alignment-variants

A pipeline to align (map) reads against a reference genome, and call variants based on the alignment.

Pipeline

Reads are trimmed and filtered using fastp, then mapped against the reference genome using bwa-mem (short reads) or minimap2 (long reads). Alignment metrics are generated by samtools and qualimap.

Variants are called using freebayes.

flowchart TD
  ref[ref.fa]
  fastq_short[fastq_short]
  fastq_long[fastq_long]
  fastq_short --> fastp(fastp)
  fastq_long --> nanoq_pre_filter("nanoq (pre-filter)")
  fastq_long --> filtlong(filtlong)
  filtlong --> nanoq_post_filter("nanoq (post-filter)")
  ref --> bwa(bwa_mem)
  fastp --> bwa
  bwa --> alignments[alignments]
  ref --> minimap2(minimap2)
  filtlong --> minimap2
  minimap2 --> alignments
  alignments --> qualimap(qualimap_bamqc)
  alignments --> samtools_stats(samtools_stats)
  alignments --> freebayes(freebayes)
  alignments --> samtools_mpileup(samtools_mpileup)
  samtools_mpileup --> generate_low_coverage_bed(generate_low_coverage_bed)
  samtools_mpileup --> percent_coverage_by_depth(percent_coverage_by_depth)
  qualimap --> combined_alignment_qc(combined_alignment_qc)
  samtools_stats --> combined_alignment_qc

Loading

Usage

nextflow run BCCDC-PHL/alignment-variants \
  --ref /path/to/ref.fa \
  --fastq_input /path/to/fastqs \
  --outdir /path/to/outputs

Including the --collect_outputs flag will add the collected_fastp.csv and collected_qualimap_bamqc.csv outputs, which include results for all samples collected into a single file.

nextflow run BCCDC-PHL/alignment-variants \
  --ref /path/to/ref.fa \
  --fastq_input /path/to/fastqs \
  --collect_outputs \
  --outdir /path/to/outputs

An alternative filename prefix can be set for the collected outputs using the --collected_outputs_prefix flag:

nextflow run BCCDC-PHL/alignment-variants \
  --ref /path/to/ref.fa \
  --fastq_input /path/to/fastqs \
  --collect_outputs \
  --collected_outputs_prefix 'test' \
  --outdir /path/to/outputs

...which would produce files named: test_fastp.csv and test_qualimap_bamqc.csv.

If long reads are available, they can be included with the --fastq_input_long and --align_long_reads flags:

nextflow run BCCDC-PHL/alignment-variants \
  --ref /path/to/ref.fa \
  --fastq_input /path/to/fastqs \
  --fastq_input_long /path/to/long_fastqs \
  --align_long_reads \
  --outdir /path/to/outputs

Alternatively, a samplesheet.csv file can be provided:

nextflow run BCCDC-PHL/alignment-variants \
  --ref /path/to/ref.fa \
  --samplesheet_input /path/to/samplesheet.csv \
  --outdir /path/to/outputs

The fields should include:

ID
R1
R2

...and if long reads are included, use the field:

LONG

...if separate reference sequences are to be used for each sample, a REF field can be included, with a path to the reference genome for each sample. When including the REF field in the samplesheet, the --ref flag can be omitted.

Alignment Cleaning

By default, alignments will be cleaned as described below. Alignment cleaning is optional, and can be skipped using the --skip_alignment_cleaning flag.

Immediately after alignment, the following filters are applied using samtools view with the -F 1548 flag.

This translates to removing reads that meet these criteria:

• read unmapped (0x4)
• mate unmapped (0x8)*
• read fails platform/vendor quality checks (0x200)
• read is PCR or optical duplicate (0x400)

See the explain-sam-flags site for more details on these flags.

After that, secondary and unmapped reads are removed using the -r flag of samtools fixmate

And duplicates are removed using the -r flag of samtools markdup

Outputs

output
├── SAMPLE-1
│   ├── SAMPLE-1_20231123140142_provenance.yml
│   ├── SAMPLE-1_fastp.csv
│   ├── SAMPLE-1_fastp.json
│   ├── SAMPLE-1_long.bam
│   ├── SAMPLE-1_long.bam.bai
│   ├── SAMPLE-1_short.bam
│   ├── SAMPLE-1_short.bam.bai
│   ├── SAMPLE-1_short_depths.tsv
│   ├── SAMPLE-1_short_freebayes.vcf
│   ├── SAMPLE-1_short_low_coverage_regions.bed
│   ├── SAMPLE-1_short_percent_coverage_by_depth.csv
│   ├── SAMPLE-1_short_qualimap_alignment_qc.csv
│   ├── SAMPLE-1_short_qualimap_genome_results.txt
│   └── SAMPLE-1_short_qualimap_report.pdf
├── SAMPLE-2
│   ├── SAMPLE-2_20231123140112_provenance.yml
│   ├── SAMPLE-2_fastp.csv
│   ├── SAMPLE-2_fastp.json
│   ├── SAMPLE-2_long.bam
│   ├── SAMPLE-2_long.bam.bai
│   ├── SAMPLE-2_short.bam
│   ├── SAMPLE-2_short.bam.bai
│   ├── SAMPLE-2_short_depths.tsv
│   ├── SAMPLE-2_short_freebayes.vcf
│   ├── SAMPLE-2_short_low_coverage_regions.bed
│   ├── SAMPLE-2_short_percent_coverage_by_depth.csv
│   ├── SAMPLE-2_short_qualimap_alignment_qc.csv
│   ├── SAMPLE-2_short_qualimap_genome_results.txt
│   └── SAMPLE-2_short_qualimap_report.pdf
├── collected_fastp.csv
└── collected_qualimap_bamqc.csv