RNAseq-nf

RNAseq mapping, quality control, and reads counting nextflow pipeline

Overview of pipeline workflow

Prerequisites

General prerequisites

The following programs need to be installed and in the PATH environment variable:

• fastqc
• cutadapt, which requires Python version > 2.7
• trim_galore
• RESeQC
• multiQC
• STAR
• htseq; the python script htseq-count must also be in the PATH
• nextflow

In addition, STAR requires genome indices that can be generated from a genome fasta file ref.fa and a splice junction annotation file ref.gtf using the following command:

STAR --runThreadN n --runMode genomeGenerate --genomeDir ref --genomeFastaFiles ref.fa --sjdbGTFfile ref.gtf --sjdbOverhang 99

Prerequisites for alignment with hisat2

In order to perform the optional alignment with hisat2, hisat2 must be installed:

• hisat2

In addition, indexes files .ht2 must be downloaded from generated from hisat2, or generated from a reference fasta file (e.g., reference.fa) and a GTF annotation file (e.g., reference.gtf) using the following commands:

extract_splice_sites.py reference.gtf > genome.ss
extract_exons.py reference.gtf > genome.exon
hisat2-build reference.fa --ss genome.ss --exon genome.exon genome_tran

Prerequisites for reads trimming at splice junctions

In order to perform the optional reads trimming at splice junctions, GATK must be installed:

• GATK GenomeAnalysisTK.jar

In addition, index .fai and dictionnary .dict must be generated from the fasta reference genome using the following commands:

samtools faidx ref.fa
java -jar picard.jar CreateSequenceDictionary R= ref.fa O= ref.dict

Prerequisites for base quality score recalibration

• GATK GenomeAnalysisTK.jar
• GATK bundle VCF files with lists of indels and SNVs (recommended: 1000 genomes indels, Mills gold standard indels VCFs, dbsnp VCF)
• bed file with intervals to be considered

Usage

To run the pipeline on a series of paired-end fastq files (with suffixes _1 and _2) in folder fastq, and a reference genome with indexes in folder ref_genome, one can type:

nextflow run iarcbioinfo/RNAseq-nf --input_folder fastq --gendir ref_genome --suffix1 _1 --suffix2 _2

Use hisat2 for mapping

To use the reads trimming at splice junctions step, you must add the --hisat2 option, specify the path to the folder containing the hisat2 index files, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/RNAseq-nf --input_folder fastq --suffix1 _1 --suffix2 _2 --hisat2 --hisat2_idx /home/user/reference/genome_tran 

Enable reads trimming at splice junctions

To use the reads trimming at splice junctions step, you must add the --sjtrim option, specify the path to the folder containing the GenomeAnalysisTK jar file, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/RNAseq-nf --input_folder fastq --gendir ref_genome --suffix1 _1 --suffix2 _2 --sjtrim --GATK_folder /home/user/GATK 

Enable Base Quality Score Recalibration

To use the base quality score recalibration step, you must add the --bqsr option, specify the path to the folder containing the GenomeAnalysisTK jar file, the path to the GATK bundle folder for your reference genome, specify the path to the bed file with intervals to be considered, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/RNAseq-nf --input_folder fastq --gendir ref_genome --suffix1 _1 --suffix2 _2 --bqsr --GATK_folder /home/user/GATK --GATK_bundle /home/user/GATKbundle --intervals intervals.bed

All parameters

PARAMETER	DEFAULT	DESCRIPTION
--help	null	print usage and optional parameters
--input_folder	.	input folder
--output_folder	.	output folder
--gendir	ref	reference genome folder
--cpu	4	number of CPUs
--mem	50	memory for mapping
--memOther	2	memory for QC and counting
--fastq_ext	fq.gz	extension of fastq files
--suffix1	_1	suffix for second element of read files pair
--suffix2	_2	suffix for second element of read files pair
--output_folder	.	output folder for aligned BAMs
--annot_gtf	Homo_sapiens.GRCh38.79.gtf	annotation GTF file
--annot_gff	Homo_sapiens.GRCh38.79.gff	annotation GFF file
--fasta_ref	ref.fa	reference genome fasta file for GATK
--GATK_folder	GATK	folder with jar file GenomeAnalysisTK.jar
--GATK_bundle	GATK_bundle	folder with files for BQSR
--intervals	intervals.bed	bed file with intervals for BQSR
--RG	PL:ILLUMINA	string to be added to read group information in BAM file
--sjtrim	false	enable reads trimming at splice junctions
--bqsr	false	enable base quality score recalibration
--gene_bed	gene.bed	bed file with genes for RESeQC
--stranded	no	Strand information for counting with htseq [no, yes, reverse]
--stranded	no	Strand information for counting with htseq [no, yes, reverse]
--hisat2	false	use hisat2 instead of STAR for mapping
--hisat2_idx	genome_tran	index filename prefix for hisat2