Mixing pooled libraries at the same time

[namtran93]

Hello everyone,

I am a new member of SEQanswers. I am planning to run MiniSeq system for the first time. Unluckily, I have a big problem. My pooled library is a combination of 2 kinds of library, which were prepared by 2 library preps kits. The first one is Nextera XT and the second one is NEB Ultra II. The problem is that they have different lengths in index sequences. Nextera XT indexes contain 8 nucleotides, meanwhile NEB Ultra II was designed with only 6 nucleotide. How can I run those libraries at the same time, on the same flow cell. Please help me!!!

[nucacidhunter]

Easiest way is to add two bases to NEB index from adapter sequences and run indexing read for 8 cycles. If you are able to use bcl2fastq software you can also demultiplex at two stages once for Nextera index and once for NEB by masking cycle 7 and 8 of index.

[namtran93]

Re:nucacidhunter

Easiest way is to add two bases to NEB index from adapter sequences and run indexing read for 8 cycles. If you are able to use bcl2fastq software you can also demultiplex at two stages once for Nextera index and once for NEB by masking cycle 7 and 8 of index.

Thank you for you ideal. As your suggestion, the bases of "the modified indexes" are identical. Is it possible? The expert of Illumina told me that in particular cycle (used to read indexes), the signal must be different at least 1 of total indexes.

[nucacidhunter]

Yu need to write down Nextera indexes and NEB ones with two additional bases from adapter and look at nucleotide diversity in each position of 8 cycles. Ideally you should have different nucleotides in each position but even if you have at least one of each bases for green laser (G, T) and red laser (A, C) in each cycle it should be fine. Example (N:A, C, G, T):

N1N2N3N4N5N6N7N8
N1N2N3N4N5N6N7N8
N1N2N3N4N5N6N7N8
N1N2N3N4N5N6N7N8

If N7 and N8 are identical bases you have the option of demultiplexing using 6 index bases if they are divergent enough.

[namtran93]

Yu need to write down Nextera indexes and NEB ones with two additional bases from adapter and look at nucleotide diversity in each position of 8 cycles. Ideally you should have different nucleotides in each position but even if you have at least one of each bases for green laser (G, T) and red laser (A, C) in each cycle it should be fine. Example (N:A, C, G, T):

N1N2N3N4N5N6N7N8
N1N2N3N4N5N6N7N8
N1N2N3N4N5N6N7N8
N1N2N3N4N5N6N7N8

If N7 and N8 are identical bases you have the option of demultiplexing using 6 index bases if they are divergent enough.

Thanks for your reply. I will design my custom indexes as your suggestion. I hope everything will be good.