Question: How to demultiplex dual-index NextSeq data for random index 2?
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gravatar for heir_of_isildur88
2.7 years ago by
heir_of_isildur8830 wrote:

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.

sequencing next-gen • 1.9k views
ADD COMMENTlink written 2.7 years ago by heir_of_isildur8830

Did you ever find a way to do this ?

ADD REPLYlink written 2.6 years ago by moranr270

How many unique indexes do you expect in second position?

ADD REPLYlink written 2.6 years ago by genomax89k

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

ADD REPLYlink written 2.6 years ago by moranr270

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 )

ADD REPLYlink modified 2.6 years ago • written 2.6 years ago by genomax89k

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

ADD REPLYlink written 2.6 years ago by moranr270

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

ADD REPLYlink written 2.6 years ago by genomax89k

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

ADD REPLYlink written 2.6 years ago by moranr270
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