Workflow: Trim Galore RNA-Seq pipeline paired-end

Fetched 2023-01-04 15:15:52 GMT

The original [BioWardrobe's](https://biowardrobe.com) [PubMed ID:26248465](https://www.ncbi.nlm.nih.gov/pubmed/26248465) **RNA-Seq** basic analysis for a **pair-end** experiment. A corresponded input [FASTQ](http://maq.sourceforge.net/fastq.shtml) file has to be provided. Current workflow should be used only with the single-end RNA-Seq data. It performs the following steps: 1. Trim adapters from input FASTQ files 2. Use STAR to align reads from input FASTQ files according to the predefined reference indices; generate unsorted BAM file and alignment statistics file 3. Use fastx_quality_stats to analyze input FASTQ files and generate quality statistics files 4. Use samtools sort to generate coordinate sorted BAM(+BAI) file pair from the unsorted BAM file obtained on the step 1 (after running STAR) 5. Generate BigWig file on the base of sorted BAM file 6. Map input FASTQ files to predefined rRNA reference indices using Bowtie to define the level of rRNA contamination; export resulted statistics to file 7. Calculate isoform expression level for the sorted BAM file and GTF/TAB annotation file using GEEP reads-counting utility; export results to file

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Inputs

ID Type Title Doc
threads Integer (Optional) Number of threads

Number of threads for those steps that support multithreading

clip_3p_end Integer (Optional) Clip from 3p end

Number of bases to clip from the 3p end

clip_5p_end Integer (Optional) Clip from 5p end

Number of bases to clip from the 5p end

exclude_chr String (Optional) Chromosome to be excluded in rpkm calculation

Chromosome to be excluded in rpkm calculation

minimum_rpkm Float (Optional) Minimum RPKM for Gene Body Average Tag Density Plot

Minimum RPKM for Gene Body Average Tag Density Plot

annotation_file File [GTF] Annotation file

GTF or TAB-separated annotation file

chrom_length_file File [Textual format] Chromosome length file

Chromosome length file

fastq_file_upstream File [FASTQ] FASTQ read 1 input file

Reads data in a FASTQ format, received after paired end sequencing

star_indices_folder Directory STAR indices folder

Path to STAR generated indices

bowtie_indices_folder Directory BowTie Ribosomal Indices

Path to Bowtie generated indices

fastq_file_downstream File [FASTQ] FASTQ read 2 input file

Reads data in a FASTQ format, received after paired end sequencing

Steps

ID Runs Label Doc
get_stat
../tools/collect-statistics-rna-seq.cwl (CommandLineTool)

Tool processes and combines log files generated by Trimgalore, Bowtie, Samtools and MACS2.

`get_output_prefix` function returns output file prefix equal to `output_prefix`+`_collected_statistics_report` (if this input is provided) or generated on the base of bowtie log basename with `_collected_statistics_report` extension.

trim_fastq
../tools/trimgalore.cwl (CommandLineTool)

Tool runs Trimgalore - the wrapper around Cutadapt and FastQC to consistently apply adapter and quality trimming to FastQ files.

`default_log_name` function returns names for generated log files (for both paired-end and single-end cases). `trim_galore` itself doesn't support setting custom names for output files.

For paired-end data processing both `input_file_pair` and `paired` should be set. If either of them is not set, the other one becomes unset automatically.

If input trigger was set to false, skip running trimaglore and return unchanged input files

bypass_trim
../tools/bypass-trimgalore-pe.cwl (CommandLineTool)

If the number of reads in the trimmed_fastq_file_1 is less then min_reads_count, tool will return original_fastq_file_1/2 and nulls as selected_report_file_1/2. Otherwise, the trimmed_fastq_file_1/2 and trimming_report_file_1/2 will be returned. Might be usefull in case of trimgalore removed all reads from the original_fastq_file_1/2.

star_aligner
../tools/star-alignreads.cwl (CommandLineTool)

Tool runs STAR alignReads.

`default_output_name_prefix` function returns output files prefix if `outFileNamePrefix` is not set. By default prefix is equal to basename of `readFilesIn`.

bam_to_bigwig

Workflow converts input BAM file into bigWig and bedGraph files.

Input BAM file should be sorted by coordinates (required by `bam_to_bedgraph` step).

If `split` input is not provided use true by default. Default logic is implemented in `valueFrom` field of `split` input inside `bam_to_bedgraph` step to avoid possible bug in cwltool with setting default values for workflow inputs.

`scale` has higher priority over the `mapped_reads_number`. The last one is used to calculate `-scale` parameter for `bedtools genomecov` (step `bam_to_bedgraph`) only in a case when input `scale` is not provided. All logic is implemented inside `bedtools-genomecov.cwl`.

`bigwig_filename` defines the output name only for generated bigWig file. `bedgraph_filename` defines the output name for generated bedGraph file and can influence on generated bigWig filename in case when `bigwig_filename` is not provided.

