file  src2, basic:boolean  merge_lanes)[Source: v1.3.0]

Convert FASTQ files to paired-end reads.

Input arguments

src1
label:Mate1
type:list:data:file
src2
label:Mate2
type:list:data:file
merge_lanes
label:Merge lanes
type:basic:boolean
description:Merge paired-end sample data split into multiple sequencing lanes into a single pair of FASTQ files.
default:False

Output results

fastq
label:Reads file (mate 1)
type:list:basic:file
fastq2
label:Reads file (mate 2)
type:list:basic:file
fastqc_url
label:Quality control with FastQC (Upstream)
type:list:basic:file:html
fastqc_url2
label:Quality control with FastQC (Downstream)
type:list:basic:file:html
fastqc_archive
label:Download FastQC archive (Upstream)
type:list:basic:file
fastqc_archive2
label:Download FastQC archive (Downstream)
type:list:basic:file

Convert files to reads (single-end)

data:reads:fastq:singlefiles-to-fastq-single (list:data:file  src, basic:boolean  merge_lanes)[Source: v1.3.0]

Convert FASTQ files to single-end reads.

Input arguments

src
label:Reads
type:list:data:file
description:Sequencing reads in FASTQ format
merge_lanes
label:Merge lanes
type:basic:boolean
description:Merge sample data split into multiple sequencing lanes into a single FASTQ file.
default:False

Output results

fastq
label:Reads file
type:list:basic:file
fastqc_url
label:Quality control with FastQC
type:list:basic:file:html
fastqc_archive
label:Download FastQC archive
type:list:basic:file

Cuffdiff 2.2

data:differentialexpression:cuffdiffcuffdiff (list:data:cufflinks:cuffquant  case, list:data:cufflinks:cuffquant  control, list:basic:string  labels, data:annotation  annotation, data:genome:fasta  genome, basic:boolean  multi_read_correct, basic:decimal  fdr, basic:string  library_type, basic:string  library_normalization, basic:string  dispersion_method)[Source: v2.2.1]

Cuffdiff finds significant changes in transcript expression, splicing, and promoter use. You can use it to find differentially expressed genes and transcripts, as well as genes that are being differentially regulated at the transcriptional and post-transcriptional level. See [here](http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/) and [here](https://software.broadinstitute.org/cancer/software/genepattern/modules/docs/Cuffdiff/7) for more information.

Input arguments

case
label:Case samples
type:list:data:cufflinks:cuffquant
control
label:Control samples
type:list:data:cufflinks:cuffquant
labels
label:Group labels
type:list:basic:string
description:Define labels for each sample group.
default:['control', 'case']
annotation
label:Annotation (GTF/GFF3)
type:data:annotation
description:A transcript annotation file produced by cufflinks, cuffcompare, or other tool.
genome
label:Run bias detection and correction algorithm
type:data:genome:fasta
description:Provide Cufflinks with a multifasta file (genome file) via this option to instruct it to run a bias detection and correction algorithm which can significantly improve accuracy of transcript abundance estimates.
required:False
multi_read_correct
label:Do initial estimation procedure to more accurately weight reads with multiple genome mappings
type:basic:boolean
default:False
fdr
label:Allowed FDR
type:basic:decimal
description:The allowed false discovery rate. The default is 0.05.
default:0.05
library_type
label:Library type
type:basic:string
description:In cases where Cufflinks cannot determine the platform and protocol used to generate input reads, you can supply this information manually, which will allow Cufflinks to infer source strand information with certain protocols. The available options are listed below. For paired-end data, we currently only support protocols where reads are point towards each other: fr-unstranded - Reads from the left-most end of the fragment (in transcript coordinates) map to the transcript strand, and the right-most end maps to the opposite strand; fr-firststrand - Same as above except we enforce the rule that the right-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during first strand synthesis is sequenced; fr-secondstrand - Same as above except we enforce the rule that the left-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during second strand synthesis is sequenced.
default:fr-unstranded
choices:
  • fr-unstranded: fr-unstranded
  • fr-firststrand: fr-firststrand
  • fr-secondstrand: fr-secondstrand
library_normalization
label:Library normalization method
type:basic:string
description:You can control how library sizes (i.e. sequencing depths) are normalized in Cufflinks and Cuffdiff. Cuffdiff has several methods that require multiple libraries in order to work. Library normalization methods supported by Cufflinks work on one library at a time.
default:geometric
choices:
  • geometric: geometric
  • classic-fpkm: classic-fpkm
  • quartile: quartile
dispersion_method
label:Dispersion method
type:basic:string
description:Cuffdiff works by modeling the variance in fragment counts across replicates as a function of the mean fragment count across replicates. Strictly speaking, models a quantitity called dispersion - the variance present in a group of samples beyond what is expected from a simple Poisson model of RNA_Seq. You can control how Cuffdiff constructs its model of dispersion in locus fragment counts. Each condition that has replicates can receive its own model, or Cuffdiff can use a global model for all conditions. All of these policies are identical to those used by DESeq (Anders and Huber, Genome Biology, 2010).
default:pooled
choices:
  • pooled: pooled
  • per-condition: per-condition
  • blind: blind
  • poisson: poisson

Output results

raw
label:Differential expression (gene level)
type:basic:file
de_json
label:Results table (JSON)
type:basic:json
de_file
label:Results table (file)
type:basic:file
transcript_diff_exp
label:Differential expression (transcript level)
type:basic:file
tss_group_diff_exp
label:Differential expression (primary transcript)
type:basic:file
cds_diff_exp
label:Differential expression (coding sequence)
type:basic:file
cuffdiff_output
label:Cuffdiff output
type:basic:file
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string

Cuffmerge

data:annotation:cuffmergecuffmerge (list:data:cufflinks:cufflinks  expressions, list:data:annotation:gtf  gtf, data:annotation  gff, data:genome:fasta  genome, basic:integer  threads)[Source: v1.3.1]

Cufflinks includes a script called Cuffmerge that you can use to merge together several Cufflinks assemblies. It also handles running Cuffcompare for you, and automatically filters a number of transfrags that are probably artfifacts. The main purpose of Cuffmerge is to make it easier to make an assembly GTF file suitable for use with Cuffdiff. See [here](http://cole-trapnell-lab.github.io/cufflinks/cuffmerge/) for more information.

Input arguments

expressions
label:Cufflinks transcripts (GTF)
type:list:data:cufflinks:cufflinks
required:False
gtf
label:Annotation files (GTF)
type:list:data:annotation:gtf
description:Annotation files you wish to merge together with Cufflinks produced annotation files (e.g. upload Cufflinks annotation GTF file)
required:False
gff
label:Reference annotation (GTF/GFF3)
type:data:annotation
description:An optional “reference” annotation GTF. The input assemblies are merged together with the reference GTF and included in the final output.
required:False
genome
label:Reference genome
type:data:genome:fasta
description:This argument should point to the genomic DNA sequences for the reference. If a directory, it should contain one fasta file per contig. If a multifasta file, all contigs should be present. The merge script will pass this option to cuffcompare, which will use the sequences to assist in classifying transfrags and excluding artifacts (e.g. repeats). For example, Cufflinks transcripts consisting mostly of lower-case bases are classified as repeats. Note that <seq_dir> must contain one fasta file per reference chromosome, and each file must be named after the chromosome, and have a .fa or .fasta extension
required:False
threads
label:Use this many processor threads
type:basic:integer
description:Use this many threads to align reads. The default is 1.
default:1

Output results

annot
label:Merged GTF file
type:basic:file
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Cuffnorm

data:cuffnormcuffnorm (list:data:cufflinks:cuffquant  cuffquant, data:annotation  annotation, basic:boolean  useERCC)[Source: v2.1.3]

Cufflinks includes a program, Cuffnorm, that you can use to generate tables of expression values that are properly normalized for library size. Cuffnorm takes a GTF2/GFF3 file of transcripts as input, along with two or more SAM, BAM, or CXB files for two or more samples. See [here](http://cole-trapnell-lab.github.io/cufflinks/cuffnorm/) for more information. Replicate relation needs to be defined for Cuffnorm to account for replicates. If the replicate relation is not defined, each sample will be treated individually.

Input arguments

cuffquant
label:Cuffquant expression file
type:list:data:cufflinks:cuffquant
annotation
label:Annotation (GTF/GFF3)
type:data:annotation
description:A transcript annotation file produced by cufflinks, cuffcompare, or other source.
useERCC
label:ERCC spike-in normalization
type:basic:boolean
description:Use ERRCC spike-in controls for normalization.
default:False

Output results

genes_count
label:Genes count
type:basic:file
genes_fpkm
label:Genes FPKM
type:basic:file
genes_attr
label:Genes attr table
type:basic:file
isoform_count
label:Isoform count
type:basic:file
isoform_fpkm
label:Isoform FPKM
type:basic:file
isoform_attr
label:Isoform attr table
type:basic:file
cds_count
label:CDS count
type:basic:file
cds_fpkm
label:CDS FPKM
type:basic:file
cds_attr
label:CDS attr table
type:basic:file
tss_groups_count
label:TSS groups count
type:basic:file
tss_groups_fpkm
label:TSS groups FPKM
type:basic:file
tss_attr
label:TSS attr table
type:basic:file
run_info
label:Run info
type:basic:file
raw_scatter
label:FPKM exp scatter plot
type:basic:file
boxplot
label:Boxplot
type:basic:file
fpkm_exp_raw
label:FPKM exp raw
type:basic:file
replicate_correlations
label:Replicate correlatios plot
type:basic:file
fpkm_means
label:FPKM means
type:basic:file
exp_fpkm_means
label:Exp FPKM means
type:basic:file
norm_scatter
label:FKPM exp scatter normalized plot
type:basic:file
required:False
fpkm_exp_norm
label:FPKM exp normalized
type:basic:file
required:False
spike_raw
label:Spike raw
type:basic:file
required:False
spike_norm
label:Spike normalized
type:basic:file
required:False
R_data
label:All R normalization data
type:basic:file
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Cuffquant 2.2

data:cufflinks:cuffquantcuffquant (data:alignment:bam  alignment, data:annotation  annotation, data:genome:fasta  genome, data:annotation:gtf  mask_file, basic:string  library_type, basic:boolean  multi_read_correct)[Source: v1.3.1]

Cuffquant allows you to compute the gene and transcript expression profiles and save these profiles to files that you can analyze later with Cuffdiff or Cuffnorm. See [here](http://cole-trapnell-lab.github.io/cufflinks/manual/) for more information.

