Process definitions

ATAC-Seq

data:workflow:atacseqworkflow-atac-seq (data:reads:fastq  reads, data:index:bowtie2  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)

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:index:bowtie2

promoter

label:Promoter regions BED file type:data:bed description:BED file containing promoter regions (TSS+-1000 bp 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 4 bp and reads on “-” strand by 5 bp. 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 1000 bp for small local region (–slocal), and 10000 bp 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 1000 bp for small local region (–slocal), and 10000 bp 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: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 tag. If set, MACS will be terminated if paired-peak model has 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 to true, 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. 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 ()

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 ()

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 ()

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 ()

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 ()

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:seq:nucleotide  genome, data:index:bwa  bwa_index, 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)

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 sequence (FASTA) type:data:seq:nucleotide

bwa_index

label:Genome index (BWA) type:data:index:bwa

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:index:bwa  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)

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:index:bwa

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

Alleyoop UTR Rates

data:alleyoop:utrrates:alleyoop-utr-rates (data:seq:nucleotide  ref_seq, data:bed  regions, data:alignment:bam:slamdunk  slamdunk, basic:integer  read_length)

Run Alleyoop utrrates.

Input arguments

ref_seq

label:FASTA file containig sequences for aligning type:data:seq:nucleotide required:True hidden:False

regions

label:BED file with coordinates of regions of interest type:data:bed required:True hidden:False

slamdunk

label:Slamdunk results type:data:alignment:bam:slamdunk required:True hidden:False

read_length

label:Maximum read length type:basic:integer description:Maximum length of reads in the input FASTQ file required:True hidden:False default:150

Output results

report

label:Tab-separated file containing conversion rates on each region of interest type:basic:file required:True hidden:False

plot

label:Region of interest conversion rate plot type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

Alleyoop collapse

data:alleyoop:collapse:alleyoop-collapse (data:alignment:bam:slamdunk  slamdunk, basic:string  source)

Run Alleyoop collapse tool on Slamdunk results.

Input arguments

slamdunk

label:Slamdunk results type:data:alignment:bam:slamdunk required:True hidden:False

source

label:Gene ID source type:basic:string required:True hidden:False default:ENSEMBL choices:

• ENSEMBL: ENSEMBL
• UCSC: UCSC

Output results

tcount

label:Count report containing SLAMSeq statistics type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

Alleyoop rates

data:alleyoop:rates:alleyoop-rates (data:seq:nucleotide  ref_seq, data:alignment:bam:slamdunk  slamdunk)

Run Alleyoop rates.

Input arguments

ref_seq

label:FASTA file containig sequences for aligning type:data:seq:nucleotide required:True hidden:False

slamdunk

label:Slamdunk results type:data:alignment:bam:slamdunk required:True hidden:False

Output results

report

label:Tab-separated file containing the overall conversion rates type:basic:file required:True hidden:False

plot

label:Overall conversion rate plot file type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

Alleyoop snpeval

data:alleyoop:snpeval:alleyoop-snpeval (data:seq:nucleotide  ref_seq, data:bed  regions, data:alignment:bam:slamdunk  slamdunk, basic:integer  read_length)

Run Alleyoop snpeval.

Input arguments

ref_seq

label:FASTA file containig sequences for aligning type:data:seq:nucleotide required:True hidden:False

regions

label:BED file with coordinates of regions of interest type:data:bed required:True hidden:False

slamdunk

label:Slamdunk results type:data:alignment:bam:slamdunk required:True hidden:False

read_length

label:Maximum read length type:basic:integer description:Maximum length of reads in the input FASTQ file required:True hidden:False default:150

Output results

report

label:Tab-separated file with read counts, T>C read counts and SNP indication type:basic:file required:True hidden:False

plot

label:SNP evaluation plot type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

Alleyoop summary

data:alleyoop:summary:alleyoop-summary (list:data:alignment:bam:slamdunk  slamdunk)

Run Alleyoop summary.

Input arguments

slamdunk

label:Slamdunk results type:list:data:alignment:bam:slamdunk required:True hidden:False

Output results

report

label:Tab-separated file with mapping statistics type:basic:file required:True hidden:False

plot_data

label:PCA values of the samples based on T>C read counts in regions of interest. type:basic:file required:False hidden:False

plot

label:PCA plot of the samples based on T>C read counts in regions of interest. type:basic:file required:False hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

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)

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)

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:seq:nucleotide  genome, data:annotation:gtf  annotation, data:alignment:bam:star  alignment_star, data:alignment:bam  alignment, data:bed  input_bed_junctions)

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:seq:nucleotide

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)

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)

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)

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)

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)

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)

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:index: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:index:star  rrna_reference, data:index:star  globin_reference)

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:index: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:index:star description:Reference sequence index prepared by STAR aligner indexing tool.

qc.globin_reference

label:Indexed Globin reference sequence type:data:index: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:index: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:index:star  rrna_reference, data:index:star  globin_reference)

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:index: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:index:star description:Reference sequence index prepared by STAR aligner indexing tool.

qc.globin_reference

label:Indexed Globin reference sequence type:data:index: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:index:star  star_index, list:data:seq:nucleotide  adapters, data:annotation  annotation, basic:string  stranded)

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:index: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:index:star  star_index, list:data:seq:nucleotide  adapters, data:annotation  annotation, basic:string  stranded)

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:index: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:index: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:index:star  rrna_reference, data:index:star  globin_reference)

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:index: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:index:star description:Reference sequence index prepared by STAR aligner indexing tool.

qc.globin_reference

label:Indexed Globin reference sequence type:data:index: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:index: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:index:star  rrna_reference, data:index:star  globin_reference)

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:index: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:index:star description:Reference sequence index prepared by STAR aligner indexing tool.

qc.globin_reference

label:Indexed Globin reference sequence type:data:index: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:index:star  genome, data:annotation  annotation, data:index:star  rrna_reference, data:index: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: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)

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:index: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:index:star description:Reference sequence index prepared by STAR aligner indexing tool.

globin_reference

label:Indexed Globin reference sequence type:data:index: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.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 in selective alignment mode and 0 otherwise. required:False