All workflow inputs and outputs don't have `format` field to avoid format incompatibility errors when workflow is used as subworkflow.

get_gene_body
../tools/plugin-plot-rna.cwl (CommandLineTool)
Gene body average tag density plot and RPKM distribution histogram

Runs R script to produce gene body average tag density plot and RPKM distribution histogram Doesn't fail even when we couldn't produce any plots

bowtie_aligner
../tools/bowtie-alignreads.cwl (CommandLineTool)

Tool maps input raw reads files to reference genome using Bowtie.

`default_output_filename` function returns default name for SAM output and log files. In case when `sam` and `output_filename` inputs are not set, default filename will have `.sam` extension but format may not correspond SAM specification. To set output filename manually use `output_filename` input. Default output filename is based on `output_filename` or basename of `upstream_filelist`, `downstream_filelist` or `crossbow_filelist` file (if array, the first file in array is taken). If function is called without argenments and `output_filename` input is set, it will be returned from the function.

For single-end input data any of the `upstream_filelist` or `downstream_filelist` inputs can be used.

Log filename (`log_file` output) is generated by `default_output_filename` function with ex='.bw'

`indices_folder` defines folder to contain Bowtie indices. Based on the first found file with `rev.1.ebwt` or `rev.1.ebwtl` extension, bowtie index prefix is returned from input's `valueFrom` field.

group_isoforms
../tools/group-isoforms.cwl (CommandLineTool)

Tool runs get_gene_n_tss.R script to group isoforms by gene and common TSS

rename_upstream
../tools/rename.cwl (CommandLineTool)

Tool renames `source_file` to `target_filename`. Input `target_filename` should be set as string. If it's a full path, only basename will be used. If BAI file is present, it will be renamed too

rpkm_calculation
../tools/geep.cwl (CommandLineTool)
geep

Tool calculates RPKM values grouped by isoforms or genes.

`default_output_prefix` function returns default prefix based on `bam_file` basename, if `output_prefix` is not provided.

rename_downstream
../tools/rename.cwl (CommandLineTool)

Tool renames `source_file` to `target_filename`. Input `target_filename` should be set as string. If it's a full path, only basename will be used. If BAI file is present, it will be renamed too

get_annotation_gtf
../tools/ucsc-genepredtogtf.cwl (CommandLineTool)

genePredToGtf - Convert genePred table or file to gtf

get_bam_statistics
../tools/samtools-stats.cwl (CommandLineTool)

Generates statistics for the input BAM file.

samtools_sort_index
../tools/samtools-sort-index.cwl (CommandLineTool)

Tool to sort and index input BAM/SAM/CRAM. If input `trigger` is set to `true` or isn't set at all (`true` is used by default), run `samtools sort` and `samtools index`, return sorted BAM and BAI/CSI index file. If input `trigger` is set to `false`, return unchanged `sort_input` (BAM/SAM/CRAM) and index (BAI/CSI, if provided in `secondaryFiles`) files.

Trigger logic is implemented in two bash scripts set by default as `bash_script_sort` and `bash_script_index` inputs. For both of then, if the first argument $0 (which is `trigger` input) is true, run `samtools sort/index` with the rest of the arguments. If $0 is not true, skip `samtools sort/index` and return `sort_input` and `secondaryFiles` (if provided).

Input `trigger` is Boolean, but returns String, because of `valueFrom` field. The `valueFrom` is used, because if `trigger` is false, cwl-runner doesn't append this argument at all to the the `baseCommand` - new feature of CWL v1.0.2. Alternatively, `prefix` field could be used, but it causes changing in script logic.

If using `sort_output_filename`, the output file extension should be `*.bam`, because `samtools sort` defines the output file format on the base of the file extension. If `*.sam` is sed as output filename, it cannot be usefully indexed by `samtools index`.

`default_bam` function is used to generate output filename for `samtools sort` if input `sort_output_filename` is not set or when `trigger` is false and we need to return `sort_input` and `secondaryFiles` (if provided) files. Output filename is generated on the base of `sort_input` basename with `.bam` extension by default.

`ext` function is used to return the index file extension (BAI/CSI) based on `csi` and `bai` inputs according to the following logic `csi` && `bai` => BAI !`csi` && !`bai ` => BAI `csi` && !`bai ` => CSI

extract_fastq_upstream
../tools/extract-fastq.cwl (CommandLineTool)

Tool to decompress input FASTQ file(s). If several FASTQ files are provided, they will be concatenated in the order that corresponds to files in input. Bash script's logic: - disable case sensitive glob check - check if root name of input file already include '.fastq' or '.fq' extension. If yes, set DEFAULT_EXT to \"\", otherwise use '.fastq' - check file type, decompress if needed - return 1, if file type is not recognized This script also works of input file doesn't have any extension at all

extract_fastq_downstream
../tools/extract-fastq.cwl (CommandLineTool)