Input arguments

alignment
label:Aligned reads
type:data:alignment:bam
annotation
label:Annotation (GTF/GFF3)
type:data:annotation
genome
label:Run bias detection and correction algorithm
type:data:genome:fasta
description:Provide Cufflinks with a multifasta file (genome file) via this option to instruct it to run a bias detection and correction algorithm which can significantly improve accuracy of transcript abundance estimates.
required:False
mask_file
label:Mask file
type:data:annotation:gtf
description:Ignore all reads that could have come from transcripts in this GTF file. We recommend including any annotated rRNA, mitochondrial transcripts other abundant transcripts you wish to ignore in your analysis in this file. Due to variable efficiency of mRNA enrichment methods and rRNA depletion kits, masking these transcripts often improves the overall robustness of transcript abundance estimates.
required:False
library_type
label:Library type
type:basic:string
description:In cases where Cufflinks cannot determine the platform and protocol used to generate input reads, you can supply this information manually, which will allow Cufflinks to infer source strand information with certain protocols. The available options are listed below. For paired-end data, we currently only support protocols where reads are point towards each other: fr-unstranded - Reads from the left-most end of the fragment (in transcript coordinates) map to the transcript strand, and the right-most end maps to the opposite strand; fr-firststrand - Same as above except we enforce the rule that the right-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during first strand synthesis is sequenced; fr-secondstrand - Same as above except we enforce the rule that the left-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during second strand synthesis is sequenced.
default:fr-unstranded
choices:
  • fr-unstranded: fr-unstranded
  • fr-firststrand: fr-firststrand
  • fr-secondstrand: fr-secondstrand
multi_read_correct
label:Do initial estimation procedure to more accurately weight reads with multiple genome mappings
type:basic:boolean
description:Run an initial estimation procedure that weights reads mapping to multiple locations more accurately.
default:False

Output results

cxb
label:Abundances (.cxb)
type:basic:file
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string

Cuffquant results

data:cufflinks:cuffquantupload-cxb (basic:file  src, basic:string  source, basic:string  species, basic:string  build, basic:string  feature_type)[Source: v1.2.1]

Upload Cuffquant results file (.cxb)

Input arguments

src
label:Cuffquant file
type:basic:file
description:Upload Cuffquant results file. Supported extention: *.cxb
required:True
validate_regex:\.(cxb)$
source
label:Gene ID database
type:basic:string
choices:
  • AFFY: AFFY
  • DICTYBASE: DICTYBASE
  • ENSEMBL: ENSEMBL
  • NCBI: NCBI
  • UCSC: UCSC
species
label:Species
type:basic:string
description:Species latin name.
choices:
  • Homo sapiens: Homo sapiens
  • Mus musculus: Mus musculus
  • Rattus norvegicus: Rattus norvegicus
  • Dictyostelium discoideum: Dictyostelium discoideum
  • Odocoileus virginianus texanus: Odocoileus virginianus texanus
  • Solanum tuberosum: Solanum tuberosum
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string
default:gene
choices:
  • gene: gene
  • transcript: transcript
  • exon: exon

Output results

cxb
label:Cuffquant results
type:basic:file
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string

Custom master file

data:masterfile:ampliconupload-master-file (basic:file  src, basic:string  panel_name)[Source: v1.1.1]

This should be a tab delimited file (*.txt). Please check the [example](http://genial.is/amplicon-masterfile) file for details.

Input arguments

src
label:Master file
type:basic:file
validate_regex:\.txt(|\.gz|\.bz2|\.tgz|\.tar\.gz|\.tar\.bz2|\.zip|\.rar|\.7z)$
panel_name
label:Panel name
type:basic:string

Output results

master_file
label:Master file
type:basic:file
bedfile
label:BED file (merged targets)
type:basic:file
nomergebed
label:BED file (nonmerged targets)
type:basic:file
olapfreebed
label:BED file (overlap-free targets)
type:basic:file
primers
label:Primers
type:basic:file
panel_name
label:Panel name
type:basic:string

Cutadapt (Diagenode CATS, paired-end)

data:reads:fastq:paired:cutadaptcutadapt-custom-paired (data:reads:fastq:paired  reads)[Source: v1.1.2]

Cutadapt process configured to be used with the Diagenode CATS kits.

Input arguments

reads
label:NGS reads
type:data:reads:fastq:paired

Output results

fastq
label:Reads file (forward)
type:list:basic:file
fastq2
label:Reads file (reverse)
type:list:basic:file
report
label:Cutadapt report
type:basic:file
fastqc_url
label:Quality control with FastQC (forward)
type:list:basic:file:html
fastqc_url2
label:Quality control with FastQC (reverse)
type:list:basic:file:html
fastqc_archive
label:Download FastQC archive (forward)
type:list:basic:file
fastqc_archive2
label:Download FastQC archive (reverse)
type:list:basic:file

Cutadapt (Diagenode CATS, single-end)

data:reads:fastq:single:cutadaptcutadapt-custom-single (data:reads:fastq:single  reads)[Source: v1.1.2]

Cutadapt process configured to be used with the Diagenode CATS kits.

Input arguments

reads
label:NGS reads
type:data:reads:fastq:single

Output results

fastq
label:Reads file
type:list:basic:file
report
label:Cutadapt report
type:basic:file
fastqc_url
label:Quality control with FastQC
type:list:basic:file:html
fastqc_archive
label:Download FastQC archive
type:list:basic:file

Cutadapt (paired-end)

data:reads:fastq:paired:cutadaptcutadapt-paired (data:reads:fastq:paired  reads, data:seq:nucleotide  mate1_5prime_file, data:seq:nucleotide  mate1_3prime_file, data:seq:nucleotide  mate2_5prime_file, data:seq:nucleotide  mate2_3prime_file, list:basic:string  mate1_5prime_seq, list:basic:string  mate1_3prime_seq, list:basic:string  mate2_5prime_seq, list:basic:string  mate2_3prime_seq, basic:integer  times, basic:decimal  error_rate, basic:integer  min_overlap, basic:boolean  match_read_wildcards, basic:integer  nextseq_trim, basic:integer  leading, basic:integer  trailing, basic:integer  crop, basic:integer  headcrop, basic:integer  minlen, basic:integer  max_n, basic:string  pair_filter)[Source: v2.2.0]

Cutadapt finds and removes adapter sequences, primers, poly-A tails and other types of unwanted sequence from high-throughput sequencing reads. More information about Cutadapt can be found [here](http://cutadapt.readthedocs.io/en/stable/).

Input arguments

reads
label:Select sample(s)
type:data:reads:fastq:paired
adapters.mate1_5prime_file
label:5 prime adapter file for Mate 1
type:data:seq:nucleotide
required:False
adapters.mate1_3prime_file
label:3 prime adapter file for Mate 1
type:data:seq:nucleotide
required:False
adapters.mate2_5prime_file
label:5 prime adapter file for Mate 2
type:data:seq:nucleotide
required:False
adapters.mate2_3prime_file
label:3 prime adapter file for Mate 2
type:data:seq:nucleotide
required:False
adapters.mate1_5prime_seq
label:5 prime adapter sequence for Mate 1
type:list:basic:string
required:False
adapters.mate1_3prime_seq
label:3 prime adapter sequence for Mate 1
type:list:basic:string
required:False
adapters.mate2_5prime_seq
label:5 prime adapter sequence for Mate 2
type:list:basic:string
required:False
adapters.mate2_3prime_seq
label:3 prime adapter sequence for Mate 2
type:list:basic:string
required:False
adapters.times
label:Times
type:basic:integer
description:Remove up to COUNT adapters from each read.
default:1
adapters.error_rate
label:Error rate
type:basic:decimal
description:Maximum allowed error rate (no. of errors divided by the length of the matching region).
default:0.1
adapters.min_overlap
label:Minimal overlap
type:basic:integer
description:Minimum overlap for an adapter match.
default:3
adapters.match_read_wildcards
label:Match read wildcards
type:basic:boolean
description:Interpret IUPAC wildcards in reads.
default:False
modify_reads.nextseq_trim
label:NextSeq-specific quality trimming
type:basic:integer
description:NextSeq-specific quality trimming (each read). Trims also dark cycles appearing as high-quality G bases. This option is mutually exclusive with the use of regular (-g) quality trimming.
required:False
modify_reads.leading
label:Quality on 5 prime
type:basic:integer
description:Remove low quality bases from 5 prime. Specifies the minimum quality required to keep a base.
required:False
modify_reads.trailing
label:Quality on 3 prime
type:basic:integer
description:Remove low quality bases from the 3 prime. Specifies the minimum quality required to keep a base.
required:False
modify_reads.crop
label:Crop
type:basic:integer
description:Cut the specified number of bases from the end of the reads.
required:False
modify_reads.headcrop
label:Headcrop
type:basic:integer
description:Cut the specified number of bases from the start of the reads.
required:False
filtering.minlen
label:Min length
type:basic:integer
description:Drop the read if it is below a specified.
required:False
filtering.max_n
label:Max numebr of N-s
type:basic:integer
description:Discard reads having more ‘N’ bases than specified.
required:False
filtering.pair_filter
label:Which of the reads have to match the filtering criterion
type:basic:string
description:Which of the reads in a paired-end read have to match the filtering criterion in order for the pair to be filtered.
default:any
choices:
  • Any of the reads in a paired-end read have to match the filtering criterion: any
  • Both of the reads in a paired-end read have to match the filtering criterion: both

