Advice needed for --use-bases-mask option (bcl2fastq)

[lfaller]

Hi all,

A colleague in the lab asked me to demultiplex a recent NextSeq run. She loaded it with samples prepared from two libraries. One library had single indices and one had dual indices. She also prepared two sample sheets for me to use.

I was able to use the sample sheet for the single-index samples successfully but running bcl2fastq again, this time using the sample sheet for the dual-index samples, did not work (all reads ended up in two large "Undetermined" fastq files).

Someone suggested I use the --use-bases-mask option for bcl2fastq but I am not entirely sure how to set it up.

The information in the RunInfo.xml file is as follows:

...
    <Reads>
      <Read Number="1" NumCycles="147" IsIndexedRead="N" />
      <Read Number="2" NumCycles="12" IsIndexedRead="Y" />
      <Read Number="3" NumCycles="12" IsIndexedRead="Y" />
      <Read Number="4" NumCycles="147" IsIndexedRead="N" />
    </Reads>
    <FlowcellLayout LaneCount="4" SurfaceCount="2" SwathCount="3" TileCount="12" SectionPerLane="3" LanePerSection="2">
      <TileSet TileNamingConvention="FiveDigit">
        <Tiles>
          <Tile>1_11101</Tile>
          <Tile>1_21101</Tile>
...

The indices my colleague gave me in the sample sheet are eight bases long so I tried this approach:

--use-bases-mask Y147,I8nnnn,I8nnnn,Y147

However, as before, all reads ended up in the Undetermined files.

My questions:

1) Is using --use-bases-mask the best approach?
2) I read somewhere on here that the sample sheets can be combined so that bcl2fastq only needs to be run once -- is that a preferable approach?

By the way, for all of this I am using bcl2fastq v2.17.1.14.

Thank you in advance, any advice would be appreciated!

[fanli]

Originally Posted by lfaller

--use-bases-mask Y147,I8nnnn,I8nnnn,Y147

However, as before, all reads ended up in the Undetermined files.

Maybe try looking in the index read files and see if the 8-base barcodes are indeed at the beginning of the index reads.

--create-fastq-for-index-reads

Originally Posted by lfaller

1) Is using --use-bases-mask the best approach?

Seems appropriate to me.

[kcchan]

Did you remember to reverse complement the index2s?

[lfaller]

Thank you for suggestion!

I went back to the lab tech and asked her to take a critical look at the indices. She regenerated the file using the Illumina Experiment Manager and now it worked. I believe she messed up the file she gave me the first time around...