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