Output results

fastq
label:Reads file (forward)
type:list:basic:file
fastq2
label:Reads file (reverse)
type:list:basic:file
report
label:Cutadapt report
type:basic:file
fastqc_url
label:Quality control with FastQC (forward)
type:list:basic:file:html
fastqc_url2
label:Quality control with FastQC (reverse)
type:list:basic:file:html
fastqc_archive
label:Download FastQC archive (forward)
type:list:basic:file
fastqc_archive2
label:Download FastQC archive (reverse)
type:list:basic:file

Cutadapt (single-end)

data:reads:fastq:single:cutadaptcutadapt-single (data:reads:fastq:single  reads, data:seq:nucleotide  up_primers_file, data:seq:nucleotide  down_primers_file, list:basic:string  up_primers_seq, list:basic:string  down_primers_seq, basic:integer  polya_tail, basic:integer  min_overlap, basic:integer  nextseq_trim, basic:integer  leading, basic:integer  trailing, basic:integer  crop, basic:integer  headcrop, basic:integer  minlen, basic:integer  max_n, basic:boolean  match_read_wildcards, basic:integer  times, basic:decimal  error_rate)[Source: v2.0.0]

Cutadapt finds and removes adapter sequences, primers, poly-A tails and other types of unwanted sequence from high-throughput sequencing reads. More information about Cutadapt can be found [here](http://cutadapt.readthedocs.io/en/stable/).

Input arguments

reads
label:Select sample(s)
type:data:reads:fastq:single
adapters.up_primers_file
label:5 prime adapter file
type:data:seq:nucleotide
required:False
adapters.down_primers_file
label:3 prime adapter file
type:data:seq:nucleotide
required:False
adapters.up_primers_seq
label:5 prime adapter sequence
type:list:basic:string
required:False
adapters.down_primers_seq
label:3 prime adapter sequence
type:list:basic:string
required:False
adapters.polya_tail
label:Poly-A tail
type:basic:integer
description:Length of poly-A tail, example - AAAN -> 3, AAAAAN -> 5
required:False
adapters.min_overlap
label:Minimal overlap
type:basic:integer
description:Minimum overlap for an adapter match
default:3
modify_reads.nextseq_trim
label:NextSeq-specific quality trimming
type:basic:integer
description:NextSeq-specific quality trimming (each read). Trims also dark cycles appearing as high-quality G bases. This option is mutually exclusive with the use of regular (-g) quality trimming.
required:False
modify_reads.leading
label:Quality on 5 prime
type:basic:integer
description:Remove low quality bases from 5 prime. Specifies the minimum quality required to keep a base. This option is mutually exclusive with the use of NextSeq-specific quality trimming.
required:False
modify_reads.trailing
label:Quality on 3 prime
type:basic:integer
description:Remove low quality bases from the 3 prime. Specifies the minimum quality required to keep a base. This option is mutually exclusive with the use of NextSeq-specific quality trimming.
required:False
modify_reads.crop
label:Crop
type:basic:integer
description:Cut the read to a specified length by removing bases from the end
required:False
modify_reads.headcrop
label:Headcrop
type:basic:integer
description:Cut the specified number of bases from the start of the read
required:False
filtering.minlen
label:Min length
type:basic:integer
description:Drop the read if it is below a specified length
required:False
filtering.max_n
label:Max numebr of N-s
type:basic:integer
description:Discard reads having more ‘N’ bases than specified.
required:False
filtering.match_read_wildcards
label:Match read wildcards
type:basic:boolean
description:Interpret IUPAC wildcards in reads.
required:False
default:False
filtering.times
label:Times
type:basic:integer
description:Remove up to COUNT adapters from each read.
default:1
filtering.error_rate
label:Error rate
type:basic:decimal
description:Maximum allowed error rate (no. of errors divided by the length of the matching region).
default:0.1

Output results

fastq
label:Reads file
type:list:basic:file
report
label:Cutadapt report
type:basic:file
fastqc_url
label:Quality control with FastQC
type:list:basic:file:html
fastqc_archive
label:Download FastQC archive
type:list:basic:file

Cutadapt - STAR - FeatureCounts (3’ mRNA-Seq, single-end)

data:workflow:quant:featurecounts:singleworkflow-cutadapt-star-fc-quant-single (data:reads:fastq:single  reads, data:genomeindex:star  star_index, data:annotation  annotation, data:genomeindex:star  rrna_reference, data:genomeindex:star  globin_reference, basic:boolean  show_advanced, basic:integer  quality_cutoff, basic:integer  n_reads, basic:integer  seed, basic:decimal  fraction, basic:boolean  two_pass)[Source: v1.0.1]

This 3’ mRNA-Seq pipeline is comprised of QC, preprocessing, alignment and quantification steps. Reads are preprocessed by __Cutadapt__ which removes adapters, trims reads for quality from the 3’-end, and discards reads that are too short after trimming. Preprocessed reads are aligned by __STAR__ aligner. For read-count quantification, the __FeatureCounts__ tool is used. QoRTs QC and Samtools idxstats tools are used to report alignment QC metrics. Additional QC steps operate on downsampled reads and include an alignment of input reads to the rRNA/globin reference sequences. The reported alignment rate is used to asses the rRNA/globin sequence depletion rate.

Input arguments

reads
label:Select sample(s)
type:data:reads:fastq:single
star_index
label:Genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
annotation
label:Annotation
type:data:annotation
description:Genome annotation file (GTF).
rrna_reference
label:Indexed rRNA reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
globin_reference
label:Indexed Globin reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
cutadapt.quality_cutoff
label:Reads quality cutoff
type:basic:integer
description:Trim low-quality bases from 3’ end of each read before adapter removal. The use of this option will override the use of NextSeq/NovaSeq-specific trim option.
required:False
downsampling.n_reads
label:Number of reads
type:basic:integer
default:1000000
downsampling.seed
label:Seed
type:basic:integer
default:11
downsampling.fraction
label:Fraction
type:basic:decimal
description:Use the fraction of reads in range [0.0, 1.0] from the original input file instead of the absolute number of reads. If set, this will override the “Number of reads” input parameter.
required:False
downsampling.two_pass
label:2-pass mode
type:basic:boolean
description:Enable two-pass mode when down-sampling. Two-pass mode is twice as slow but with much reduced memory.
default:False

Output results

Cutadapt - STAR - FeatureCounts - basic QC (3’ mRNA-Seq, single-end)

data:workflow:quant:featurecounts:singleworkflow-cutadapt-star-fc-quant-wo-depletion-single (data:reads:fastq:single  reads, data:genomeindex:star  star_index, data:annotation  annotation, basic:boolean  show_advanced, basic:integer  quality_cutoff)[Source: v1.0.0]

This 3’ mRNA-Seq pipeline is comprised of QC, preprocessing, alignment and quantification steps. Reads are preprocessed by __Cutadapt__ which removes adapters, trims reads for quality from the 3’-end, and discards reads that are too short after trimming. Preprocessed reads are aligned by __STAR__ aligner. For read-count quantification, the __FeatureCounts__ tool is used. QoRTs QC and Samtools idxstats tools are used to report alignment QC metrics.

Input arguments

reads
label:Select sample(s)
type:data:reads:fastq:single
star_index
label:Genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
annotation
label:Annotation
type:data:annotation
description:Genome annotation file (GTF).
show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
cutadapt.quality_cutoff
label:Reads quality cutoff
type:basic:integer
description:Trim low-quality bases from 3’ end of each read before adapter removal. The use of this option will override the use of NextSeq/NovaSeq-specific trim option.
required:False

Output results

Cutadapt - STAR - HTSeq-count (paired-end)

data:workflow:rnaseq:htseqworkflow-custom-cutadapt-star-htseq-paired (data:reads:fastq:paired  reads, data:genomeindex:star  genome, data:annotation:gtf  gff, basic:string  stranded, basic:boolean  advanced, basic:boolean  noncannonical, basic:boolean  chimeric, basic:integer  chimSegmentMin, basic:boolean  quantmode, basic:boolean  singleend, basic:boolean  gene_counts, basic:string  outFilterType, basic:integer  outFilterMultimapNmax, basic:integer  outFilterMismatchNmax, basic:decimal  outFilterMismatchNoverLmax, basic:integer  alignSJoverhangMin, basic:integer  alignSJDBoverhangMin, basic:integer  alignIntronMin, basic:integer  alignIntronMax, basic:integer  alignMatesGapMax, basic:string  mode, basic:string  feature_class, basic:string  id_attribute, basic:boolean  name_ordered)[Source: v1.0.1]

This RNA-seq pipeline is comprised of three steps, preprocessing, alignment, and quantification. First, reads are preprocessed by __cutadapt__ which finds and removes adapter sequences, primers, poly-A tails and other types of unwanted sequence from high-throughput sequencing reads. Next, preprocessed reads are aligned by __STAR__ aligner. At the time of implementation, STAR is considered a state-of-the-art tool that consistently produces accurate results from diverse sets of reads, and performs well even with default settings. For more information see [this comparison of RNA-seq aligners](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792058/). Finally, aligned reads are summarized to genes by __HTSeq-count__. Compared to featureCounts, HTSeq-count is not as computationally efficient. All three tools in this workflow support parallelization to accelerate the analysis.

