Question: Demultiplex Novaseq run with mixed samples containing either UDI alone or UMI-UDI
0
gravatar for diwasri
28 days ago by
diwasri0
diwasri0 wrote:

I have the raw basecall files from a Novaseq run that was run using the XP workflow. In each of the lanes I have 8 samples with UDI only and 8 samples with xGen UMI-UDI. I need to demultiplex the samples and generate fastq files. The command I am trying to use is

/root/bin/bcl2fastq --input-dir /pathtoNovaseqRun/Data/Intensities/BaseCalls \
               --runfolder-dir /pathtoNovaseqRun/ \
               --output-dir /pathto/output/files \
               --sample-sheet /pathtoNovaseqRun/sample_sheet.csv \
               --stats-dir /pathtoNovaseqRun/Stats \
               --reports-dir /pathtoNovaseqRun/Reports \
               --use-bases-mask Y51,I8Y9,I8,Y51 \
               --mask-short-adapter-reads 0 \
               --create-fastq-for-index-reads \
               --ignore-missing-bcl \
               --no-lane-splitting

This gives me the following error

ERROR: bcl2fastq::common::Exception: 2020-Sep-23 21:29:20: Success (0): /root/bin/bcl2fastq/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

A small excerpt from the samplesheet is linked

Untitled
How can I demultiplex this set of data?

Thank you!!

demultiplexing rna-seq umi • 120 views
ADD COMMENTlink modified 28 days ago by genomax91k • written 28 days ago by diwasri0
1
gravatar for genomax
28 days ago by
genomax91k
United States
genomax91k wrote:

You have to use a specific method for IDT xGen UDI-UMI data demultiplexing. It is described in this tech note. You can create a samplesheet for the UDI only samples and demultiplex them separately.

ADD COMMENTlink modified 28 days ago • written 28 days ago by genomax91k

Thank you. So, to clarify, I will need to have separate sample sheets for the samples with UDI only and for those with the xGen UDI-UMI and then process them separately.

For the UDI only samples, can I use the code originally mentioned with the exception of the --use-bases-mask changed to Y51,I8,I8,Y51

ADD REPLYlink written 28 days ago by diwasri0

You should use Y51,I8n*,I8,Y51. Assuming index 1 has been sequenced to 17 bp,

ADD REPLYlink written 28 days ago by genomax91k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1314 users visited in the last hour