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]. 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 (4) corresponds to the default used in Zakeri et al. 2017 and larger values imply a more fine-grained factorization. If range factorization is enabled, a common value to select for this parameter is 4. A value of 0 signifies the use of basic rich equivalence classes. 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:index:star  genome, data:annotation  annotation, data:index:star  rrna_reference, data:index: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: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)

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:index: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:index:star description:Reference sequence index prepared by STAR aligner indexing tool.

globin_reference

label:Indexed Globin reference sequence type:data:index: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.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 in selective alignment mode and 0 otherwise. required:False

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]. 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 (4) corresponds to the default used in Zakeri et al. 2017 and larger values imply a more fine-grained factorization. If range factorization is enabled, a common value to select for this parameter is 4. A value of 0 signifies the use of basic rich equivalence classes. 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)

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

BEDPE file

data:bedpe:upload-bedpe (basic:file  src, basic:string  species, basic:string  build)

Upload BEDPE files.

Input arguments

src

label:Select BEDPE file to upload type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False 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 required:True hidden:False

Output results

bedpe

label:BEDPE file type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

BWA ALN

data:alignment:bam:bwaalnalignment-bwa-aln (data:index:bwa  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)

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:index:bwa

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:index:bwa  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)

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:index:bwa

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:index:bwa  genome, data:reads:fastq  reads, basic:integer  match, basic:integer  missmatch, basic:integer  gap_o, basic:integer  gap_e)

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:index:bwa

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

BWA genome index

data:index:bwa:bwa-index (data:seq:nucleotide  ref_seq)

Create BWA genome index.

Input arguments

ref_seq

label:Reference sequence (nucleotide FASTA) type:data:seq:nucleotide required:True hidden:False

Output results

index

label:BWA index type:basic:dir required:True hidden:False

fastagz

label:FASTA file (compressed) type:basic:file required:True hidden:False

fasta

label:FASTA file type:basic:file required:True hidden:False

fai

label:FASTA file index type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

Bam split

data:alignment:bam:primarybam-split (data:alignment:bam  bam, data:sam:header  header, data:sam:header  header2)

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

Bamclipper

data:alignment:bam:bamclipped:bamclipper (data:alignment:bam  alignment, data:bedpe  bedpe, basic:boolean  skip)

Remove primer sequence from BAM alignments by soft-clipping. This process is a wrapper for bamclipper which can be found at https://github.com/tommyau/bamclipper.

Input arguments

alignment

label:Alignment BAM file type:data:alignment:bam required:True hidden:False

bedpe

label:BEDPE file type:data:bedpe required:False hidden:False

skip

label:Skip Bamclipper step type:basic:boolean description:Use this option to skip Bamclipper step. required:True hidden:False default:False

Output results

bam

label:Clipped BAM file type:basic:file required:True hidden:False

bai

label:Index of clipped BAM file type:basic:file required:True hidden:False

stats

label:Alignment statistics type:basic:file required:True hidden:False

bigwig

label:BigWig file type:basic:file required:False hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

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)

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)

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

BaseQualityScoreRecalibrator

data:alignment:bam:bqsr:bqsr (data:alignment:bam  bam, data:seq:nucleotide  reference, list:data:variants:vcf  known_sites, data:bed  intervals, basic:string  read_group, basic:string  validation_stringency)

A two pass process of BaseRecalibrator and ApplyBQSR from GATK. See GATK website for more information on BaseRecalibrator. It is possible to modify read group using GATK’s AddOrReplaceGroups through Replace read groups in BAM (``read_group``) input field.

Input arguments

bam

label:BAM file containing reads type:data:alignment:bam required:True hidden:False

reference

label:Reference genome file type:data:seq:nucleotide required:True hidden:False

known_sites

label:List of known sites of variation type:list:data:variants:vcf required:True hidden:False

intervals

label:One or more genomic intervals over which to operate. type:data:bed description:This field is optional, but it can speed up the process by restricting calculations to specific genome regions. required:False hidden:False

read_group

label:Replace read groups in BAM type:basic:string description:Replace read groups in a BAM file.This argument enables the user to replace all read groups in the INPUT file with a single new read group and assign all reads to this read group in the OUTPUT BAM file. Addition or replacement is performed using Picard’s AddOrReplaceReadGroups tool. Input should take the form of -name=value delimited by a ”;”, e.g. “-ID=1;-LB=GENIALIS;-PL=ILLUMINA;-PU=BARCODE;-SM=SAMPLENAME1”. See tool’s documentation for more information on tag names. Note that PL, LB, PU and SM are require fields. See caveats of rewriting read groups in the documentation. required:True hidden:False default:

validation_stringency

label:Validation stringency type:basic:string description:Validation stringency for all SAM files read by this program. Setting stringency to SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded. Default is STRICT. This setting is used in BaseRecalibrator and ApplyBQSR processes. required:True hidden:False default:STRICT choices:

• STRICT: STRICT
• LENIENT: LENIENT
• SILENT: SILENT

Output results

bam

label:Base quality score recalibrated BAM file type:basic:file required:True hidden:False

bai

label:Index of base quality score recalibrated BAM file type:basic:file required:True hidden:False

stats

label:Alignment statistics type:basic:file required:True hidden:False

bigwig

label:BigWig file type:basic:file required:False hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

recal_table

label:Recalibration tabled type:basic:file required:True hidden:False

BaseSpace file

data:filebasespace-file-import (basic:string  file_id, basic:secret  access_token_secret)

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

Bedtools bamtobed

data:bedpe:bedtools-bamtobed (data:alignment:bam  alignment)

Takes in a BAM file and calculates a normalization factor in BEDPE format. Done by sorting with Samtools and transformed with Bedtools.

Input arguments

alignment

label:Alignment BAM file type:data:alignment:bam required:True hidden:False

Output results

bedpe

label:BEDPE file type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

Bisulfite conversion rate

data:wgbs:bsrate:bs-conversion-rate (data:alignment:bam:walt  mr, basic:boolean  skip, data:seq:nucleotide  sequence, basic:boolean  count_all, basic:integer  read_length, basic:decimal  max_mismatch, basic:boolean  a_rich)

Estimate bisulfite conversion rate in a control set. The program bsrate included in [Methpipe] (https://github.com/smithlabcode/methpipe) will estimate the bisulfite conversion rate.