Input arguments

reads
label:NGS reads
type:data:reads:fastq:paired
genome
label:Indexed reference genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool
gff
label:Annotation (GFF)
type:data:annotation:gtf
stranded
label:Assay type
type:basic:string
description:In strand non-specific assay a read is considered overlapping with a feature regardless of whether it is mapped to the same or the opposite strand as the feature. In strand-specific forward assay and single reads, the read has to be mapped to the same strand as the feature. For paired-end reads, the first read has to be on the same strand and the second read on the opposite strand. In strand-specific reverse assay these rules are reversed.
default:no
choices:
  • Strand non-specific: no
  • Strand-specific forward: yes
  • Strand-specific reverse: reverse
advanced
label:Advanced
type:basic:boolean
default:False
star.noncannonical
label:Remove non-cannonical junctions (Cufflinks compatibility)
type:basic:boolean
description:It is recommended to remove the non-canonical junctions for Cufflinks runs using –outFilterIntronMotifs RemoveNoncanonical.
default:False
star.detect_chimeric.chimeric
label:Detect chimeric and circular alignments
type:basic:boolean
description:To switch on detection of chimeric (fusion) alignments (in addition to normal mapping), –chimSegmentMin should be set to a positive value. Each chimeric alignment consists of two “segments”. Each segment is non-chimeric on its own, but the segments are chimeric to each other (i.e. the segments belong to different chromosomes, or different strands, or are far from each other). Both segments may contain splice junctions, and one of the segments may contain portions of both mates. –chimSegmentMin parameter controls the minimum mapped length of the two segments that is allowed. For example, if you have 2x75 reads and used –chimSegmentMin 20, a chimeric alignment with 130b on one chromosome and 20b on the other will be output, while 135 + 15 won’t be.
default:False
star.detect_chimeric.chimSegmentMin
label:–chimSegmentMin
type:basic:integer
disabled:!star.detect_chimeric.chimeric
default:20
star.t_coordinates.quantmode
label:Output in transcript coordinates
type:basic:boolean
description:With –quantMode TranscriptomeSAM option STAR will output alignments translated into transcript coordinates in the Aligned.toTranscriptome.out.bam file (in addition to alignments in genomic coordinates in Aligned.*.sam/bam files). These transcriptomic alignments can be used with various transcript quantification software that require reads to be mapped to transcriptome, such as RSEM or eXpress.
default:False
star.t_coordinates.singleend
label:Allow soft-clipping and indels
type:basic:boolean
description:By default, the output satisfies RSEM requirements: soft-clipping or indels are not allowed. Use –quantTranscriptomeBan Singleend to allow insertions, deletions ans soft-clips in the transcriptomic alignments, which can be used by some expression quantification software (e.g. eXpress).
disabled:!star.t_coordinates.quantmode
default:False
star.t_coordinates.gene_counts
label:Count reads
type:basic:boolean
description:With –quantMode GeneCounts option STAR will count number reads per gene while mapping. A read is counted if it overlaps (1nt or more) one and only one gene. Both ends of the paired-end read are checked for overlaps. The counts coincide with those produced by htseq-count with default parameters. ReadsPerGene.out.tab file with 4 columns which correspond to different strandedness options: column 1: gene ID; column 2: counts for unstranded RNA-seq; column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes); column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse).
disabled:!star.t_coordinates.quantmode
default:False
star.filtering.outFilterType
label:Type of filtering
type:basic:string
description:Normal: standard filtering using only current alignment; BySJout: keep only those reads that contain junctions that passed filtering into SJ.out.tab
default:Normal
choices:
  • Normal: Normal
  • BySJout: BySJout
star.filtering.outFilterMultimapNmax
label:–outFilterMultimapNmax
type:basic:integer
description:Read alignments will be output only if the read maps fewer than this value, otherwise no alignments will be output (default: 10).
required:False
star.filtering.outFilterMismatchNmax
label:–outFilterMismatchNmax
type:basic:integer
description:Alignment will be output only if it has fewer mismatches than this value (default: 10).
required:False
star.filtering.outFilterMismatchNoverLmax
label:–outFilterMismatchNoverLmax
type:basic:decimal
description:Max number of mismatches per pair relative to read length: for 2x100b, max number of mismatches is 0.06*200=8 for the paired read.
required:False
star.alignment.alignSJoverhangMin
label:–alignSJoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for spliced alignments (default: 5).
required:False
star.alignment.alignSJDBoverhangMin
label:–alignSJDBoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for annotated (sjdb) spliced alignments (default: 3).
required:False
star.alignment.alignIntronMin
label:–alignIntronMin
type:basic:integer
description:Minimum intron size: genomic gap is considered intron if its length >= alignIntronMin, otherwise it is considered Deletion (default: 21).
required:False
star.alignment.alignIntronMax
label:–alignIntronMax
type:basic:integer
description:Maximum intron size, if 0, max intron size will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
star.alignment.alignMatesGapMax
label:–alignMatesGapMax
type:basic:integer
description:Maximum gap between two mates, if 0, max intron gap will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
htseq.mode
label:Mode
type:basic:string
description:Mode to handle reads overlapping more than one feature. Possible values for <mode> are union, intersection-strict and intersection-nonempty
default:union
choices:
  • union: union
  • intersection-strict: intersection-strict
  • intersection-nonempty: intersection-nonempty
htseq.feature_class
label:Feature class
type:basic:string
description:Feature class (3rd column in GFF file) to be used. All other features will be ignored.
default:exon
htseq.id_attribute
label:ID attribute
type:basic:string
description:GFF attribute to be used as feature ID. Several GFF lines with the same feature ID will be considered as parts of the same feature. The feature ID is used to identity the counts in the output table.
default:gene_id
htseq.name_ordered
label:Use name-ordered BAM file for counting reads
type:basic:boolean
description:Use name-sorted BAM file for reads quantification. Improves compatibility with larger BAM files, but requires more computational time.
required:False
default:False

Output results

Cutadapt - STAR - HTSeq-count (single-end)

data:workflow:rnaseq:htseqworkflow-custom-cutadapt-star-htseq-single (data:reads:fastq:single  reads, data:genomeindex:star  genome, data:annotation:gtf  gff, basic:string  stranded, basic:boolean  advanced, basic:boolean  noncannonical, basic:boolean  chimeric, basic:integer  chimSegmentMin, basic:boolean  quantmode, basic:boolean  singleend, basic:boolean  gene_counts, basic:string  outFilterType, basic:integer  outFilterMultimapNmax, basic:integer  outFilterMismatchNmax, basic:decimal  outFilterMismatchNoverLmax, basic:integer  alignSJoverhangMin, basic:integer  alignSJDBoverhangMin, basic:integer  alignIntronMin, basic:integer  alignIntronMax, basic:integer  alignMatesGapMax, basic:string  mode, basic:string  feature_class, basic:string  id_attribute, basic:boolean  name_ordered)[Source: v1.0.1]

This RNA-seq pipeline is comprised of three steps, preprocessing, alignment, and quantification. First, reads are preprocessed by __cutadapt__ which finds and removes adapter sequences, primers, poly-A tails and other types of unwanted sequence from high-throughput sequencing reads. Next, preprocessed reads are aligned by __STAR__ aligner. At the time of implementation, STAR is considered a state-of-the-art tool that consistently produces accurate results from diverse sets of reads, and performs well even with default settings. For more information see [this comparison of RNA-seq aligners](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792058/). Finally, aligned reads are summarized to genes by __HTSeq-count__. Compared to featureCounts, HTSeq-count is not as computationally efficient. All three tools in this workflow support parallelization to accelerate the analysis.

