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RNAseq-nf

RNAseq mapping, quality control, and reads counting nextflow pipeline

Overview of pipeline workflow

Scheme of alignment/realignment Workflow

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_fo lder . input folder
--output_f older . 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_ex t 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_f older . output folder for aligned BAMs
--annot_gt f Homo_sapiens.GR Ch38.79.gtf annotation GTF file
--annot_gf f Homo_sapiens.GR Ch38.79.gff annotation GFF file
--fasta_re f ref.fa reference genome fasta file for GATK
--GATK_fol der GATK folder with jar file GenomeAnalysis TK.jar
--GATK_bun dle 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_i dx genome_tran index filename prefix for hisat2