secondary_rewriter

Some aligners such as minimap2 do not write the SEQ and QUAL fields to secondary alignments (https://github.com/lh3/minimap2/issues/458) making it hard to analyze them (for example, SNPs will not be visible in a genome browser for secondary alignments). This program adds these back, referring to the primary alignment to get the SEQ and QUAL, and adding them to the secondaries. This is a three-pass program. The first pass collects the secondary alignments into an external file the second adds the SEQ and QUAL fields to the external file, and then the third pass inserts the secondary alignments in place.

Install

First install rust, probably with rustup https://rustup.rs/

Then

cargo install secondary_rewriter

Usage

This small shell script automates the multi-step pipeline (supports BAM or CRAM)

```

!/bin/bash

write_secondaries.sh

usage

./write_secondaries.sh

e.g.

./write_secondaries.sh input.cram ref.fa output.cram 16 32G

THR=${4:-4} MEM=${5:-16G}

samtools view -@$THR -h $1 -T $2 -f256 > sec.txt samtools view -@$THR -h $1 -T $2 -F256 | secondary_rewriter --secondaries sec.txt | samtools sort -@$THR -m $MEM - -o $3

```

The two-pass strategy works as follows

1. First pass: output ALL secondary alignments (reads with flag 256) to a external file
2. Second pass: read secondary alignments from external file into memory, and then scan original SAM/BAM/CRAM to add SEQ and QUAL fields on the primary alignments to the secondary alignments, and pipe to samtools sort (needed because all the new secondary reads with BAM/CRAM at the end)

This seems laborious, but the strategy avoids loading the entire SAM/BAM/CRAM into memory and a full samtools sort of the entire file. The samtools sort is the most expensive part of the process, and you can consider tuning it or using other programs like sambamba too https://www.basepairtech.com/blog/sorting-bam-files-samtools-vs-sambamba/ (sambamba does not support CRAM afaik)

Help

```

secondary_rewriter 0.1.6 Adds SEQ and QUAL fields to secondary alignments from the primary alignment

USAGE: secondary_rewriter [OPTIONS]

OPTIONS: -h, --help Print help information -s, --secondaries -V, --version Print version information

```