Input arguments

reads
label:NGS reads
type:data:reads:fastq:single
genome
label:Indexed reference genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool
gff
label:Annotation (GFF)
type:data:annotation:gtf
stranded
label:Assay type
type:basic:string
description:In strand non-specific assay a read is considered overlapping with a feature regardless of whether it is mapped to the same or the opposite strand as the feature. In strand-specific forward assay and single reads, the read has to be mapped to the same strand as the feature. For paired-end reads, the first read has to be on the same strand and the second read on the opposite strand. In strand-specific reverse assay these rules are reversed.
default:no
choices:
  • Strand non-specific: no
  • Strand-specific forward: yes
  • Strand-specific reverse: reverse
advanced
label:Advanced
type:basic:boolean
default:False
star.noncannonical
label:Remove non-cannonical junctions (Cufflinks compatibility)
type:basic:boolean
description:It is recommended to remove the non-canonical junctions for Cufflinks runs using –outFilterIntronMotifs RemoveNoncanonical.
default:False
star.detect_chimeric.chimeric
label:Detect chimeric and circular alignments
type:basic:boolean
description:To switch on detection of chimeric (fusion) alignments (in addition to normal mapping), –chimSegmentMin should be set to a positive value. Each chimeric alignment consists of two “segments”. Each segment is non-chimeric on its own, but the segments are chimeric to each other (i.e. the segments belong to different chromosomes, or different strands, or are far from each other). Both segments may contain splice junctions, and one of the segments may contain portions of both mates. –chimSegmentMin parameter controls the minimum mapped length of the two segments that is allowed. For example, if you have 2x75 reads and used –chimSegmentMin 20, a chimeric alignment with 130b on one chromosome and 20b on the other will be output, while 135 + 15 won’t be.
default:False
star.detect_chimeric.chimSegmentMin
label:–chimSegmentMin
type:basic:integer
disabled:!star.detect_chimeric.chimeric
default:20
star.t_coordinates.quantmode
label:Output in transcript coordinates
type:basic:boolean
description:With –quantMode TranscriptomeSAM option STAR will output alignments translated into transcript coordinates in the Aligned.toTranscriptome.out.bam file (in addition to alignments in genomic coordinates in Aligned.*.sam/bam files). These transcriptomic alignments can be used with various transcript quantification software that require reads to be mapped to transcriptome, such as RSEM or eXpress.
default:False
star.t_coordinates.singleend
label:Allow soft-clipping and indels
type:basic:boolean
description:By default, the output satisfies RSEM requirements: soft-clipping or indels are not allowed. Use –quantTranscriptomeBan Singleend to allow insertions, deletions ans soft-clips in the transcriptomic alignments, which can be used by some expression quantification software (e.g. eXpress).
disabled:!star.t_coordinates.quantmode
default:False
star.t_coordinates.gene_counts
label:Count reads
type:basic:boolean
description:With –quantMode GeneCounts option STAR will count number reads per gene while mapping. A read is counted if it overlaps (1nt or more) one and only one gene. Both ends of the paired-end read are checked for overlaps. The counts coincide with those produced by htseq-count with default parameters. ReadsPerGene.out.tab file with 4 columns which correspond to different strandedness options: column 1: gene ID; column 2: counts for unstranded RNA-seq; column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes); column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse).
disabled:!star.t_coordinates.quantmode
default:False
star.filtering.outFilterType
label:Type of filtering
type:basic:string
description:Normal: standard filtering using only current alignment; BySJout: keep only those reads that contain junctions that passed filtering into SJ.out.tab
default:Normal
choices:
  • Normal: Normal
  • BySJout: BySJout
star.filtering.outFilterMultimapNmax
label:–outFilterMultimapNmax
type:basic:integer
description:Read alignments will be output only if the read maps fewer than this value, otherwise no alignments will be output (default: 10).
required:False
star.filtering.outFilterMismatchNmax
label:–outFilterMismatchNmax
type:basic:integer
description:Alignment will be output only if it has fewer mismatches than this value (default: 10).
required:False
star.filtering.outFilterMismatchNoverLmax
label:–outFilterMismatchNoverLmax
type:basic:decimal
description:Max number of mismatches per pair relative to read length: for 2x100b, max number of mismatches is 0.06*200=8 for the paired read.
required:False
star.alignment.alignSJoverhangMin
label:–alignSJoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for spliced alignments (default: 5).
required:False
star.alignment.alignSJDBoverhangMin
label:–alignSJDBoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for annotated (sjdb) spliced alignments (default: 3).
required:False
star.alignment.alignIntronMin
label:–alignIntronMin
type:basic:integer
description:Minimum intron size: genomic gap is considered intron if its length >= alignIntronMin, otherwise it is considered Deletion (default: 21).
required:False
star.alignment.alignIntronMax
label:–alignIntronMax
type:basic:integer
description:Maximum intron size, if 0, max intron size will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
star.alignment.alignMatesGapMax
label:–alignMatesGapMax
type:basic:integer
description:Maximum gap between two mates, if 0, max intron gap will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
htseq.mode
label:Mode
type:basic:string
description:Mode to handle reads overlapping more than one feature. Possible values for <mode> are union, intersection-strict and intersection-nonempty
default:union
choices:
  • union: union
  • intersection-strict: intersection-strict
  • intersection-nonempty: intersection-nonempty
htseq.feature_class
label:Feature class
type:basic:string
description:Feature class (3rd column in GFF file) to be used. All other features will be ignored.
default:exon
htseq.id_attribute
label:ID attribute
type:basic:string
description:GFF attribute to be used as feature ID. Several GFF lines with the same feature ID will be considered as parts of the same feature. The feature ID is used to identity the counts in the output table.
default:gene_id
htseq.name_ordered
label:Use name-ordered BAM file for counting reads
type:basic:boolean
description:Use name-sorted BAM file for reads quantification. Improves compatibility with larger BAM files, but requires more computational time.
required:False
default:False

Output results

Cutadapt - STAR - RSEM (Diagenode CATS, paired-end)

data:workflow:rnaseq:rsemworkflow-custom-cutadapt-star-rsem-paired (data:reads:fastq:paired  reads, data:genomeindex:star  star_index, data:index:expression  expression_index, basic:string  stranded, basic:boolean  advanced, basic:boolean  noncannonical, basic:boolean  chimeric, basic:integer  chimSegmentMin, basic:boolean  quantmode, basic:boolean  singleend, basic:boolean  gene_counts, basic:string  outFilterType, basic:integer  outFilterMultimapNmax, basic:integer  outFilterMismatchNmax, basic:decimal  outFilterMismatchNoverLmax, basic:integer  alignSJoverhangMin, basic:integer  alignSJDBoverhangMin, basic:integer  alignIntronMin, basic:integer  alignIntronMax, basic:integer  alignMatesGapMax)[Source: v1.0.2]

This RNA-seq pipeline is configured to be used with the Diagenode CATS RNA-seq kits. It is comprised of three steps, preprocessing, alignment, and quantification. First, reads are preprocessed by cutadapt which finds and removes adapter sequences, primers, poly-A tails and other types of unwanted sequence from high-throughput sequencing reads. Next, preprocessed reads are aligned by STAR aligner. Finally, RSEM estimates gene and isoform expression levels from the aligned reads.

Input arguments

reads
label:NGS reads
type:data:reads:fastq:paired
star_index
label:STAR genome index
type:data:genomeindex:star
expression_index
label:Gene expression indices
type:data:index:expression
stranded
label:Assay type
type:basic:string
description:In strand non-specific assay a read is considered overlapping with a feature regardless of whether it is mapped to the same or the opposite strand as the feature. In strand-specific forward assay and single reads, the read has to be mapped to the same strand as the feature. For paired-end reads, the first read has to be on the same strand and the second read on the opposite strand. In strand-specific reverse assay these rules are reversed.
default:no
choices:
  • Strand non-specific: no
  • Strand-specific forward: yes
  • Strand-specific reverse: reverse
advanced
label:Advanced
type:basic:boolean
default:False
star.noncannonical
label:Remove non-cannonical junctions (Cufflinks compatibility)
type:basic:boolean
description:It is recommended to remove the non-canonical junctions for Cufflinks runs using –outFilterIntronMotifs RemoveNoncanonical.
default:False
star.detect_chimeric.chimeric
label:Detect chimeric and circular alignments
type:basic:boolean
description:To switch on detection of chimeric (fusion) alignments (in addition to normal mapping), –chimSegmentMin should be set to a positive value. Each chimeric alignment consists of two “segments”. Each segment is non-chimeric on its own, but the segments are chimeric to each other (i.e. the segments belong to different chromosomes, or different strands, or are far from each other). Both segments may contain splice junctions, and one of the segments may contain portions of both mates. –chimSegmentMin parameter controls the minimum mapped length of the two segments that is allowed. For example, if you have 2x75 reads and used –chimSegmentMin 20, a chimeric alignment with 130b on one chromosome and 20b on the other will be output, while 135 + 15 won’t be.
default:False
star.detect_chimeric.chimSegmentMin
label:–chimSegmentMin
type:basic:integer
disabled:!star.detect_chimeric.chimeric
default:20
star.t_coordinates.quantmode
label:Output in transcript coordinates
type:basic:boolean
description:With –quantMode TranscriptomeSAM option STAR will output alignments translated into transcript coordinates in the Aligned.toTranscriptome.out.bam file (in addition to alignments in genomic coordinates in Aligned.*.sam/bam files). These transcriptomic alignments can be used with various transcript quantification software that require reads to be mapped to transcriptome, such as RSEM or eXpress.
default:True
star.t_coordinates.singleend
label:Allow soft-clipping and indels
type:basic:boolean
description:By default, the output satisfies RSEM requirements: soft-clipping or indels are not allowed. Use –quantTranscriptomeBan Singleend to allow insertions, deletions ans soft-clips in the transcriptomic alignments, which can be used by some expression quantification software (e.g. eXpress).
disabled:!star.t_coordinates.quantmode
default:False
star.t_coordinates.gene_counts
label:Count reads
type:basic:boolean
description:With –quantMode GeneCounts option STAR will count number reads per gene while mapping. A read is counted if it overlaps (1nt or more) one and only one gene. Both ends of the paired-end read are checked for overlaps. The counts coincide with those produced by htseq-count with default parameters. ReadsPerGene.out.tab file with 4 columns which correspond to different strandedness options: column 1: gene ID; column 2: counts for unstranded RNA-seq; column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes); column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse).
disabled:!star.t_coordinates.quantmode
default:False
star.filtering.outFilterType
label:Type of filtering
type:basic:string
description:Normal: standard filtering using only current alignment; BySJout: keep only those reads that contain junctions that passed filtering into SJ.out.tab
default:Normal
choices:
  • Normal: Normal
  • BySJout: BySJout
star.filtering.outFilterMultimapNmax
label:–outFilterMultimapNmax
type:basic:integer
description:Read alignments will be output only if the read maps fewer than this value, otherwise no alignments will be output (default: 10).
required:False
star.filtering.outFilterMismatchNmax
label:–outFilterMismatchNmax
type:basic:integer
description:Alignment will be output only if it has fewer mismatches than this value (default: 10).
required:False
star.filtering.outFilterMismatchNoverLmax
label:–outFilterMismatchNoverLmax
type:basic:decimal
description:Max number of mismatches per pair relative to read length: for 2x100b, max number of mismatches is 0.06*200=8 for the paired read.
required:False
star.alignment.alignSJoverhangMin
label:–alignSJoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for spliced alignments (default: 5).
required:False
star.alignment.alignSJDBoverhangMin
label:–alignSJDBoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for annotated (sjdb) spliced alignments (default: 3).
required:False
star.alignment.alignIntronMin
label:–alignIntronMin
type:basic:integer
description:Minimum intron size: genomic gap is considered intron if its length >= alignIntronMin, otherwise it is considered Deletion (default: 21).
required:False
star.alignment.alignIntronMax
label:–alignIntronMax
type:basic:integer
description:Maximum intron size, if 0, max intron size will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
star.alignment.alignMatesGapMax
label:–alignMatesGapMax
type:basic:integer
description:Maximum gap between two mates, if 0, max intron gap will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False

