trnamaprecal

This software is created to use Oxford Nanopore RNA004 flowcells for tRNA sequecin and contains two programs.

Use bwamapbam to align the Dorado output BAM using BWA and keep the modification tags generated by Dorado. Unfortunately BWA is currently affected by a bug that writes an incorrect SAM header to it's output, which confuses our SAM parser. See this github issue. use the patch provided in this repository to fix BWA.

After alignment use trnamapqrecal to adjust the mapping qualities to account for the low distance reference sequences. This program will assign > q0 mapping qualities to reads that have high enough alignment scores for the primary alignment compared to the alternative mappings given the actual distance of the reference seqeunces.

Usage

Bam alignment

Usage: bwamapbam [OPTIONS] --reference <REFERENCE> --bam <BAM> --out <OUT>


Options:
      --reference <REFERENCE>  
      --bam <BAM>              
      --out <OUT>              
      --bwa <BWA>              [default: bwa]
      --args <ARGS>            [default: "-t8 -Y -h30,200 -W13 -k6 -xont2d -T20 -u"]
  -h, --help                   Print help

Use --bwa to provide an alternative BWA executable path. BWA arguments are optimal for human tRNA reference mapping. Use the --args to customize the BWA command line arguments, the -u is required to generate the XB tag this software relies on. The reference should be the location of the BWA indexed .fa file.

Mapping quality recalibration

Usage: trnamapqrecal [OPTIONS] --reference <REFERENCE> --bam <BAM> --out <OUT>

Options:
      --reference <REFERENCE>  
      --bam <BAM>              
      --out <OUT>              
      --counts <COUNTS>        
      --qs <QS>                [default: 0]
  -h, --help                   Print help

If --counts is provided the tRNA reference counts are output grouped by mapping quality. You can use --counts in combination with --qs to filter on ONT read quality (qs tag should be in BAM records). --out should be a BAM file, this output is not filtered for read quality! You can use a samtools expression to filter the bam file.

When running modkit on the recalibrated bam you should probably filter for mapq and qs using a samtools expression:

samtools view -q1 -e '[qs] > 4' in.bam -o out.bam

Citation

The paper for which this software was implemented is under submission

Adva Kochavi, Arno Velds, Roderick Beijersbergen, Reuven Agami. (2025). Exploiting nanopore sequencing advances for tRNA sequencing of human cancer models