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

samtools view file.bam | secondary_rewriter --pass1 > sec.txt samtools view file.bam | secondary_rewriter --pass2 --secondaries sec.txt > sec2.txt samtools view -h file.bam | secondary_rewriter --pass3 --secondaries sec2.txt | samtools view - -o out.bam

This small shell script automates this (supports CRAM)

```

!/bin/bash

write_secondaries.sh

usage

./write_secondaries.sh

e.g.

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

THR=${4:-4}

samtools view -@$THR -h $1 -T $2 | secondaryrewriter --pass1 > sec.txt echo Done pass 1 samtools view -@$THR -h $1 -T $2 | secondaryrewriter --pass2 --secondaries sec.txt > sec2.txt echo Done pass 2 samtools view -@$THR -h $1 -T $2 | secondary_rewriter --pass3 --secondaries sec2.txt.gz | samtools view -@$THR -T $2 - -o $3 echo Done pass 3

```

The three-pass strategy works as follows

1. First pass: output ALL secondary alignments (reads with flag 256) to a external file
2. Second pass: reading 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 that are stored in a hashmap
3. Third pass: the secondary alignments with the new SEQ and QUAL fields are re-inserted in place into the SAM file, and re-encoded. By re-inserting them in-place, it avoids a full samtools sort on the output which is disk/memory/cpu intensive.

This seems laborious, but the three-pass strategy avoids loading the entire SAM/BAM/CRAM into memory

Help

```

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

USAGE: secondary_rewriter [OPTIONS]

OPTIONS: -h, --help Print help information --pass1 --pass2 --pass3 -s, --secondaries -V, --version Print version information

```