Output results

Cutadapt - STAR - RSEM (Diagenode CATS, single-end)

data:workflow:rnaseq:rsemworkflow-custom-cutadapt-star-rsem-single (data:reads:fastq:single  reads, data:genomeindex:star  star_index, data:index:expression  expression_index, basic:string  stranded, basic:boolean  advanced, basic:boolean  noncannonical, basic:boolean  chimeric, basic:integer  chimSegmentMin, basic:boolean  quantmode, basic:boolean  singleend, basic:boolean  gene_counts, basic:string  outFilterType, basic:integer  outFilterMultimapNmax, basic:integer  outFilterMismatchNmax, basic:decimal  outFilterMismatchNoverLmax, basic:integer  alignSJoverhangMin, basic:integer  alignSJDBoverhangMin, basic:integer  alignIntronMin, basic:integer  alignIntronMax, basic:integer  alignMatesGapMax)[Source: v1.0.2]

This RNA-seq pipeline is configured to be used with the Diagenode CATS RNA-seq kits. It is comprised of three steps, preprocessing, alignment, and quantification. First, reads are preprocessed by cutadapt which finds and removes adapter sequences, primers, poly-A tails and other types of unwanted sequence from high-throughput sequencing reads. Next, preprocessed reads are aligned by STAR aligner. Finally, RSEM estimates gene and isoform expression levels from the aligned reads.

Input arguments

reads
label:NGS reads
type:data:reads:fastq:single
star_index
label:STAR genome index
type:data:genomeindex:star
expression_index
label:Gene expression indices
type:data:index:expression
stranded
label:Assay type
type:basic:string
description:In strand non-specific assay a read is considered overlapping with a feature regardless of whether it is mapped to the same or the opposite strand as the feature. In strand-specific forward assay and single reads, the read has to be mapped to the same strand as the feature. For paired-end reads, the first read has to be on the same strand and the second read on the opposite strand. In strand-specific reverse assay these rules are reversed.
default:no
choices:
  • Strand non-specific: no
  • Strand-specific forward: yes
  • Strand-specific reverse: reverse
advanced
label:Advanced
type:basic:boolean
default:False
star.noncannonical
label:Remove non-cannonical junctions (Cufflinks compatibility)
type:basic:boolean
description:It is recommended to remove the non-canonical junctions for Cufflinks runs using –outFilterIntronMotifs RemoveNoncanonical.
default:False
star.detect_chimeric.chimeric
label:Detect chimeric and circular alignments
type:basic:boolean
description:To switch on detection of chimeric (fusion) alignments (in addition to normal mapping), –chimSegmentMin should be set to a positive value. Each chimeric alignment consists of two “segments”. Each segment is non-chimeric on its own, but the segments are chimeric to each other (i.e. the segments belong to different chromosomes, or different strands, or are far from each other). Both segments may contain splice junctions, and one of the segments may contain portions of both mates. –chimSegmentMin parameter controls the minimum mapped length of the two segments that is allowed. For example, if you have 2x75 reads and used –chimSegmentMin 20, a chimeric alignment with 130b on one chromosome and 20b on the other will be output, while 135 + 15 won’t be.
default:False
star.detect_chimeric.chimSegmentMin
label:–chimSegmentMin
type:basic:integer
disabled:!star.detect_chimeric.chimeric
default:20
star.t_coordinates.quantmode
label:Output in transcript coordinates
type:basic:boolean
description:With –quantMode TranscriptomeSAM option STAR will output alignments translated into transcript coordinates in the Aligned.toTranscriptome.out.bam file (in addition to alignments in genomic coordinates in Aligned.*.sam/bam files). These transcriptomic alignments can be used with various transcript quantification software that require reads to be mapped to transcriptome, such as RSEM or eXpress.
default:True
star.t_coordinates.singleend
label:Allow soft-clipping and indels
type:basic:boolean
description:By default, the output satisfies RSEM requirements: soft-clipping or indels are not allowed. Use –quantTranscriptomeBan Singleend to allow insertions, deletions ans soft-clips in the transcriptomic alignments, which can be used by some expression quantification software (e.g. eXpress).
disabled:!star.t_coordinates.quantmode
default:False
star.t_coordinates.gene_counts
label:Count reads
type:basic:boolean
description:With –quantMode GeneCounts option STAR will count number reads per gene while mapping. A read is counted if it overlaps (1nt or more) one and only one gene. Both ends of the paired-end read are checked for overlaps. The counts coincide with those produced by htseq-count with default parameters. ReadsPerGene.out.tab file with 4 columns which correspond to different strandedness options: column 1: gene ID; column 2: counts for unstranded RNA-seq; column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes); column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse).
disabled:!star.t_coordinates.quantmode
default:False
star.filtering.outFilterType
label:Type of filtering
type:basic:string
description:Normal: standard filtering using only current alignment; BySJout: keep only those reads that contain junctions that passed filtering into SJ.out.tab
default:Normal
choices:
  • Normal: Normal
  • BySJout: BySJout
star.filtering.outFilterMultimapNmax
label:–outFilterMultimapNmax
type:basic:integer
description:Read alignments will be output only if the read maps fewer than this value, otherwise no alignments will be output (default: 10).
required:False
star.filtering.outFilterMismatchNmax
label:–outFilterMismatchNmax
type:basic:integer
description:Alignment will be output only if it has fewer mismatches than this value (default: 10).
required:False
star.filtering.outFilterMismatchNoverLmax
label:–outFilterMismatchNoverLmax
type:basic:decimal
description:Max number of mismatches per pair relative to read length: for 2x100b, max number of mismatches is 0.06*200=8 for the paired read.
required:False
star.alignment.alignSJoverhangMin
label:–alignSJoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for spliced alignments (default: 5).
required:False
star.alignment.alignSJDBoverhangMin
label:–alignSJDBoverhangMin
type:basic:integer
description:Minimum overhang (i.e. block size) for annotated (sjdb) spliced alignments (default: 3).
required:False
star.alignment.alignIntronMin
label:–alignIntronMin
type:basic:integer
description:Minimum intron size: genomic gap is considered intron if its length >= alignIntronMin, otherwise it is considered Deletion (default: 21).
required:False
star.alignment.alignIntronMax
label:–alignIntronMax
type:basic:integer
description:Maximum intron size, if 0, max intron size will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False
star.alignment.alignMatesGapMax
label:–alignMatesGapMax
type:basic:integer
description:Maximum gap between two mates, if 0, max intron gap will be determined by (2pow(winBinNbits)*winAnchorDistNbins) (default: 0).
required:False

Output results

Cutadapt - STAR - StringTie (Corall, paired-end)

data:workflow:rnaseq:corallworkflow-corall-paired (data:reads:fastq:paired  reads, data:genomeindex:star  star_index, data:annotation  annotation, data:genomeindex:star  rrna_reference, data:genomeindex:star  globin_reference, basic:boolean  show_advanced, basic:integer  quality_cutoff, basic:integer  n_reads, basic:integer  seed, basic:decimal  fraction, basic:boolean  two_pass)[Source: v1.0.1]

RNA-seq pipeline optimized for the Lexogen Corall Total RNA-Seq Library Prep Kit. UMI-sequences are extracted from the raw reads before the reads are trimmed and quality filtered using Cutadapt. Preprocessed reads are aligned by the STAR aligner and de-duplicated using UMI-tools. Gene abundance estimates are reported by the StringTie tool. QC operates on downsampled reads and includes alignment of input reads to the rRNA/globin reference sequences. The reported alignment rate is used to asses the rRNA/globin sequence depletion rate. The analysis results and QC reports are summarized by the MultiQC.

