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Detect when i5 indices need to be rev-comp'ed #469

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tomkinsc opened this issue Apr 20, 2023 · 1 comment
Open

Detect when i5 indices need to be rev-comp'ed #469

tomkinsc opened this issue Apr 20, 2023 · 1 comment

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@tomkinsc
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A task, revcomp_i5, has been present for a while to reverse complement i5 (barcode_2) indices in sample sheet files. The illumina_demux functionality should detect when this is likely needed, and when it is the i5 indices in the supplied sample sheet(s) should be automatically rev-comp'ed before demux is run again with the modified sample sheets.

This should be possible by using the IlluminaBasecallsToSam debug options FIRST_TILE=1 and TILE_LIMIT=1 to restrict demultiplexing to a single tile, and then inspecting the count of reads in the demux metrics file written by picard: rev-comp i5 seqs if [(sum of counts in READS for samples) / (count of reads in "other" row, NNNNNNNNNN-NNNNNNNNNN)] > some low threshold.

This should have a guardrail to prevent an incorrect sample sheet from being rev-comp'ed and re-demuxed ad infinitum if it is incorrect for some other reason.

@tomkinsc
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We can also generally infer this by inspecting RunInfo.xml forIsReverseComplement="Y" for index 2. Example from a NextSeq 2000 RunInfo.xml:

<Reads>
    <Read Number="1" NumCycles="123" IsIndexedRead="N" IsReverseComplement="N"/>
    <Read Number="2" NumCycles="8" IsIndexedRead="Y" IsReverseComplement="N"/>
    <Read Number="3" NumCycles="8" IsIndexedRead="Y" IsReverseComplement="Y"/>
    <Read Number="4" NumCycles="123" IsIndexedRead="N" IsReverseComplement="N"/>
</Reads>

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