How to demultiplex dual-index NextSeq data for random index 2?

Question

[heir_of_isildur88]

Hi all,

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?

Options tried:

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.

[moranr]

Did you ever find a way to do this ?

[GenoMax]

How many unique indexes do you expect in second position?

[moranr]

Index_2 has 4 unique indexes as 4 samples are Dual index and 4 have a single index.

[GenoMax]

Easiest solution for you may be to run bcl2fastq twice. See Demultiplex dual index NextSeq run with only one barcode (this also may be useful: Demultiplexing reads from two libraries (single and dual indices) that were sequenced in the same sequencing run )

[moranr]

Thanks Genomax , i tried both post's suggestions and still not working for me. I'll explore more thanks.

[GenoMax]

Can you tell us what is not working and we can try to help?

[moranr]

Thanks, Created a question :How can I demultiplex two Library preps(Single Index and Dual Index) using bcl2fastq.