Input arguments

mr

label:Aligned reads from bisulfite sequencing type:data:alignment:bam:walt description:Bisulfite specifc alignment such as WALT is required as .mr file type is used. Duplicatesshould be removed to reduce any bias introduced by incomplete conversion on PCR duplicatereads. required:True hidden:False

skip

label:Skip Bisulfite conversion rate step type:basic:boolean description:Bisulfite conversion rate step can be skipped. required:True hidden:False default:False

sequence

label:Unmethylated control sequence type:data:seq:nucleotide description:Separate unmethylated control sequence FASTA file is required to estimate bisulfiteconversion rate. required:False hidden:False

count_all

label:Count all cytosines including CpGs type:basic:boolean required:True hidden:False default:True

read_length

label:Average read length type:basic:integer required:True hidden:False default:150

max_mismatch

label:Maximum fraction of mismatches type:basic:decimal required:False hidden:False

a_rich

label:Reads are A-rich type:basic:boolean required:True hidden:False default:False

Output results

report

label:Bisulfite conversion rate report type:basic:file required:True hidden:False

Bowtie (Dicty)

data:alignment:bam:bowtie1alignment-bowtie (data:index:bowtie  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)

An ultrafast memory-efficient short read aligner.

Input arguments

genome

label:Reference genome type:data:index:bowtie

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

Bowtie genome index

data:index:bowtie:bowtie-index (data:seq:nucleotide  ref_seq)

Create Bowtie genome index.

Input arguments

ref_seq

label:Reference sequence (nucleotide FASTA) type:data:seq:nucleotide required:True hidden:False

Output results

index

label:Bowtie index type:basic:dir required:True hidden:False

fastagz

label:FASTA file (compressed) type:basic:file required:True hidden:False

fasta

label:FASTA file type:basic:file required:True hidden:False

fai

label:FASTA file index type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

Bowtie2

data:alignment:bam:bowtie2alignment-bowtie2 (data:index:bowtie2  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:boolean  no_overlap, basic:boolean  dovetail, 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, basic:boolean  no_unal, basic:integer  bw_binsize, basic:integer  bw_timeout)

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:index:bowtie2

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

PE_options.no_overlap

label:Not concordant when mates overlap type:basic:boolean description:When true, it is considered not concordant when mates overlap at all. Defaul is false. default:False

PE_options.dovetail

label:Dovetail type:basic:boolean description:If the mates “dovetail”, that is if one mate alignment extends past the beginning of the other such that the wrong mate begins upstream, consider that to be concordant. Default: mates cannot dovetail in a concordant alignment. default:False

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_opts.no_unal

label:Suppress SAM records for unaligned reads type:basic:boolean description:When true, suppress SAM records for unaligned reads. Default is false. default:False

misc_opts.bw_binsize

label:BigWig bin size type:basic:integer description:Size of the bins, in bases, for the output of the bigwig/bedgraph file. Default is 50. default:50

misc_opts.bw_timeout

label:BigWig timeout (s) type:basic:integer description:Time, in seconds, before creation of BigWig file is stopped. Default is 480 seconds. default:480

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 genome index

data:index:bowtie2:bowtie2-index (data:seq:nucleotide  ref_seq)

Create Bowtie2 genome index.

Input arguments

ref_seq

label:Reference sequence (nucleotide FASTA) type:data:seq:nucleotide required:True hidden:False

Output results

index

label:Bowtie2 index type:basic:dir required:True hidden:False

fastagz

label:FASTA file (compressed) type:basic:file required:True hidden:False

fasta

label:FASTA file type:basic:file required:True hidden:False

fai

label:FASTA file index type:basic:file required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

Cell Ranger Count

data:scexpression:10x:cellranger-count (data:screads:10x:  reads, data:genomeindex:10x:  genome_index, basic:string  chemistry, basic:integer  trim_r1, basic:integer  trim_r2, basic:integer  expected_cells, basic:integer  force_cells)

Perform gene expression analysis. Generate single cell feature counts for a single library. https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/count

Input arguments

reads

label:10x reads data object type:data:screads:10x: required:True hidden:False

genome_index

label:10x genome index data object type:data:genomeindex:10x: required:True hidden:False

chemistry

label:Chemistry type:basic:string description:Assay configuration. By default the assay configuration is detected automatically, which is the recommended mode. You should only specify chemistry if there is an error in automatic detection. required:False hidden:False default:auto choices:

• auto: auto
• threeprime: Single Cell 3'
• fiveprime: Single Cell 5'
• SC3Pv1: Single Cell 3' v1
• SC3Pv2: Single Cell 3' v2
• SC3Pv3: Single Cell 3' v3
• C5P-PE: Single Cell 5' paired-end
• SC5P-R2: Single Cell 5' R2-only

trim_r1

label:Trim R1 type:basic:integer description:Hard-trim the input R1 sequence to this length. Note that the length includes the Barcode and UMI sequences so do not set this below 26 for Single Cell 3’ v2 or Single Cell 5’. This and “Trim R2” are useful for determining the optimal read length for sequencing. required:False hidden:False

trim_r2

label:Trim R2 type:basic:integer description:Hard-trim the input R2 sequence to this length. required:False hidden:False

expected_cells

label:Expected number of recovered cells type:basic:integer required:True hidden:False default:3000

force_cells

label:Force cell number type:basic:integer description:Force pipeline to use this number of cells, bypassing the cell detection algorithm. Use this if the number of cells estimated by Cell Ranger is not consistent with the barcode rank plot. required:False hidden:False