Input arguments

reads
label:Select sample(s)
type:data:reads:fastq:paired
star_index
label:Genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
annotation
label:Annotation
type:data:annotation
description:Genome annotation file (GTF).
rrna_reference
label:Indexed rRNA reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
globin_reference
label:Indexed Globin reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
cutadapt.quality_cutoff
label:Reads quality cutoff
type:basic:integer
description:Trim low-quality bases from 3’ end of each read before adapter removal. Use this option when processing the data generated by older Illumina machines. The use of this option will override the NextSeq/NovaSeq-specific trimming procedure which is enabled by default and is recommended for Illumina machines that utilize 2-color chemistry to encode the four bases.
required:False
downsampling.n_reads
label:Number of reads
type:basic:integer
default:1000000
downsampling.seed
label:Seed
type:basic:integer
default:11
downsampling.fraction
label:Fraction
type:basic:decimal
description:Use the fraction of reads in range [0.0, 1.0] from the orignal input file instead of the absolute number of reads. If set, this will override the “Number of reads” input parameter.
required:False
downsampling.two_pass
label:2-pass mode
type:basic:boolean
description:Enable two-pass mode when down-sampling. Two-pass mode is twice as slow but with much reduced memory.
default:False

Output results

Cutadapt - STAR - StringTie (Corall, single-end)

data:workflow:rnaseq:corallworkflow-corall-single (data:reads:fastq:single  reads, data:genomeindex:star  star_index, data:annotation  annotation, data:genomeindex:star  rrna_reference, data:genomeindex:star  globin_reference, basic:boolean  show_advanced, basic:integer  quality_cutoff, basic:integer  n_reads, basic:integer  seed, basic:decimal  fraction, basic:boolean  two_pass)[Source: v1.0.1]

RNA-seq pipeline optimized for the Lexogen Corall Total RNA-Seq Library Prep Kit. UMI-sequences are extracted from the raw reads before the reads are trimmed and quality filtered using Cutadapt. Preprocessed reads are aligned by the STAR aligner and de-duplicated using UMI-tools. Gene abundance estimates are reported by the StringTie tool. QC operates on downsampled reads and includes alignment of input reads to the rRNA/globin reference sequences. The reported alignment rate is used to asses the rRNA/globin sequence depletion rate. The analysis results and QC reports are summarized by the MultiQC.

Input arguments

reads
label:Select sample(s)
type:data:reads:fastq:single
star_index
label:Genome
type:data:genomeindex:star
description:Genome index prepared by STAR aligner indexing tool.
annotation
label:Annotation
type:data:annotation
description:Genome annotation file (GTF).
rrna_reference
label:Indexed rRNA reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
globin_reference
label:Indexed Globin reference sequence
type:data:genomeindex:star
description:Reference sequence index prepared by STAR aligner indexing tool.
show_advanced
label:Show advanced parameters
type:basic:boolean
default:False
cutadapt.quality_cutoff
label:Reads quality cutoff
type:basic:integer
description:Trim low-quality bases from 3’ end of each read before adapter removal. Use this option when processing the data generated by older Illumina machines. The use of this option will override the NextSeq/NovaSeq-specific trimming procedure which is enabled by default and is recommended for Illumina machines that utilize 2-color chemistry to encode the four bases.
required:False
downsampling.n_reads
label:Number of reads
type:basic:integer
default:1000000
downsampling.seed
label:Seed
type:basic:integer
default:11
downsampling.fraction
label:Fraction
type:basic:decimal
description:Use the fraction of reads in range [0.0, 1.0] from the orignal input file instead of the absolute number of reads. If set, this will override the “Number of reads” input parameter.
required:False
downsampling.two_pass
label:2-pass mode
type:basic:boolean
description:Enable two-pass mode when down-sampling. Two-pass mode is twice as slow but with much reduced memory.
default:False

Output results

DESeq2

data:differentialexpression:deseq2differentialexpression-deseq2 (list:data:expression  case, list:data:expression  control, basic:boolean  beta_prior, basic:boolean  count, basic:integer  min_count_sum, basic:boolean  cook, basic:decimal  cooks_cutoff, basic:boolean  independent, basic:decimal  alpha)[Source: v2.4.1]

The DESeq2 package estimates variance-mean dependence in count data from high-throughput sequencing assays and tests for differential expression based on a model using the negative binomial distribution. See [here](https://www.bioconductor.org/packages/release/bioc/manuals/DESeq2/man/DESeq2.pdf) and [here](http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html) for more information.

Input arguments

case
label:Case
type:list:data:expression
description:Case samples (replicates)
control
label:Control
type:list:data:expression
description:Control samples (replicates)
options.beta_prior
label:Beta prior
type:basic:boolean
description:Whether or not to put a zero-mean normal prior on the non-intercept coefficients.
default:False
filter.count
label:Filter genes based on expression count
type:basic:boolean
default:True
filter.min_count_sum
label:Minimum raw gene expression count summed over all samples
type:basic:integer
description:Filter genes in the expression matrix input. Remove genes where the expression count sum over all samples is below the threshold.
hidden:!filter.count
default:10
filter.cook
label:Filter genes based on Cook’s distance
type:basic:boolean
default:False
filter.cooks_cutoff
label:Threshold on Cook’s distance
type:basic:decimal
description:If one or more samples have Cook’s distance larger than the threshold set here, the p-value for the row is set to NA. If left empty, the default threshold of 0.99 quantile of the F(p, m-p) distribution is used, where p is the number of coefficients being fitted and m is the number of samples. This test excludes Cook’s distance of samples belonging to experimental groups with only two samples.
required:False
hidden:!filter.cook
filter.independent
label:Apply independent gene filtering
type:basic:boolean
default:False
filter.alpha
label:Significance cut-off used for optimizing independent gene filtering
type:basic:decimal
description:The value should be set to adjusted p-value cut-off (FDR).
hidden:!filter.independent
default:0.1

Output results

raw
label:Differential expression
type:basic:file
de_json
label:Results table (JSON)
type:basic:json
de_file
label:Results table (file)
type:basic:file
count_matrix
label:Count matrix
type:basic:file
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string

Detect library strandedness

data:strandednesslibrary-strandedness (data:reads:fastq  reads, basic:integer  read_number, data:index:salmon  salmon_index)[Source: v0.1.2]

This process uses the Salmon transcript quantification tool to automatically infer the NGS library strandedness. For more details, please see the Salmon [documentation](https://salmon.readthedocs.io/en/latest/library_type.html)

Input arguments

reads
label:Sequencing reads
type:data:reads:fastq
description:Sequencing reads in .fastq format. Both single and paired-end libraries are supported
read_number
label:Number of input reads
type:basic:integer
description:Number of sequencing reads that are subsampled from each of the original .fastq files before library strand detection
default:50000
salmon_index
label:Transcriptome index file
type:data:index:salmon
description:Transcriptome index file created using the Salmon indexing tool. cDNA (transcriptome) sequences used for index file creation must be derived from the same species as the input sequencing reads to obtain the reliable analysis results

Output results

strandedness
label:Library strandedness type
type:basic:string
description:The predicted library strandedness type. The codes U and IU indicate ‘strand non-specific’ library for single or paired-end reads, respectively. Codes SF and ISF correspond to the ‘strand-specific forward’ library, for the single or paired-end reads, respectively. For ‘strand-specific reverse’ library, the corresponding codes are SR and ISR. For more details, please see the Salmon [documentation](https://salmon.readthedocs.io/en/latest/library_type.html)
fragment_ratio
label:Compatible fragment ratio
type:basic:decimal
description:The ratio of fragments that support the predicted library strandedness type
log
label:Log file
type:basic:file
description:Analysis log file.

Dictyostelium expressions

data:expression:polyaexpression-dicty (data:alignment:bam  alignment, data:annotation:gff3  gff, data:mappability:bcm  mappable)[Source: v1.3.1]

Dictyostelium-specific pipeline. Developed by Bioinformatics Laboratory, Faculty of Computer and Information Science, University of Ljubljana, Slovenia and Shaulsky Lab, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.

Input arguments

alignment
label:Aligned sequence
type:data:alignment:bam
gff
label:Features (GFF3)
type:data:annotation:gff3
mappable
label:Mappability
type:data:mappability:bcm

Output results

exp
label:Expression RPKUM (polyA)
type:basic:file
description:mRNA reads scaled by uniquely mappable part of exons.
rpkmpolya
label:Expression RPKM (polyA)
type:basic:file
description:mRNA reads scaled by exon length.
rc
label:Read counts (polyA)
type:basic:file
description:mRNA reads uniquely mapped to gene exons.
rpkum
label:Expression RPKUM
type:basic:file
description:Reads scaled by uniquely mappable part of exons.
rpkm
label:Expression RPKM
type:basic:file
description:Reads scaled by exon length.
rc_raw
label:Read counts (raw)
type:basic:file
description:Reads uniquely mapped to gene exons.
exp_json
label:Expression RPKUM (polyA) (json)
type:basic:json
exp_type
label:Expression Type (default output)
type:basic:string
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string

Differential Expression (table)

data:differentialexpression:uploadupload-diffexp (basic:file  src, basic:string  gene_id, basic:string  logfc, basic:string  fdr, basic:string  logodds, basic:string  fwer, basic:string  pvalue, basic:string  stat, basic:string  source, basic:string  species, basic:string  build, basic:string  feature_type, list:data:expression  case, list:data:expression  control)[Source: v1.2.1]

Upload Differential Expression table.

