alndv

A nextflow (DSL 2) Whole‑Genome Short‑Read Pipeline (BWA‑MEM2 → DeepVariant → GLnexus) for small‑variant discovery from paired‑end FASTQ files.

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1. Overview

FASTQ ─► FASTQC ─► BWA‑MEM2 ──► MERGEB ─► QUALIMAP 
                                   └─► DEPTH (mosdepth)
                                   └─► DEEPVARIANT_AUTOSOMES ─┬─► per‑sample VCF
                                   |         |                └─► per‑sample gVCF
                                   |         │
                                   |         ▼
                                   |       GLnexus (cohort merge) ─► joint autosomes BCF
                                   | 
                                   └─► DEEPVARIANT_SEX ─┬─► per‑sample VCF
                                            │           └─► per‑sample gVCF
                                            │
                                            ▼
                                          GLnexus (cohort merge) ─► joint SEX BCF

Step	Tool	Purpose
FASTQC	FastQC 0.12	Raw‑read quality control
BWAMEM	bwa‑mem2 2.3 / bwa 0.7	Alignment, alt‑aware mapping, optional HLA typing
MERGEB	samtools 1.19	Merge technical replicates into a single CRAM
QUALIMAP	Qualimap 2.3	Alignment QC (streaming from CRAM via FIFO)
DEPTH	mosdepth 0.3	Depth & breadth of coverage
DEEPVARIANT	DeepVariant 1.8	SNP/indel calling (WGS model)
GLNEXUS_DEEPVARIANT	GLnexus 1.4	Joint genotyping of all gVCFs

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2. File‑tree after a successful run (default --outdir results)

results/
├── QC/
│   └── FASTQC/         # \*.html and \*.zip reports
├── BWA/
│   ├── \*.log.bwamem
│   └── HLA/            # .hla.all and logs (if --hla)
├── CRAM/
│   ├── \*.cram
│   └── \*.cram.crai    
├── DEPTH/
│   ├── \*.mosdepth.dist.txt
│   └── \*.mosdepth.summary.txt
├── qualimap/
│   └── SAMPLE/         # Qualimap HTML + tables
├── deepvariant\_persample/
│   ├── \*.vcf.gz
│   └── \*.g.vcf.gz
└── glx/
└── all.glx.bcf

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3. Quick start

nextflow run main.nf --csv reads-test.cvs --ref test.fa --debug true

Required inputs

Flag	Description
--reads	Glob pattern for paired FASTQ (sample_*{1,2}.fq.gz) or
--csv	CSV with columns sampleId,part,runDV,read1,read2
--ref	Reference FASTA indexed with .fai and .dict

Example CSV:

sampleId,part,runDV,read1,read2
sampleA,1,true,/data/sampleA_R1.fastq.gz,/data/sampleA_R2.fastq.gz
sampleA,2,true,/data/sampleA_rep2_R1.fastq.gz,/data/sampleA_rep2_R2.fastq.gz
sampleB,1,false,/data/sampleB_R1.fastq.gz,/data/sampleB_R2.fastq.gz

runDV accepts true/false, yes/no, or 1/0. When the column is omitted or left blank, the pipeline defaults to true to preserve the previous behavior.

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4. Key parameters

Parameter	Default	Notes
--alt	false	Activate alt‑aware alignment (*.alt file required)
--hla	false	Run xHLA / k8 Alt-aware typing (human data only)
--debug	false	Dry‑run style outputs; generates dummy files

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5. Process outputs

Process	Channel	Data type	Path / pattern
FASTQC	fqc	directory	<sample>-<part>.fastqc/
BWAMEM	bams	tuple	<sample>-<part>.mkdup.cram + .crai
MERGEB	mbams	tuple	<sample>.merged.cram + .crai
QUALIMAP	qualimap_results	dir	qualimap/<sample>/
DEPTH	depth	files	<sample>.depth.*
DEEPVARIANT	vcf / gvcf_file	files	<sample>.vcf.gz, <sample>.g.vcf.gz
GLNEXUS_DEEPVARIANT	bcf	file	all.glx.bcf

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6. Joint‑calling logic

All per‑sample gVCFs are collected into one tuple:

allgvcf = DEEPVARIANT.out.gvcf_file.collect()
          .map{ gvcfs -> tuple('all', gvcfs) }

The tuple ('all', [list_of_gvcfs]) feeds GLNEXUS_DEEPVARIANT, which emits all.glx.bcf (Bgzipped BCF, ready for analysis or left‑alignment/annotation).

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7. Containers & reproducibility

Every process runs inside an OCI/Singularity container pinned by digest:

• bwa-mem2_instrain_multiqc_qualimap_samtools:850f96...
• google/deepvariant:1.8.0
• biocontainers/glnexus:1.4.1--h40d77a6_0

Pull happens automatically via Nextflow Wave/Seqera Registry when -with-docker or -with-singularity is enabled.

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8. Troubleshooting

Issue	Remedy
qualimap hangs	Ensure JVM has enough heap (--java-mem-size) and CRAM reference matches --ref.
DeepVariant “invalid range”	Reference/CRAM mismatch or truncated CRAM; regenerate index.
GLnexus OOM	Lower --threads or set NX_MEM_GB via params.memory.

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9. Developer

1. Alex Di Genova

10. License

Licensed under the MIT License.
Pull requests welcome!