Undo demultiplexing

With the introduction of lima v2.5.0, it is possible to undo all demultiplexing steps for HiFi data. For this, the bioconda package contains a new lima-undo binary.

Example:

lima movie.hifi_reads.bam demux.consensusreadset.xml --hifi-preset SYMMETRIC --store-unbarcoded
lima-undo demux.consensusreadset.xml undo.bam

Let’s unroll what’s happening. In the first line, we explicitly request to store the unbarcoded reads. Without this, we would not be able to recover unbarcoded reads. The XML contains all the file paths to the BAM files. The second call is to the new lima-undo binary that takes a XML or BAM file as input and ouput.

Optionally, you can also provide multiple input BAM files with one output BAM:

lima-undo demux.bam demux.unbarcoded.bam undo.bam

This works also with split BAM files:

lima-undo demux.bc1001-bc1001.bam demux.bc1002-bc1002.bam demux.unbarcoded.bam undo.bam

How does it work?

lima v2.5.0 and later stores everything that got clipped in an internal binary structure in the ls tag. Multiple demultiplexing rounds are supported. Once lima-undo gets called, for each read the individual demultiplexing steps get reverted until the read is identical to the original HiFi read.

How can I check if undo results are correct?

How to check that the result is identical:

samtools sort --no-PG -t "zm" undo.bam -o sorted.undo.bam
samtools view --no-PG sorted.undo.bam > undo.sam
samtools view --no-PG movie.hifi_reads.bam > original.sam
diff original.sam undo.sam