Input arguments

src
label:Differential expression file
type:basic:file
description:Differential expression file. Supported file types: *.xls, *.xlsx, *.tab (tab-delimited file), *.diff. DE file must include columns with log2(fold change) and FDR or pval information. DE file must contain header row with column names. Accepts DESeq, DESeq2, edgeR and CuffDiff output files.
validate_regex:\.(xls|xlsx|tab|tab.gz|diff|diff.gz)$
gene_id
label:Gene ID label
type:basic:string
logfc
label:LogFC label
type:basic:string
fdr
label:FDR label
type:basic:string
required:False
logodds
label:LogOdds label
type:basic:string
required:False
fwer
label:FWER label
type:basic:string
required:False
pvalue
label:Pvalue label
type:basic:string
required:False
stat
label:Statistics label
type:basic:string
required:False
source
label:Gene ID database
type:basic:string
choices:
  • AFFY: AFFY
  • DICTYBASE: DICTYBASE
  • ENSEMBL: ENSEMBL
  • NCBI: NCBI
  • UCSC: UCSC
species
label:Species
type:basic:string
description:Species latin name.
choices:
  • Homo sapiens: Homo sapiens
  • Mus musculus: Mus musculus
  • Rattus norvegicus: Rattus norvegicus
  • Dictyostelium discoideum: Dictyostelium discoideum
  • Odocoileus virginianus texanus: Odocoileus virginianus texanus
  • Solanum tuberosum: Solanum tuberosum
build
label:Build
type:basic:string
description:Genome build or annotation version.
feature_type
label:Feature type
type:basic:string
default:gene
choices:
  • gene: gene
  • transcript: transcript
  • exon: exon
case
label:Case
type:list:data:expression
description:Case samples (replicates)
required:False
control
label:Control
type:list:data:expression
description:Control samples (replicates)
required:False

Output results

raw
label:Differential expression
type:basic:file
de_json
label:Results table (JSON)
type:basic:json
de_file
label:Results table (file)
type:basic:file
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string

Expression Time Course

data:etcetc-bcm (list:data:expression  expressions, basic:boolean  avg)[Source: v1.1.1]

Select gene expression data and form a time course.

Input arguments

expressions
label:RPKM expression profile
type:list:data:expression
required:True
avg
label:Average by time
type:basic:boolean
default:True

Output results

etcfile
label:Expression time course file
type:basic:file
etc
label:Expression time course
type:basic:json

Expression aggregator

data:aggregator:expressionexpression-aggregator (list:data:expression  exps, basic:string  group_by, data:aggregator:expression  expr_aggregator)[Source: v0.2.2]

Collect expression data from samples grouped by sample descriptor field. The Expression aggregator process should not be run in Batch Mode, as this will create redundant outputs. Rather, select multiple samples below for which you wish to aggregate the expression matrix.

Input arguments

exps
label:Expressions
type:list:data:expression
group_by
label:Sample descriptor field
type:basic:string
expr_aggregator
label:Expression aggregator
type:data:aggregator:expression
required:False

Output results

exp_matrix
label:Expression matrix
type:basic:file
box_plot
label:Box plot
type:basic:json
log_box_plot
label:Log box plot
type:basic:json
source
label:Gene ID database
type:basic:string
species
label:Species
type:basic:string
exp_type
label:Expression type
type:basic:string

Expression data

data:expressionupload-expression (basic:file  rc, basic:file  exp, basic:string  exp_name, basic:string  exp_type, basic:string  source, basic:string  species, basic:string  build, basic:string  feature_type)[Source: v2.2.1]

Upload expression data by providing raw expression data (read counts) and/or normalized expression data together with the associated data normalization type.

Input arguments

rc
label:Read counts (raw expression)
type:basic:file
description:Reads mapped to genomic features (raw count data). Supported extensions: .txt.gz (preferred), .tab.* or .txt.*
required:False
validate_regex:\.(txt|tab|gz)(|\.gz|\.bz2|\.tgz|\.tar\.gz|\.tar\.bz2|\.zip|\.rar|\.7z)$
exp
label:Normalized expression
type:basic:file
description:Normalized expression data. Supported extensions: .tab.gz (preferred) or .tab.*
required:False
validate_regex:\.(tab|gz)(|\.gz|\.bz2|\.tgz|\.tar\.gz|\.tar\.bz2|\.zip|\.rar|\.7z)$
exp_name
label:Expression name
type:basic:string
exp_type
label:Normalization type
type:basic:string
description:Normalization type
required:False
source
label:Gene ID source
type:basic:string
choices:
  • AFFY: AFFY
  • DICTYBASE: DICTYBASE
  • ENSEMBL: ENSEMBL
  • NCBI: NCBI
  • UCSC: UCSC
species
label:Species
type:basic:string
description:Species latin name.
choices:
  • Homo sapiens: Homo sapiens
  • Mus musculus: Mus musculus
  • Rattus norvegicus: Rattus norvegicus
  • Dictyostelium discoideum: Dictyostelium discoideum
  • Odocoileus virginianus texanus: Odocoileus virginianus texanus
  • Solanum tuberosum: Solanum tuberosum
build
label:Build
type:basic:string
description:Genome build or annotation version.
feature_type
label:Feature type
type:basic:string
default:gene
choices:
  • gene: gene
  • transcript: transcript
  • exon: exon

Output results

exp
label:Normalized expression
type:basic:file
description:Normalized expression
rc
label:Read counts
type:basic:file
description:Reads mapped to genomic features.
required:False
exp_json
label:Expression (json)
type:basic:json
exp_type
label:Expression type
type:basic:string
exp_set
label:Expressions
type:basic:file
exp_set_json
label:Expressions (json)
type:basic:json
source
label:Gene ID source
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string

Expression data (Cuffnorm)

data:expressionupload-expression-cuffnorm (basic:file  exp, data:cufflinks:cuffquant  cxb, basic:string  exp_type)[Source: v1.4.1]

Upload expression data by providing Cuffnorm results.

Input arguments

exp
label:Normalized expression
type:basic:file
cxb
label:Cuffquant analysis
type:data:cufflinks:cuffquant
description:Cuffquant analysis.
exp_type
label:Normalization type
type:basic:string
default:Cuffnorm

Output results

exp
label:Normalized expression
type:basic:file
description:Normalized expression
rc
label:Read counts
type:basic:file
description:Reads mapped to genomic features.
required:False
exp_json
label:Expression (json)
type:basic:json
exp_type
label:Expression type
type:basic:string
exp_set
label:Expressions
type:basic:file
exp_set_json
label:Expressions (json)
type:basic:json
source
label:Gene ID source
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type
type:basic:string

Expression data (STAR)

data:expression:starupload-expression-star (basic:file  rc, basic:string  stranded, basic:string  source, basic:string  species, basic:string  build, basic:string  feature_type)[Source: v1.3.1]

Upload expression data by providing STAR aligner results.

Input arguments

rc
label:Read counts (raw expression)
type:basic:file
description:Reads mapped to genomic features (raw count data). Supported extensions: .txt.gz (preferred), .tab.* or .txt.*
validate_regex:\.(txt|tab|gz)(|\.gz|\.bz2|\.tgz|\.tar\.gz|\.tar\.bz2|\.zip|\.rar|\.7z)$
stranded
label:Is data from a strand specific assay?
type:basic:string
description:For stranded=no, a read is considered overlapping with a feature regardless of whether it is mapped to the same or the opposite strand as the feature. For stranded=yes and single-end reads, the read has to be mapped to the same strand as the feature. For paired-end reads, the first read has to be on the same strand and the second read on the opposite strand. For stranded=reverse, these rules are reversed.
default:yes
choices:
  • yes: yes
  • no: no
  • reverse: reverse
source
label:Gene ID source
type:basic:string
choices:
  • AFFY: AFFY
  • DICTYBASE: DICTYBASE
  • ENSEMBL: ENSEMBL
  • NCBI: NCBI
  • UCSC: UCSC
species
label:Species
type:basic:string
description:Species latin name.
choices:
  • Homo sapiens: Homo sapiens
  • Mus musculus: Mus musculus
  • Rattus norvegicus: Rattus norvegicus
  • Dictyostelium discoideum: Dictyostelium discoideum
  • Odocoileus virginianus texanus: Odocoileus virginianus texanus
  • Solanum tuberosum: Solanum tuberosum
build
label:Build
type:basic:string
description:Genome build or annotation version.
feature_type
label:Feature type
type:basic:string
default:gene
choices:
  • gene: gene
  • transcript: transcript
  • exon: exon

Output results

rc
label:Read counts (raw data)
type:basic:file
description:Reads mapped to genomic features.
exp
label:Expression data
type:basic:file
exp_json
label:Expression (json)
type:basic:json
exp_type
label:Expression type
type:basic:string
exp_set
label:Expressions
type:basic:file
exp_set_json
label:Expressions (json)
type:basic:json
source
label:Gene ID source
type:basic:string
species
label:Species
type:basic:string
build
label:Build
type:basic:string
feature_type
label:Feature type