Tool to decompress input FASTQ file(s). If several FASTQ files are provided, they will be concatenated in the order that corresponds to files in input. Bash script's logic: - disable case sensitive glob check - check if root name of input file already include '.fastq' or '.fq' extension. If yes, set DEFAULT_EXT to \"\", otherwise use '.fastq' - check file type, decompress if needed - return 1, if file type is not recognized This script also works of input file doesn't have any extension at all

htseq_count_gene_expression
../tools/htseq-count.cwl (CommandLineTool)
HTSeq: Analysing high-throughput sequencing data

For convenience to use in the workflow that sort and index BAM files by coordinate this tools expects coordinate sorted and indexed BAM file as input. For single-read dat it won't influence on anything, for paired-end the more memory will be used to keep reads while looking for their proper pairs (see --max-reads-in-buffer parameter).

Current limitations: - only one `--additional-attr` is supported - skip `--nprocesses` parameter as it's not helpful when we use only one input BAM file

fastx_quality_stats_upstream
../tools/fastx-quality-stats.cwl (CommandLineTool)

Tool calculates statistics on the base of FASTQ file quality scores. If `output_filename` is not provided call function `default_output_filename` to return default output file name generated as `input_file` basename + `.fastxstat` extension.

fastx_quality_stats_downstream
../tools/fastx-quality-stats.cwl (CommandLineTool)

Tool calculates statistics on the base of FASTQ file quality scores. If `output_filename` is not provided call function `default_output_filename` to return default output file name generated as `input_file` basename + `.fastxstat` extension.

Outputs

ID Type Label Doc
bigwig File [bigWig] BigWig file

Generated BigWig file

bowtie_log File [Textual format] Bowtie alignment log

Bowtie alignment log file

rpkm_genes File [TSV] RPKM, grouped by gene name

Calculated rpkm values, grouped by gene name

bambai_pair File [BAM] Coordinate sorted BAM alignment file (+index BAI)

Coordinate sorted BAM file and BAI index file

star_sj_log File (Optional) [Textual format] STAR sj log

STAR SJ.out.tab

get_stat_log File (Optional) [YAML] YAML formatted combined log

YAML formatted combined log

star_out_log File (Optional) [Textual format] STAR log out

STAR Log.out

rpkm_isoforms File [CSV] RPKM, grouped by isoforms

Calculated rpkm values, grouped by isoforms

star_final_log File [Textual format] STAR final log

STAR Log.final.out

rpkm_common_tss File [TSV] RPKM, grouped by common TSS

Calculated rpkm values, grouped by common TSS

star_stdout_log File (Optional) [Textual format] STAR stdout log

STAR Log.std.out

gene_body_report File (Optional) [TSV] Gene body average tag density plot for all isoforms longer than 1000 bp

Gene body average tag density plot for all isoforms longer than 1000 bp in TSV format

get_stat_markdown File (Optional) [TIDE TXT] Markdown formatted combined log

Markdown formatted combined log

star_progress_log File (Optional) [Textual format] STAR progress log

STAR Log.progress.out

gene_body_plot_pdf File (Optional) [PDF] Gene body average tag density plot for all isoforms longer than 1000 bp

Gene body average tag density plot for all isoforms longer than 1000 bp in PDF format

insert_size_report File [TSV] Insert size distribution report

Insert size distribution report (right after alignment and sorting)

get_formatted_stats File (Optional) [Textual format] BioWardrobe compatible log

Processed and combined Bowtie & STAR aligner and GEEP logs

trim_report_upstream File TrimGalore report FASTQ 1

TrimGalore generated log for FASTQ 1

bam_statistics_report File [Textual format] BAM statistics report

BAM statistics report (right after alignment and sorting)

htseq_count_stderr_log File [Textual format] HTSeq: stderr log

HTSeq: stderr log

htseq_count_stdout_log File [Textual format] HTSeq: stdout log

HTSeq: stdout log

trim_report_downstream File TrimGalore report FASTQ 2

TrimGalore generated log for FASTQ 2

fastx_statistics_upstream File [Textual format] FASTQ 1 statistics

fastx_quality_stats generated FASTQ 1 quality statistics file

rpkm_distribution_plot_pdf File (Optional) [PDF] RPKM distribution plot for isoforms

RPKM distribution plot for isoforms in PDF format

fastx_statistics_downstream File [Textual format] FASTQ 2 statistics

fastx_quality_stats generated FASTQ 2 quality statistics file

htseq_count_gene_expression_file File [TSV] HTSeq: read counts grouped by gene_id

HTSeq: read counts grouped by gene_id

Permalink: https://w3id.org/cwl/view/git/b1a5dabeeeb9079b30b2871edd9c9034a1e00c1c/workflows/trim-rnaseq-pe.cwl