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
- Nextflow : for common installation procedures see the IARC-nf repository.
Basic fastq alignment
- bwa2 (default) or bwa
- samblaster
- sambamba
BAM files realignment
- samtools
Adapter sequence trimming
- AdapterRemoval
ALT contigs handling
- the k8 javascript execution shell (e.g., available in the bwakit archive); must be in the PATH
- 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
- Qualimap.
- Multiqc.
Base quality score recalibration
- GATK4; wrapper ‘gatk’ must be in the path
- 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”)
| Type |
Description |
| –input_folder |
a folder with fastq files or bam files |
Parameters
| 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) |
| 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 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
评论