Output results

matrix_filtered

label:Matrix (filtered) type:basic:file required:True hidden:False

genes_filtered

label:Genes (filtered) type:basic:file required:True hidden:False

barcodes_filtered

label:Barcodes (filtered) type:basic:file required:True hidden:False

matrix_raw

label:Matrix (raw) type:basic:file required:True hidden:False

genes_raw

label:Genes (raw) type:basic:file required:True hidden:False

barcodes_raw

label:Barcodes (raw) type:basic:file required:True hidden:False

report

label:Report type:basic:file:html required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

source

label:Gene ID source type:basic:string required:True hidden:False

Cell Ranger Mkref

data:genomeindex:10x:cellranger-mkref (data:seq:nucleotide:  genome, data:annotation:gtf:  annotation)

Reference preparation tool for 10x Genomics Cell Ranger. Build a Cell Ranger-compatible reference from genome FASTA and gene GTF files. https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references

Input arguments

genome

label:Reference genome type:data:seq:nucleotide: required:True hidden:False

annotation

label:Annotation type:data:annotation:gtf: required:True hidden:False

Output results

genome_index

label:Indexed genome type:basic:dir required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

source

label:Gene ID source type:basic:string required:True hidden:False

ChIP-Seq (Gene Score)

data:chipseq:genescorechipseq-genescore (data:chipseq:peakscore  peakscore, basic:decimal  fdr, basic:decimal  pval, basic:decimal  logratio)

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)

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, data:bed  promoter, basic:boolean  advanced, 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, data:bed  blacklist, basic:boolean  calculate_enrichment, basic:integer  profile_window, basic:string  shift_size)

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.

promoter

label:Promoter regions BED file type:data:bed description:BED file containing promoter regions (TSS+-1000 bp for example). Needed to get the number of peaks and reads mapped to promoter regions. required:False

advanced

label:Show advanced options type:basic:boolean description:Inspect and modify parameters. default:False

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:True

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 4 bp and reads on “-” strand by 5 bp. 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 1000 bp for small local region (–slocal), and 10000 bp 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 1000 bp for small local region (–slocal), and 10000 bp 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: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 tag. If set, MACS will be terminated if paired-peak model has 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 to true, 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. 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

chipqc_settings.blacklist

label:Blacklist regions type:data:bed description:BED file containing genomic regions that should be excluded from the analysis. required:False

chipqc_settings.calculate_enrichment

label:Calculate enrichment type:basic:boolean description:Calculate enrichment of signal in known genomic annotation. By default annotation is provided from the TranscriptDB package specified by genome bulid which should match one of the supported annotations (hg19, hg38, hg18, mm10, mm9, rn4, ce6, dm3). If annotation is not supported the analysis is skipped. default:False

chipqc_settings.profile_window

label:Window size type:basic:integer description:An integer indicating the width of the window used for peak profiles. Peaks will be centered on their summits and include half of the window size upstream and half downstream of this point. default:400

chipqc_settings.shift_size

label:Shift size type:basic:string description:Vector of values to try when computing optimal shift sizes. It should be specifeird as consecutive numbers vector with start:end default:1:300

Output results

ChIP-seq (MACS2-ROSE2)

data:chipseq:batch:macs2macs2-rose2-batch (list:data:alignment:bam  alignments, data:bed  promoter, basic:boolean  advanced, 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:boolean  use_filtered_bam, basic:integer  tss, basic:integer  stitch, data:bed  mask, data:bed  blacklist, basic:boolean  calculate_enrichment, basic:integer  profile_window, basic:string  shift_size)

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.

promoter

label:Promoter regions BED file type:data:bed description:BED file containing promoter regions (TSS+-1000 bp for example). Needed to get the number of peaks and reads mapped to promoter regions. required:False

advanced

label:Show advanced options type:basic:boolean description:Inspect and modify parameters. default:False

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:True

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 4 bp and reads on “-” strand by 5 bp. 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 1000 bp for small local region (–slocal), and 10000 bp 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 1000 bp for small local region (–slocal), and 10000 bp 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: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 tag. If set, MACS will be terminated if paired-peak model has 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 to true, 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. 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.use_filtered_bam

label:Use Filtered BAM File type:basic:boolean description:Use filtered BAM file from a MACS2 object to rank enhancers by. default: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

chipqc_settings.blacklist

label:Blacklist regions type:data:bed description:BED file containing genomic regions that should be excluded from the analysis. required:False

chipqc_settings.calculate_enrichment

label:Calculate enrichment type:basic:boolean description:Calculate enrichment of signal in known genomic annotation. By default annotation is provided from the TranscriptDB package specified by genome bulid which should match one of the supported annotations (hg19, hg38, hg18, mm10, mm9, rn4, ce6, dm3). If annotation is not supported the analysis is skipped. default:False

chipqc_settings.profile_window

label:Window size type:basic:integer description:An integer indicating the width of the window used for peak profiles. Peaks will be centered on their summits and include half of the window size upstream and half downstream of this point. default:400

chipqc_settings.shift_size

label:Shift size type:basic:string description:Vector of values to try when computing optimal shift sizes. It should be specifeird as consecutive numbers vector with start:end default:1:300

Output results

Chemical Mutagenesis

data:workflow:chemutworkflow-chemut (basic:string  analysis_type, data:seq:nucleotide  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)

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:seq:nucleotide

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

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

ChipQC

data:chipqc:chipqc (data:alignment:bam  alignment, data:chipseq:callpeak  peaks, data:bed  blacklist, basic:boolean  calculate_enrichment, basic:integer  quality_threshold, basic:integer  profile_window, basic:string  shift_size)

Calculate quality control metrics for ChIP-seq samples. The analysis is based on ChIPQC package which computs a variety of quality control metrics and statistics, and provides plots and a report for assessment of experimental data for further analysis.

