I have a question here and I could not find any sufficient information about this. Hope someone is able to help.
I have a set of NextSeq data sequenced pair-end, dual-indexed whereby the index2 is a 8bp random index. The cycle run is 76, 8, 8 and 76 for R1, I1, I2, R2 respectively.
I wanted to demultiplex my reads according to sample by index 1 then from there, demultiplex the reads in each sample by index 2.
I tried various options as described below but to no avail, could anyone please help me out?
1) bcl2fastq using --use-bases-mask Y, I8, I8, Y and sample sheet containing info on I2 as NNNNNNNN
--- with this I got all my reads as Undetermined reads and I could not separate them by index 1
2) bcl2fastq using --use-bases-mask Y, I8, Y8, Y and sample sheet does not contain info on I2
--- I was hoping this could work but all i got for R2 (which I presume would be my index 2 in this case) are all Ns
Please advice on how I can demultiplex my reads by 1st index then 2nd index. Thank you very much.