bcl2fastq with unique dual indexing

Question

[ginlucks]

Hello, I am trying demultiplexing and converting to fastq illumina sequencing with bcl2fastq or spaceranger, but I am retrieving only 2 big fastq files named "Undetermined" (R1 R2) and other fastq very small.

This is part of the SampleSheet.csv file I am using

[Data]
Sample_ID,Sample_Name,I7_Index_ID,index,I5_Index_ID2,index2
268-PP,268-PP,i7_UDIP01,TGGATCGA,i5_UDIP01,ACCTAGCT
268-PPA,268-PPA,i7_UDIP02,CAAGCTAG,i5_UDIP02,GTTCGATC
268-PFR_2,268-PFR,i7_UDIP03,GTACCAAG,i5_UDIP03,CATGGTTC
268-PFR,268-PFR,i7_UDIP05,TCGTGGTA,i5_UDIP05,AGCACCAT
268-PAN_,268-PAN,i7_UDIP04,ACGTTGGA,i5_UDIP04,TGCAACCT
268-PL,268-PL,i7_UDIP06,CTACAACG,i5_UDIP06,GATGTTGC
268-BP,268-BP,i7_UDIP07,GTTCAAGG,i5_UDIP07,CAAGTTCC
268-PAN,268-PAN,i7_UDIP08,ACCTGGAA,i5_UDIP08,TGGACCTT
736-PV,736-PV,i7_UDIP09,TCTTAGCG,i5_UDIP09,AGAATCGC
736-PG,736-PG,i7_UDIP10,CTCCGATA,i5_UDIP10,GAGGCTAT

and this is the command

bcl2fastq --runfolder-dir ./IlluminaRuns \
--sample-sheet ./IlluminaRuns/UDI_adapters.csv \
--output-dir ./IlluminaRuns/demultiplexed \
-r 20 -p 20 -w 20 --create-fastq-for-index-reads

Any idea about what could be the problem?

Answer 1

[GenoMax]

In cases like this more often than not your samplesheet contains wrong indexes, especially in terms of them being reverse complement of what sequencer sees. You can use the code in my answer here: Demultiplexing reads with index present in the labels

Once you figure out the combinations you need, edit the samplesheet and re-demultiplex.

[ginlucks]

Hi, thanks for answering. I did the reverse complement of I5 index (just a simple R script) because I saw that in the resulting undetermined fastq that only one index (I7) could be found...but I did not know if it was correct. Now I am more confident with that. Thanks!