Input arguments

alignment

label:Aligned reads type:data:alignment:bam required:True hidden:False

peaks

label:Called peaks type:data:chipseq:callpeak required:True hidden:False

blacklist

label:Blacklist regions type:data:bed description:BED file containing genomic regions that should be excluded from the analysis. required:False hidden:False

calculate_enrichment

label:Calculate enrichment type:basic:boolean description:Calculate enrichment of signal in known genomic annotation. By default annotation is provided from the TranscriptDB package specified by genome bulid which should match one of the supported annotations (hg19, hg38, hg18, mm10, mm9, rn4, ce6, dm3). If annotation is not supported the analysis is skipped. required:True hidden:False default:False

advanced.quality_threshold

label:Mapping quality threshold type:basic:integer description:Only reads with mapping quality scores above this threshold will be used for some statistics. required:True hidden:False default:15

advanced.profile_window

label:Window size type:basic:integer description:An integer indicating the width of the window used for peak profiles. Peaks will be centered on their summits and include half of the window size upstream and half downstream of this point. required:True hidden:False default:400

advanced.shift_size

label:Shift size type:basic:string description:Vector of values to try when computing optimal shift sizes. It should be specifeird as consecutive numbers vector with start:end required:True hidden:False default:1:300

Output results

report_folder

label:ChipQC report folder type:basic:dir required:True hidden:False

ccplot

label:Cross coverage score plot type:basic:file required:True hidden:False

coverage_histogram

label:SSD metric plot type:basic:file required:True hidden:False

peak_profile

label:Peak profile plot type:basic:file required:True hidden:False

peaks_barplot

label:Barplot of reads in peaks type:basic:file required:True hidden:False

peaks_density_plot

label:Density plot of reads in peaks type:basic:file required:True hidden:False

enrichment_heatmap

label:Heatmap of reads in genomic features type:basic:file required:False hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

Convert GFF3 to GTF

data:annotation:gtfgff-to-gtf (data:annotation:gff3  annotation)

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)

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)

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:cuffdiff:cuffdiff (list:data:cufflinks:cuffquant  case, list:data:cufflinks:cuffquant  control, list:basic:string  labels, data:annotation  annotation, data:seq:nucleotide  genome, basic:boolean  multi_read_correct, basic:boolean  create_sets, basic:decimal  gene_logfc, basic:decimal  gene_fdr, basic:decimal  fdr, basic:string  library_type, basic:string  library_normalization, basic:string  dispersion_method)

Run Cuffdiff 2.2 analysis. 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 required:True hidden:False

control

label:Control samples type:list:data:cufflinks:cuffquant required:True hidden:False

labels

label:Group labels type:list:basic:string description:Define labels for each sample group. required:True hidden:False default:['control', 'case']

annotation

label:Annotation (GTF/GFF3) type:data:annotation description:A transcript annotation file produced by cufflinks, cuffcompare, or other tool. required:True hidden:False

genome

label:Run bias detection and correction algorithm type:data:seq:nucleotide 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 hidden:False

multi_read_correct

label:Do initial estimation procedure to more accurately weight reads with multiple genome mappings type:basic:boolean required:True hidden:False default:False

create_sets

label:Create gene sets type:basic:boolean description:After calculating differential gene expressions create gene sets for up-regulated genes, down-regulated genes and all genes. required:True hidden:False default:False

gene_logfc

label:Log2 fold change threshold for gene sets type:basic:decimal description:Genes above Log2FC are considered as up-regulated and genes below -Log2FC as down-regulated. required:True hidden:!create_sets default:1.0

gene_fdr

label:FDR threshold for gene sets type:basic:decimal required:True hidden:!create_sets default:0.05

fdr

label:Allowed FDR type:basic:decimal description:The allowed false discovery rate. The default is 0.05. required:True hidden:False 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 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. required:True hidden:False 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. required:True hidden:False 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). required:True hidden:False default:pooled choices:

• pooled: pooled
• per-condition: per-condition
• blind: blind
• poisson: poisson

Output results

raw

label:Differential expression type:basic:file required:True hidden:False

de_json

label:Results table (JSON) type:basic:json required:True hidden:False

de_file

label:Results table (file) type:basic:file required:True hidden:False

transcript_diff_exp

label:Differential expression (transcript level) type:basic:file required:True hidden:False

tss_group_diff_exp

label:Differential expression (primary transcript) type:basic:file required:True hidden:False

cds_diff_exp

label:Differential expression (coding sequence) type:basic:file required:True hidden:False

cuffdiff_output

label:Cuffdiff output type:basic:file required:True hidden:False

source

label:Gene ID database type:basic:string required:True hidden:False

species

label:Species type:basic:string required:True hidden:False

build

label:Build type:basic:string required:True hidden:False

feature_type

label:Feature type type:basic:string required:True hidden:False

Cufflinks 2.2

data:cufflinks:cufflinkscufflinks (data:alignment:bam  alignment, data:annotation  annotation, data:seq:nucleotide  genome, data:annotation:gtf  mask_file, basic:string  library_type, basic:string  annotation_usage, basic:boolean  multi_read_correct)

Cufflinks assembles transcripts, estimates their abundances, and tests for differential expression and regulation in RNA-Seq samples. It accepts aligned RNA-Seq reads and assembles the alignments into a parsimonious set of transcripts. Cufflinks then estimates the relative abundances of these transcripts based on how many reads support each one, taking into account biases in library preparation protocols. See [here](http://cole-trapnell-lab.github.io/cufflinks/) for more information.

Input arguments

alignment

label:Aligned reads type:data:alignment:bam

annotation

label:Annotation (GTF/GFF3) type:data:annotation required:False

genome

label:Run bias detection and correction algorithm type:data:seq:nucleotide 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

annotation_usage

label:Instruct Cufflinks how to use the provided annotation (GFF/GTF) file type:basic:string description:GTF-guide - tells Cufflinks to use the supplied reference annotation (GFF) to guide RABT assembly. Reference transcripts will be tiled with faux-reads to provide additional information in assembly. Output will include all reference transcripts as well as any novel genes and isoforms that are assembled. –GTF - tells Cufflinks to use the supplied reference annotation (a GFF file) to estimate isoform expression. It will not assemble novel transcripts, and the program will ignore alignments not structurally compatible with any reference transcript. default:--GTF-guide choices:

• Use supplied reference annotation to guide RABT assembly (–GTF-guide): --GTF-guide
• Use supplied reference annotation to estimate isoform expression (–GTF): --GTF

