Demultiplexing same barcodes for different lanes

Question

[gessthomsxavier7]

I'm trying to demultiplex an illumina Novaseq XP worflow S2 flowcell run with a samplesheet in which same barcodes are present for samples in different lanes an example

Sample_ID,Sample_Name,Sample_Plate,Sample_Well,Index_Plate_Well,I7_Index_ID,index,I5_Index_ID,index2,Sample_Project,Description
LIB423,Sample1,A01,D701,ATTACTCG,D501,TATAGCCT,,Lane1
LIB503,Sample20,A01,D701,ATTACTCG,D501,TATAGCCT,,Lane2

using

bcl2fastq -R /mnt/NovaNAS/NovaSeq/190428_A00452_0029_BHJWFFDMXX -p 56 -o . -l TRACE

as expected bcl2fastq gave barcode collision error

2019-04-30 06:07:30 [1360880] ERROR: bcl2fastq::common::Exception: 2019-Apr-30 06:07:30: Success (0): /TeamCityBuildAgent/work/556afd631a5b66d8/src/cxx/lib/layout/BarcodeCollisionDetector.cpp(187): Throw in function void bcl2fastq::layout::BarcodeCollisionDetector::handleCollision(const value_type&, const value_type&)
Dynamic exception type: boost::exception_detail::clone_impl<bcl2fastq::layout::BarcodeCollisionError>
std::exception::what: Identical barcode found for multiple samples in the sample sheet: ATTACTCG+TATAGCCT

Is there any way to demultiplex using this samplesheet or should I create seperate samplesheet and run twice.

[ngocttb]

i have a same problem, do you fix it?

Answer 1

[Devon Ryan]

You appear to have put two samples on the same lane with the same barcode. I have no idea how you could expect that to work, but you'll have to delete one and accept that they'll be mixed together. That's unless the Lane1 and Lane2 aren't meant to be lanes, in which case they should be in the first column ("Lane") as 1 and 2. Your sheet is generally malformed and I'm surprised that bcl2fastq was able to salvage as much as it did. In general, don't make these sheets by hand unless you really know what you're doing, use IEM.

Answer 2

[GenoMax]

illumina Novaseq XP worflow S2 flowcell run

This is the critical bit. It is an XP flowcell which allows one to have lanes. You just need to have your samplesheet contain these columns

Lane,Sample_ID,Sample_Name,Sample_Plate,Sample_Well,I7_Index_ID,index,I5_Index_ID,index2,Sample_Project,Description
1,LIB423,......
2,LIB423,.....

If you wish to get a single file per sample then use --no-lane-splitting option.