Workflow Type: Galaxy

RNA-seq paired-end Workflow

Inputs dataset

  • The workflow needs a list of dataset pairs of fastqsanger.

  • As well as a gtf file with genes

  • Optional, but recommended: a gtf file with regions to exclude from normalization in Cufflinks.

    • For instance a gtf that masks chrM for the mm10 genome:
chrM	chrM_gene	exon	0	16299	.	+	.	gene_id "chrM_gene_plus"; transcript_id "chrM_tx_plus"; exon_id "chrM_ex_plus";
chrM	chrM_gene	exon	0	16299	.	-	.	gene_id "chrM_gene_minus"; transcript_id "chrM_tx_minus"; exon_id "chrM_ex_minus";

Inputs values

  • adapter sequences: this depends on the library preparation. Usually classical RNA libraries are Truseq and ISML (relatively new Illumina library) is Nextera. If you don't know, use FastQC to determine if it is Truseq or Nextera. If the read length is relatively short (50bp), there is probably no adapter.
  • reference_genome: this field will be adapted to the genomes available for STAR
  • strandness: For stranded RNA, reverse means that the read is complementary to the coding sequence, forward means that the read is in the same orientation as the coding sequence. This will help you to get from STAR only the counts corresponding to your library preparation. This is also used for the stranded coverage and for FPKM computation with cufflinks.

Processing

  • The workflow will remove adapters and low quality bases and filter out any read smaller than 15bp
  • The filtered reads are mapped with STAR with ENCODE parameters (for long RNA-seq but I use it for short also). STAR is also used to count reads per gene.
  • A multiQC is run to have an overview of the QC. This can also be used to get the strandness.
  • FPKM values for reads and transcripts are computed with cufflinks using correction for multi-mapped reads.
  • The BAM is filtered to keep only uniquely mapped reads (tag NH:i:1).
  • Coverage unstranded, and each strand independently is computed with bedtools and normalized to the number of million uniquely mapped reads (in order to compute stranded coverage the BAM is modified so second mate in pairs matches orientation of the first mate in pairs).
  • The three coverage files are converted to bigwig.

Warning

  • The coverage stranded output depends on the strandness of the library:
    • If you have an unstranded library, stranded coverages are useless
    • If you have a forward stranded library, the label matches the orientation of the first read in pairs.
    • If you have a reverse stranded library, the label matches the orientation of the second read in pairs.

Inputs

ID Name Description Type
PE fastq input PE fastq input Should be a list of paired-end RNA-seq fastqs
  • File[]
forward_adapter forward_adapter Please use: For R1: - For Nextera: CTGTCTCTTATACACATCTCCGAGCCCACGAGAC - For TrueSeq: GATCGGAAGAGCACACGTCTGAACTCCAGTCAC or AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC
  • string
gtf gtf gtf compatible with the reference_genome. Mind the UCSC/Ensembl differences in chromosome naming
  • File
gtf with regions to exclude from FPKM normalization gtf with regions to exclude from FPKM normalization Could be a gtf with for example one entry for the chrM forward and one entry for the chrM reverse
  • File?
reference_genome reference_genome reference_genome
  • string
reverse_adapter reverse_adapter Please use: For R2: - For Nextera: CTGTCTCTTATACACATCTGACGCTGCCGACGA - For TruSeq: GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT or AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
  • string
strandness strandness For stranded RNA, reverse means that the read is complementary to the coding sequence, forward means that the read is in the same orientation as the coding sequence
  • string

Steps

ID Name Description
7 Cutadapt (remove adapter + bad quality bases) toolshed.g2.bx.psu.edu/repos/lparsons/cutadapt/cutadapt/4.0+galaxy1
8 get reference_genome as text parameter toolshed.g2.bx.psu.edu/repos/iuc/compose_text_param/compose_text_param/0.1.1
9 awk command from strand toolshed.g2.bx.psu.edu/repos/iuc/map_param_value/map_param_value/0.1.1
10 bedtools orientation for forward coverage toolshed.g2.bx.psu.edu/repos/iuc/map_param_value/map_param_value/0.1.1
11 bedtools orientation for reverse coverage toolshed.g2.bx.psu.edu/repos/iuc/map_param_value/map_param_value/0.1.1
12 Get cufflinks strandess parameter toolshed.g2.bx.psu.edu/repos/iuc/map_param_value/map_param_value/0.1.1
13 STAR: map and count toolshed.g2.bx.psu.edu/repos/iuc/rgrnastar/rna_star/2.7.8a+galaxy0
14 MultiQC toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.11+galaxy0
15 Extract gene counts toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_awk_tool/1.1.2
16 Keep only uniquely mapped reads toolshed.g2.bx.psu.edu/repos/devteam/bamtools_filter/bamFilter/2.5.1+galaxy0
17 get scaling factor This step get 1 / millions of uniquely mapped reads toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_awk_tool/1.1.2
18 Compute FPKM toolshed.g2.bx.psu.edu/repos/devteam/cufflinks/cufflinks/2.2.1.3
19 revertR2orientationInBam toolshed.g2.bx.psu.edu/repos/lldelisle/revertr2orientationinbam/revertR2orientationInBam/0.0.2
20 convert dataset to parameter param_value_from_file
21 Scaled Coverage both strands combined toolshed.g2.bx.psu.edu/repos/iuc/bedtools/bedtools_genomecoveragebed/2.30.0
22 Scaled Coverage positive toolshed.g2.bx.psu.edu/repos/iuc/bedtools/bedtools_genomecoveragebed/2.30.0
23 Scaled Coverage negative toolshed.g2.bx.psu.edu/repos/iuc/bedtools/bedtools_genomecoveragebed/2.30.0
24 convert both strands coverage to bigwig wig_to_bigWig
25 convert positive coverage to bigwig wig_to_bigWig
26 convert negative coverage to bigwig wig_to_bigWig

Outputs

ID Name Description Type
mapped-reads mapped-reads n/a
  • File
output_log output_log n/a
  • File
reads_per_gene from STAR reads_per_gene from STAR n/a
  • File
MultiQC webpage MultiQC webpage n/a
  • File
MultiQC on input dataset(s): Stats MultiQC on input dataset(s): Stats n/a
  • File
HTS count like output HTS count like output n/a
  • File
genes_expression genes_expression n/a
  • File
transcripts_expression transcripts_expression n/a
  • File
both strands coverage both strands coverage n/a
  • File
positive strand coverage positive strand coverage n/a
  • File
negative strand coverage negative strand coverage n/a
  • File

Version History

v1.1 (latest) Created 19th Nov 2024 at 03:02 by WorkflowHub Bot

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v0.9 Created 7th Oct 2024 at 16:33 by WorkflowHub Bot

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v0.5 Created 16th Sep 2023 at 03:01 by WorkflowHub Bot

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v0.4.1 Created 15th Sep 2023 at 03:01 by WorkflowHub Bot

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v0.1 (earliest) Created 25th Oct 2022 at 03:01 by WorkflowHub Bot

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help Creators and Submitter
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  • Lucille Delisle
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Created: 25th Oct 2022 at 03:01

Last updated: 17th Jan 2023 at 03:01

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