alignment-nf

Nextflow pipeline for BAM realignment or fastq alignment

Description

Nextflow pipeline to perform BAM realignment or fastq alignment and QC, with/without local indel realignment and base quality score recalibration.

Dependencies

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

Basic fastq alignment

2. bwa2 (default) or bwa
3. samblaster
4. sambamba

BAM files realignment

5. samtools

Adapter sequence trimming

6. AdapterRemoval

ALT contigs handling

7. the k8 javascript execution shell (e.g., available in the bwakit archive); must be in the PATH
8. javascript bwa-postalt.js and the additional fasta reference .alt file from bwakit must be in the same directory as the reference genome file.

QC

9. Qualimap.
10. Multiqc.

Base quality score recalibration

11. GATK4; wrapper 'gatk' must be in the path
12. GATK bundle VCF files with lists of indels and SNVs (recommended: Mills gold standard indels VCFs, dbsnp VCF), and corresponding tabix indexes (.tbi)

A conda receipe, and docker and singularity containers are available with all the tools needed to run the pipeline (see "Usage")

Input

Type	Description
--input_folder	a folder with fastq files or bam files

Parameters

• Mandatory

Name	Example value	Description
--ref	hg19.fasta	genome reference with its index files (.fai, .sa, .bwt, .ann, .amb, .pac, and .dict; in the same directory)

• Optional

Name	Default value	Description
--input_file	null	Input file (comma-separated) with 4 columns: SM (sample name), RG (read group ID), pair1 (first fastq of the pair), and pair2 (second fastq of the pair).
--output_folder	.	Output folder for aligned BAMs
--cpu	8	number of CPUs
--cpu_BQSR	2	number of CPUs for GATK base quality score recalibration
--mem	32	memory
--mem_BQSR	10	memory for GATK base quality score recalibration
--RG	PL:ILLUMINA	sequencing information for aligned (for bwa)
--fastq_ext	fastq.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
--bed		bed file with interval list
--snp_vcf	dbsnp.vcf	path to SNP VCF from GATK bundle (default : dbsnp.vcf)
--indel_vcf	Mills_1000G_indels.vcf	path to indel VCF from GATK bundle (default : Mills_1000G_indels.vcf)
--postaltjs	bwa-postalt.js"	path to postalignment javascript bwa-postalt.js
--feature_file	null	Path to feature file for qualimap
--multiqc_config	null	config yaml file for multiqc
--adapterremoval_opt	null	Command line options for AdapterRemoval
--bwa_mem	bwa-mem2 mem	bwa-mem command; use "bwa mem" to switch to regular bwa-mem (both are in the docker and singularity containers)

• Flags

Flags are special parameters without value.

Name	Description
--help	print usage and optional parameters
--trim	enable adapter sequence trimming
--recalibration	perform quality score recalibration (GATK)
--alt	enable alternative contig handling (for reference genome hg38)
--bwa_option_M	Trigger the -M option in bwa and the corresponding compatibility option in samblaster (marks shorter split hits as secondary)

Usage

To run the pipeline on a series of fastq or BAM files in folder input and a fasta reference file hg19.fasta, one can type:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output

To run the pipeline without singularity just remove "-profile singularity". Alternatively, one can run the pipeline using a docker container (-profile docker) the conda receipe containing all required dependencies (-profile conda).

Use bwa-mem instead of bwa-mem2

To use bwa-mem, one can type:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output --bwa_mem "bwa mem"

Enable adapter trimming

To use the adapter trimming step, you must add the --trim option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output --trim

Enable ALT mode

To use the alternative contigs handling mode, you must provide the path to an ALT aware genome reference (e.g., hg38) AND add the --alt option, as well as satisfy the above-mentionned requirements. For example:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output --postaltjs /user/bin/bwa-0.7.15/bwakit/bwa-postalt.js --alt

Enable base quality score recalibration

To use the base quality score recalibration step, you must provide the path to 2 GATK bundle VCF files with lists of known snps and indels, respectively, AND add the --recalibration option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output --snp_vcf GATKbundle/dbsnp.vcf.gz --indel_vcf GATKbundle/Mills_1000G_indels.vcf.gz --recalibration

Output

Type	Description
BAM/	folder with BAM and BAI files of alignments or realignments
QC/BAM/multiqc_qualimap_flagstat_*report.html	multiQC report for qualimap and samtools flagstat (duplicates)
QC/BAM/multiqc_qualimap_flagstat_*report_data	data used for the multiQC report
QC/qualimap/file_BQSRecalibrated.stats.txt	qualimap summary file
QC/qualimap/file_BQSRecalibrated/	qualimap files
QC/BAM/BQSR/	GATK base quality score recalibration outputs (tables and pdf comparing scores before/after recalibration)

Directed Acyclic Graph

FAQ

Why did Indel realignment disappear from version 1.0?

Indel realignment was removed following new GATK best practices for pre-processing.

Contributions

Name	Email	Description
Nicolas Alcala*	AlcalaN@fellows.iarc.fr	Developer to contact for support
Catherine Voegele	VoegeleC@iarc.fr	Tester
Vincent Cahais	CahaisV@iarc.fr	Tester
Alexis Robitaille	RobitailleA@students.iarc.fr	Tester