RNAseq-nf

Nextflow pipeline for RNA seq processing

Decription

Nextflow pipeline for RNA sequencing mapping, quality control, reads counting, and unsupervised analysis

Dependencies

1. Nextflow: for common installation procedures see the IARC-nf repository.

2. fastqc

3. RESeQC

4. multiQC

5. STAR

6. htseq; the python script htseq-count must also be in the PATH

A singularity container is available with all the tools needed to run the pipeline (see "Usage")

References

A bundle with reference genome and corresponding annotations for STAR is available at https://data.broadinstitute.org/Trinity/CTAT_RESOURCE_LIB/.

Alternatively, STAR genome indices 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

You can provide a config file to customize the multiqc report (see https://multiqc.info/docs/#configuring-multiqc).

Reads adapter trimming with cutadapt

In order to perform the optional adapter trimming of reads before mapping the following software must be installed:

• cutadapt version > 1.15, which requires Python version > 2.7
• trim_galore

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

Reads trimming at splice junctions

In order to perform the optional reads trimming at splice junctions, GATK4 must be installed:

• GATK

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

Base quality score recalibration

In order to perform the optional base quality score recalibration, several files are required:

• GATK4 must be in the PATH variable
• GATK bundle VCF files with lists of indels and SNVs (recommended: 1000 genomes indels, dbsnp VCF)
• bed file with intervals to be considered

Input

Type	Description
--input_folder	Folder containing BAM or fastq files to be aligned
--input_file	Input tabulation-separated values file with columns SM (sample name), RG (read group), pair1 (first fastq pair file), and pair2 (second fastq pair file)

Note that there are two input methods: folder and file. Although the input folder method is the easiest because it does not require to create an input file with the right format, the input file mode is recommended in cases when a single sample has multiple paired files (e.g., due to multiplexed sequencing); in that case, users should have one line per pair of file and put a same SM identifier so that the workflow can group them into the same output bam file. For example:

SM RG pair1 pair2
sample1		sample1_1.fq.gz	sample1_2.fq.gz
sample2	RG1	sample2_RG1_1.fq.gz	sample2_RG1_2.fq.gz
sample2	RG2	sample2_RG2_1.fq.gz	sample2_RG2_2.fq.gz

Parameters

• Mandatory

Name	Example value	Description
--ref_folder	ref	Folder with genome reference files (with index)
--gtf	Homo_sapiens.GRCh38.79.gtf	Annotation GTF file
--bed	gene.bed	bed file with genes for RESeQC (interval list)

• Optional

Name	Default value	Description
--cpu	4	Number of cpu used by bwa mem and sambamba
--cpu_gatk	1	Number of CPUs for GATK processes (SJ trimming and BQSR)
--cpu_trim	15	Number of CPUs for reads trimming (cutadapt)
--mem	50	Size of memory used for mapping (in GB)
--mem_QC	2	Size of memory used for QC and cutadapt (in GB)
--fastq_ext	fq.gz	Extension of fastq files
--suffix1	_1	Suffix of fastq files 1 (first element of read files pair - should include the 1st underscore)
--suffix2	_2	Suffix of fastq files 2(second element of read files pair - should include the 1st underscore)
--output_folder	.	Output folder
--ref	ref.fa	Reference fasta file (with index) for GATK
--snp_vcf	dbsnp.vcf	Path to SNP VCF from GATK bundle
--indel_vcf	Mills_100G_indels.vcf	Path to indel VCF from GATK bundle
--STAR_mapqUnique	255	STAR default mapping quality for unique mappers
--RG	PL:ILLUMINA	Samtools read group specification
--stranded	no	Strand information for counting with htseq [no, yes, reverse]
--hisat2_idx	genome_tran	hisat2 index file prefix
--htseq_maxreads	30000000	Maximum number of reads taken into account by htseq-count
--multiqc_config	null	Config yaml file for multiqc

• Flags

Name	Description
--help	print usage and optional parameters
--cutadapt	enable adapter and quality reads trimming before alignment
--sjtrim	enable reads trimming at splice junctions
--hisat2	use hisat2 instead of STAR for mapping
--recalibration	perform quality score recalibration (GATK)

Usage

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

nextflow run iarcbioinfo/RNAseq-nf -r v2.4 -profile singularity --input_folder fastq --ref_folder ref_genome --gtf ref.gtf --bed ref.bed

To run the pipeline using conda instead of singularity, replace "-profile singularity" by "-profile conda". To run with your own local software installation, just remove "-profile singularity".

