`cmuts align`¶

Purpose¶

Aligning raw sequencing data against a reference library.

Requires¶

The sequenced reads, either in FASTQ or unaligned BAM format
The FASTA library

As well as both minimap2 and samtools on the path, and vsearch if processing paired-end input.

Usage¶

One invocation handles one sample. Pass a single FASTQ file for single-end reads,

cmuts align -f references.fasta -o treated.bam -x map-ont treated.fastq

or two for paired-end reads.

cmuts align -f references.fasta -o treated.bam -x sr treated_R1.fastq.gz treated_R2.fastq.gz

PacBio and some nanopore instruments deliver reads as an unaligned BAM rather than a FASTQ, which is passed the same way.

cmuts align -f references.fasta -o treated.bam -x map-hifi treated.hifi_reads.bam

Presets¶

The required -x argument names the platform the reads came from, which is passed on to minimap2 for alignment purposes. It must be one of the following.

Preset	Platform
`sr`	Illumina, MGI, Complete Genomics
`map-ont`	Oxford Nanopore
`map-hifi`	PacBio HiFi
`map-pb`	PacBio CLR
`map-iclr`	Illumina Complete Long Reads
`lr:hq`	Any long read platform with an error rate below one percent

Paired-end inputs are refused unless the preset is sr.

Unaligned BAM¶

The program reads only the name, the bases, and the base qualities from an unaligned BAM. Per-base tags such as the PacBio ip and pw fields, or the base modification calls in MM and ML, are lost. If a record has no sequence, has no base qualities, or is marked as secondary, supplementary, or reverse, the program exits and no output is written.

Paired-end data in unaligned BAM format is not supported. BAM files which are already aligned are refused.

Checksums¶

The output header contains the MD5 checksum of each reference in the M5 field of its @SQ line. The checksum is used by cmuts hmm to ensure alignment and processing use the same FASTA.

Merging¶

Paired-end input is merged with vsearch before alignment. A pair whose mates do not overlap cannot be merged and is discarded. vsearch reports how many pairs it merged on standard error.

Sorting¶

The alignments are sorted by samtools sort, with -t and -m controlling the number of threads and the maximum memory per thread.

CLI Options¶

Arguments¶

Argument	Description
`READS`	reads to align, as FASTQ or unaligned BAM
`MATE`	the second file of a pair, merged with the first

Input and output¶

Option	Description
`-f, --fasta FASTA`	reference sequences (required)
`-o, --output BAM`	write sorted alignments to this file (required)
`--overwrite`	replace the output file if it already exists

Alignment¶

Option	Description
`-x, --preset PRESET`	minimap2 preset for the sequencing technology (sr, map-ont, map-hifi, map-pb, map-iclr, lr:hq; required)

Performance¶

Option	Description
`-t, --threads N`	threads for merging, alignment and sorting (default 1)
`-m, --memory MiB`	memory each sorting thread holds before it writes to disk (default 768)

Information¶

Option	Description
`-h, --help`	show this help and exit
`-V, --version`	show the version and exit

Advanced¶

Accepted, and left out of --help.

Option	Description
`--dump-options`	describe every argument as JSON and exit

cmuts align¶