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

transcripts

label:Assembled transcript isoforms type:basic:file

isoforms_fpkm_tracking

label:Isoforms FPKM tracking type:basic:file

genes_fpkm_tracking

label:Genes FPKM tracking type:basic:file

skipped_loci

label:Skipped loci type:basic:file

source

label:Gene ID database type:basic:string

species

label:Species type:basic:string

build

label:Build type:basic:string

Cuffmerge

data:annotation:cuffmergecuffmerge (list:data:cufflinks:cufflinks  expressions, list:data:annotation:gtf  gtf, data:annotation  gff, data:seq:nucleotide  genome, basic:integer  threads)

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:seq:nucleotide 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)

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:seq:nucleotide  genome, data:annotation:gtf  mask_file, basic:string  library_type, basic:boolean  multi_read_correct)

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:seq:nucleotide 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)

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)

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

Cut & Run

data:workflow:cutnrunworkflow-cutnrun (data:reads:fastq:paired  reads, basic:integer  quality, basic:integer  nextseq, basic:string  phred, basic:integer  min_length, basic:integer  max_n, basic:boolean  retain_unpaired, basic:integer  unpaired_len_1, basic:integer  unpaired_len_2, basic:integer  clip_r1, basic:integer  clip_r2, basic:integer  three_prime_r1, basic:integer  three_prime_r2, list:basic:string  adapter, list:basic:string  adapter_2, data:seq:nucleotide  adapter_file_1, data:seq:nucleotide  adapter_file_2, basic:string  universal_adapter, basic:integer  stringency, basic:decimal  error_rate, basic:integer  trim_5, basic:integer  trim_3, data:index:bowtie2  genome, basic:string  mode, basic:string  speed, basic:boolean  discordantly, basic:boolean  rep_se, basic:integer  minins, basic:integer  maxins, basic:boolean  no_overlap, basic:boolean  dovetail, basic:boolean  no_unal, data:index:bowtie2  genome, basic:string  mode, basic:string  speed, basic:boolean  discordantly, basic:boolean  rep_se, basic:integer  minins, basic:integer  maxins, basic:boolean  no_overlap, basic:boolean  dovetail, basic:boolean  no_unal, basic:string  format, basic:decimal  pvalue, basic:string  duplicates, basic:boolean  bedgraph, basic:integer  min_frag_length, basic:integer  max_frag_length, basic:decimal  scale, basic:integer  bw_binsize, basic:integer  bw_timeout)

Analysis of samples processed for high resolution mapping of DNA binding sites using targeted nuclease strategy. The process is named CUT&RUN which stands for Cleavage Under Target and Release Using Nuclease. Workflow includes steps of trimming the reads with trimgalore, aligning them using bowtie2 to target species genome as well as a spike-in genome. Aligned reads are processed to produce bigwig files to be viewed in a genome browser. Peaks are called using MACS2. Fragmenting of reads is performed using alignmentSieve from deeptools package.

Input arguments

reads

label:Input reads type:data:reads:fastq:paired

options_trimming.quality_trim.quality

label:Quality cutoff type:basic:integer description:Trim low-quality ends from reads based on Phred score. required:False

options_trimming.quality_trim.nextseq

label:NextSeq/NovaSeq trim cutoff type:basic:integer description:NextSeq/NovaSeq-specific quality trimming. Trims also dark cycles appearing as high-quality G bases. This will set a specific quality cutoff, but qualities of G bases are ignored. This can not be used with Quality cutoff and will override it. required:False

options_trimming.quality_trim.phred

label:Phred score encoding type:basic:string description:Use either ASCII+33 quality scores as Phred scores (Sanger/Illumina 1 .9+ encoding) or ASCII+64 quality scores (Illumina 1.5 encoding) for quality trimming. default:--phred33 choices:

• ASCII+33: --phred33
• ASCII+64: --phred64

options_trimming.quality_trim.min_length

label:Minimum length after trimming type:basic:integer description:Discard reads that became shorter than selected length because of either quality or adapter trimming. Both reads of a read-pair need to be longer than specified length to be printed out to validated paired-end files. If only one read became too short there is the possibility of keeping such unpaired single-end reads with Retain unpaired. A value of 0 disables filtering based on length. default:20

options_trimming.quality_trim.max_n

label:Maximum number of Ns type:basic:integer description:Read exceeding this limit will result in the entire pair being removed from the trimmed output files. required:False

options_trimming.quality_trim.retain_unpaired

label:Retain unpaired reads after trimming type:basic:boolean description:If only one of the two paired-end reads “became too short, the longer read will be written. default:False

options_trimming.quality_trim.unpaired_len_1

label:Unpaired read length cutoff of mate 1 type:basic:integer hidden:!quality_trim.retain_unpaired default:35

options_trimming.quality_trim.unpaired_len_2

label:Unpaired read length cutoff for mate 2 type:basic:integer hidden:!quality_trim.retain_unpaired default:35

options_trimming.quality_trim.clip_r1

label:Trim bases from 5’ end of read 1 type:basic:integer description:This may be useful if the qualities were very poor, or if there is some sort of unwanted bias at the 5’ end. required:False

options_trimming.quality_trim.clip_r2

label:Trim bases from 5’ end of read 2 type:basic:integer description:This may be useful if the qualities were very poor, or if there is some sort of unwanted bias at the 5’ end. For paired-end bisulfite sequencing, it is recommended to remove the first few bp because the end-repair reaction may introduce a bias towards low methylation. required:False

options_trimming.quality_trim.three_prime_r1

label:Trim bases from 3’ end of read 1 type:basic:integer description:Remove bases from the 3’ end of read 1 after adapter/quality trimming has been performed. This may remove some unwanted bias from the 3’ end that is not directly related to adapter sequence or basecall quality. required:False

options_trimming.quality_trim.three_prime_r2

label:Trim bases from 3’ end of read 2 type:basic:integer description:Remove bases from the 3’ end of read 2 after adapter/quality trimming has been performed. This may remove some unwanted bias from the 3’ end that is not directly related to adapter sequence or basecall quality. required:False

options_trimming.adapter_trim.adapter

label:Read 1 adapter sequence type:list:basic:string description:Adapter sequences to be trimmed. Also see universal adapters field for predefined adapters. This is mutually exclusive with read 1 adapters file and universal adapters. required:False