Single-end fastq mode

Default is adapted to paired-end libraries. To use single-end libraries as input, you must specify the option "--suffix2 null".

nextflow run iarcbioinfo/RNAseq-nf -r v2.4 -profile singularity --input_folder fastq --ref_folder ref_genome --gtf ref.gtf --bed ref.bed --suffix2 null

If using "--input_file", you must additionally set the values in column "pair2" to "NO_fastq2". For example the following file input.txt:

SM RG pair1 pair2
sample1		sample1.fq.gz	NO_fastq2
sample2	RG1	sample2_RG1.fq.gz	NO_fastq2
sample2	RG2	sample2_RG2.fq.gz	NO_fastq2

can be processed with

nextflow run iarcbioinfo/RNAseq-nf -r v2.4 -profile singularity --input_file input.txt --ref_folder ref_genome --gtf ref.gtf --bed ref.bed --suffix2 null

Use hisat2 for mapping

To use hisat2 instead of STAR for the reads mapping, you must add the --hisat2 option, specify the path to the folder containing the hisat2 index files (genome_tran.1.ht2 to genome_tran.8.ht2), as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/RNAseq-nf --input_folder fastq --ref_folder ref_genome --gtf ref.gtf --bed ref.bed --hisat2 --hisat2_idx genome_tran 

Note that parameter '--hisat2_idx' is the prefix of the index files, not the entire path to .ht2 files.

Enable reads trimming at splice junctions

To use the reads trimming at splice junctions step, you must add the --sjtrim option as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/RNAseq-nf --input_folder fastq --ref_folder ref_genome --gtf ref.gtf --bed ref.bed --sjtrim

Enable Base Quality Score Recalibration

To use the base quality score recalibration step, you must add the --recalibration option, specify the path to the known snps and indels from the GATK bundle, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/RNAseq-nf --input_folder fastq --ref_folder ref_genome --gtf ref.gtf --bed ref.bed --recalibration --snp_vcf GATK_bundle/dbsnp_146.hg38.vcf.gz --indel_vcf GATK_bundle/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz

Output

Type	Description
BAM/file.bam	BAM files of alignments or realignments
BAM/file.bam.bai	BAI files of alignments or realignments
BAM/STAR.file.Chimeric.SJ.out.junction	STAR chimeric junction output
BAM/STAR.file.SJ.out.tab	STAR junction tab output
counts/file_count.txt	htseq-count output file
QC/multiqc_pretrim_report.html	multiqc report before trimming
QC/multiqc_pretrim_report_data	folder with data used to compute multiqc report before trimming
QC/multiqc_posttrim_report.html	multiqc report before trimming
QC/multiqc_posttrim_report_data	folder with data used to compute multiqc report before trimming
QC/adapter_trimming/file_{12}.fq.gz_trimming_report.txt	trim_galore report
QC/adapter_trimming/file_{12}val{12}_fastqc.zip	FastQC report after trimming
QC/alignment/STAR.file.Log.final.out, STAR.file.Log.out, STAR.file.Log.progress.out	STAR logs
QC/bam/file_readdist.txt, file_clipping_profile*, file_jun_stauration*	RSeQC reports
QC/fastq/file_{12}_pretrim_fastqc.zip	FastQC report before trimming

The output_folder directory contains three subfolders: BAM, counts, and QC

Directed Acyclic Graph

With default options

With option --hisat2

With options --sjtrim and --recalibration

Contributions

Name	Email	Description
Nicolas Alcala*	AlcalaN@fellows.iarc.fr	Developer to contact for support
Noemie Leblay	LeblayN@students.iarc.fr	Tester
Alexis Robitaille	RobitailleA@students.iarc.fr	Tester