bcl2fastq error: Barcode lengths in the sample sheet do not match those in --use-bases-mask
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4 weeks ago
coteparker • 0

I have 7 samples that were sequenced on Lane 1 of an Illumina NextSeq, 3 ATAC-seq samples and 4 CITE-seq samples. The samples run on this flowcell are all sequenced as 150 + 150 bases pair-end run with 10 + 16 nucleotide indexes. I used cellranger-atac (version 2.0.0) mkfastq to successfully demultiplex the ATAC-seq samples from the bcl files. I then attempted to use cellranger (version 6.0.1) mkfastq to demultiplex the CITE-seq samples from the same bcl files but unfortunately, I keep running into an error. The output redirects me to project_id/MAKE_FASTQS_CS/MAKE_FASTQS/BCL2FASTQ_WITH_SAMPLESHEET/fork0/chnk0-u468f869r48/_stderr to find the full error output. The following are the last two lines of the message describing the error.

INFO: Mask: Y150,I10,I16,Y150

ERROR: bcl2fastq::common::Exception: 2022-May-19 15:45:20: Success (0): /TeamCityBuildAgent/work/556afd631a5b66d8/src/cxx/lib/layout/Layout.cpp(419): Throw in function void bcl2fastq::layout::validateIndexLengths(const ReadMetadataContainer&, const std::vector<long unsigned int>&, bcl2fastq::layout::LaneInfo&) Dynamic exception type: boost::exception_detail::clone_impl<bcl2fastq::common::InputDataError> std::exception::what: Barcode lengths in the sample sheet do not match those in --use-bases-mask

Unless I misunderstand the format for the mask, I believe the mask used by bcl2fastq (version 2.20.0) is correct given the fact that all of the samples in the flowcell were sequenced as 150 + 150 bases paired-end run with 10 + 16 nucleotide indexes, as described earlier. Does anyone have any idea what is going on here and have any idea how I can resolve this issue? Thanks in advance!

cellranger bcl2fastq CITE mkfastq CITE-seq • 162 views
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Perhaps you missed this warning that 10x provides:

Do not pool Chromium Single Cell ATAC libraries with other 10x libraries while sequencing

What kind of NextSeq machine was this sequenced on?

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