options_trimming.adapter_trim.adapter_2

label:Read 2 adapter sequence type:list:basic:string description:Optional adapter sequence to be trimmed off read 2 of paired-end files. This is mutually exclusive with read 2 adapters file and universal adapters. required:False

options_trimming.adapter_trim.adapter_file_1

label:Read 1 adapters file type:data:seq:nucleotide description:This is mutually exclusive with read 1 adapters and universal adapters. required:False

options_trimming.adapter_trim.adapter_file_2

label:Read 2 adapters file type:data:seq:nucleotide description:This is mutually exclusive with read 2 adapters and universal adapters. required:False

options_trimming.adapter_trim.universal_adapter

label:Universal adapters type:basic:string description:Instead of default detection use specific adapters. Use 13bp of the Illumina universal adapter, 12bp of the Nextera adapter or 12bp of the Illumina Small RNA 3’ Adapter. Selecting to trim smallRNA adapters will also lower the length value to 18bp. If the smallRNA libraries are paired-end then read 2 adapter will be set to the Illumina small RNA 5’ adapter automatically (GATCGTCGGACT) unless defined explicitly. This is mutually exclusive with manually defined adapters and adapter files. required:False choices:

• Illumina: --illumina
• Nextera: --nextera
• Illumina small RNA: --small_rna

options_trimming.adapter_trim.stringency

label:Overlap with adapter sequence required to trim type:basic:integer description:Defaults to a very stringent setting of 1, i.e. even a single base pair of overlapping sequence will be trimmed of the 3’ end of any read. default:1

options_trimming.adapter_trim.error_rate

label:Maximum allowed error rate type:basic:decimal description:Number of errors divided by the length of the matching region. Default value of 0.1. default:0.1

options_trimming.hard_trim.trim_5

label:Hard trim sequence from 3’ end type:basic:integer description:Instead of performing adapter-/quality trimming, this option will simply hard-trim sequences to bp from the 3’ end. This is incompatible with other hard trimming options. required:False

options_trimming.hard_trim.trim_3

label:Hard trim sequences from 5’ end type:basic:integer description:Instead of performing adapter-/quality trimming, this option will simply hard-trim sequences to bp from the 5’ end. This is incompatible with other hard trimming options. required:False

options_aln_species.genome

label:Species genome type:data:index:bowtie2

options_aln_species.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

options_aln_species.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 default:--very-sensitive choices:

• Very fast: --very-fast
• Fast: --fast
• Sensitive: --sensitive
• Very sensitive: --very-sensitive

options_aln_species.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

options_aln_species.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 is true (–no-mixed). default:True

options_aln_species.minins

label:Minimal distance type:basic:integer description:The minimum fragment length (–minins) for valid paired-end alignments. Value 0 imposes no minimum. default:10

options_aln_species.maxins

label:Maximal distance type:basic:integer description:The maximum fragment length (–maxins) for valid paired-end alignments. default:700

options_aln_species.no_overlap

label:Not concordant when mates overlap type:basic:boolean description:When true, it is considered not concordant when mates overlap at all. Defaul is true (–no-overlap). default:False

options_aln_species.dovetail

label:Dovetail type:basic:boolean description:If the mates “dovetail”, that is if one mate alignment extends past the beginning of the other such that the wrong mate begins upstream, consider that to be concordant. Default: mates cannot dovetail in a concordant alignment. If true, parameter –dovetail is turned on. default:False

options_aln_species.no_unal

label:Suppress SAM records for unaligned reads type:basic:boolean description:When true, suppress SAM records for unaligned reads. Default is true (–no-unal). default:True

options_aln_spikein.genome

label:Spike-in genome type:data:index:bowtie2

options_aln_spikein.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

options_aln_spikein.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 default:--very-sensitive choices:

• Very fast: --very-fast
• Fast: --fast
• Sensitive: --sensitive
• Very sensitive: --very-sensitive

options_aln_spikein.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

options_aln_spikein.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 is true (–no-mixed). default:True

options_aln_spikein.minins

label:Minimal distance type:basic:integer description:The minimum fragment length (–minins) for valid paired-end alignments. Value 0 imposes no minimum. default:10

options_aln_spikein.maxins

label:Maximal distance type:basic:integer description:The maximum fragment length (–maxins) for valid paired-end alignments. default:700

options_aln_spikein.no_overlap

label:Not concordant when mates overlap type:basic:boolean description:When true, it is considered not concordant when mates overlap at all. Defaul is true (–no-overlap). default:True

options_aln_spikein.dovetail

label:Dovetail type:basic:boolean description:If the mates “dovetail”, that is if one mate alignment extends past the beginning of the other such that the wrong mate begins upstream, consider that to be concordant. Default: mates cannot dovetail in a concordant alignment. If true, parameter –dovetail is turned on. default:False

options_aln_spikein.no_unal

label:Suppress SAM records for unaligned reads type:basic:boolean description:When true, suppress SAM records for unaligned reads. Default is true (–no-unal). default:True

options_pc.format

label:Format of tag file type:basic:string description:This specifies the format of input files. For paired-end data the format dicates how MACS2 will treat mates. If the selected format is BAM, MACS2 will only keep the left mate (5’ end) tag. However, when format BAMPE is selected, MACS2 will use actual insert sizes of pairs of reads to build fragment pileup, instead of building bimodal distribution plus and minus strand reads to predict fragment size. required:False default:BAMPE choices:

• BAM: BAM
• BAMPE: BAMPE

options_pc.pvalue

label:P-value cutoff type:basic:decimal description:The p-value cutoff. required:False default:0.001

options_pc.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. default:all choices:

• 1: 1
• auto: auto
• all: all

options_pc.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, -log10(pvalue) and -log10(qvalue) 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

options_sieve.min_frag_length

label:Minimum fragment length type:basic:integer description:The minimum fragment length needed for read/pair inclusion. This option is primarily useful in ATACseq experiments, for filtering mono- or di-nucleosome fragments. Default is 0. default:0

options_sieve.max_frag_length

label:Maximum fragment length type:basic:integer description:The maximum fragment length needed for read/pair inclusion. A value of 0 indicates no limit. Default is 0. default:0

options_scale.scale

label:Scale factor type:basic:decimal description:Magnitude of the scale factor. The scaling factor is calculated by dividing the scale with the number of features in BEDPE (scale/(number of features)). default:10000

options_misc.bw_binsize

label:BigWig bin size type:basic:integer description:Size of the bins, in bases, for the output of the bigwig/bedgraph file. Default is 50. default:50

options_misc.bw_timeout

label:BigWig timeout type:basic:integer description:Number of seconds before calculation of BigWig file is aborted. Default is 3600 seconds (1 hour). default:3600

Output results

Cutadapt (3’ mRNA-seq, single-end)

data:reads:fastq:single:cutadapt:cutadapt-3prime-single (data:reads:fastq:single  reads, basic:integer  nextseq_trim, basic:integer  quality_cutoff, basic:integer  min_len, basic:integer  min_overlap, basic:integer  times)

Process 3’ mRNA-seq datasets using Cutadapt tool.

Input arguments

reads

label:Select sample(s) type:data:reads:fastq:single required:True hidden:False

options.nextseq_trim

label:NextSeq/NovaSeq trim type:basic:integer description:NextSeq/NovaSeq-specific quality trimming. Trims also dark cycles appearing as high-quality G bases. This option is mutually exclusive with the use of standard quality-cutoff trimming and is suitable for the use with data generated by the recent Illumina machines that utilize two-color chemistry to encode the four bases. required:True hidden:False default:10

options.quality_cutoff

label: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 trim option. required:False hidden:False

options.min_len

label:Discard reads shorter than specified minimum length. type:basic:integer required:True hidden:False default:20

options.min_overlap

label:Mimimum overlap type:basic:integer description:Minimum overlap between adapter and read for an adapter to be found. required:True hidden:False default:20

options.times

label:Remove up to a specified number of adapters from each read. type:basic:integer required:True hidden:False default:2

Output results

fastq

label:Reads file. type:list:basic:file required:True hidden:False

report

label:Cutadapt report type:basic:file required:True hidden:False

fastqc_url

label:Quality control with FastQC. type:list:basic:file:html required:True hidden:False

fastqc_archive

label:Download FastQC archive. type:list:basic:file required:True hidden:False

Cutadapt (Corall RNA-Seq, paired-end)

data:reads:fastq:paired:cutadapt:cutadapt-corall-paired (data:reads:fastq:paired  reads, basic:integer  nextseq_trim, basic:integer  quality_cutoff, basic:integer  min_len, basic:integer  min_overlap)

Pre-process reads obtained using CORALL Total RNA-Seq Library Prep Kit. Trim UMI-tags from input reads and use Cutadapt to remove adapters and run QC filtering steps.

Input arguments

reads

label:Select sample(s) type:data:reads:fastq:paired required:True hidden:False

options.nextseq_trim

label:NextSeq/NovaSeq trim type:basic:integer description:NextSeq/NovaSeq-specific quality trimming. Trims also dark cycles appearing as high-quality G bases. This option is mutually exclusive with the use of standard quality-cutoff trimming and is suitable for the use with data generated by the recent Illumina machines that utilize two-color chemistry to encode the four bases. required:True hidden:False default:10

options.quality_cutoff

label: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 trim option. required:False hidden:False

options.min_len

label:Minimum read length type:basic:integer required:True hidden:False default:20

options.min_overlap

label:Mimimum overlap type:basic:integer description:Minimum overlap between adapter and read for an adapter to be found. required:True hidden:False default:20

Output results

fastq

label:Remaining mate1 reads type:list:basic:file required:True hidden:False

fastq2

label:Remaining mate2 reads type:list:basic:file required:True hidden:False

report

label:Cutadapt report type:basic:file required:True hidden:False

fastqc_url

label:Mate1 quality control with FastQC type:list:basic:file:html required:True hidden:False

fastqc_url2

label:Mate2 quality control with FastQC type:list:basic:file:html required:True hidden:False

fastqc_archive

label:Download mate1 FastQC archive type:list:basic:file required:True hidden:False

fastqc_archive2

label:Download mate2 FastQC archive type:list:basic:file required:True hidden:False

Cutadapt (Corall RNA-Seq, single-end)

data:reads:fastq:single:cutadapt:cutadapt-corall-single (data:reads:fastq:single  reads, basic:integer  nextseq_trim, basic:integer  quality_cutoff, basic:integer  min_len, basic:integer  min_overlap)

Pre-process reads obtained using CORALL Total RNA-Seq Library Prep Kit. Trim UMI-tags from input reads and use Cutadapt to remove adapters and run QC filtering steps.

Input arguments

reads

label:Select sample(s) type:data:reads:fastq:single required:True hidden:False

options.nextseq_trim

label:NextSeq/NovaSeq trim type:basic:integer description:NextSeq/NovaSeq-specific quality trimming. Trims also dark cycles appearing as high-quality G bases. This option is mutually exclusive with the use of standard quality-cutoff trimming and is suitable for the use with data generated by the recent Illumina machines that utilize two-color chemistry to encode the four bases. required:True hidden:False default:10

options.quality_cutoff

label: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 trim option. required:False hidden:False

options.min_len

label:Minimum read length type:basic:integer required:True hidden:False default:20

options.min_overlap

label:Mimimum overlap type:basic:integer description:Minimum overlap between adapter and read for an adapter to be found. required:True hidden:False default:20

Output results

fastq

label:Reads file type:list:basic:file required:True hidden:False

report

label:Cutadapt report type:basic:file required:True hidden:False

fastqc_url

label:Quality control with FastQC type:list:basic:file:html required:True hidden:False

fastqc_archive

label:Download FastQC archive type:list:basic:file required:True hidden:False

Cutadapt (Diagenode CATS, paired-end)

data:reads:fastq:paired:cutadaptcutadapt-custom-paired (data:reads:fastq:paired  reads)

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)

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)

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)

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)