Pooling of Nextera and NexteraXT libraries

[exo]

Hey all,

So i was thinking, can you actually pool both nextera and nexteraXT prepd libraries on the same cycling run?
I mean theoretically, this should be possible since both kits use the same indices and adapters (correct me if im wrong please ).
I think the only difference is that the NexteraXT is tailored for lower input and that is it - am i correct?

Let me know what you think. Im leaning towards trying it out this week.
Best,
EXo

[DRYTCYV]

Hi,
Since i don't use the beads normalization step in Nextera XT, I follow like a Nextera protocol. The difference its correlated with the genome complexity and size, p ex: Human genomes vs small and amplicons. And input sample.

[exo]

Yea, I also skip the beads normalization step in the NexteraXT protocol. But would pooling of library A (done with Nextera) and library B (done with NexteraXT) on the same seqeuncing run effect the demultiplexing of the run? Since in the sample sheet you have to chose either Nextera or xt..

[Simone78]

Several times I pooled Nextera and Nextera XT libraries and didn´t have any problem, but in both cases I started from full-length cDNA derived from single cells.

[DRYTCYV]

There's no problem with sample sheet, my last run I had 2 16S experiment and 2 regular nextera xt experiment, and I put everything like a Nextera XT assay.
In the end the only important thing its molarity.
This is the way I think..... if I'm not correct just say!

[exo]

Alright, perfect thanks for the input!

[Sergioo]

[QUOTE=exo;132982]
I mean theoretically, this should be possible since both kits use the same indices and adapters (correct me if im wrong please ).

I may be misreading your statement, but I don't think Nextera kit and Nextera XT index are interchangeable. They are different! I mistakenly once used Nextera's and Miseq failed even to start the 1st cycle of the run!
Otherwise, if libraries were prepared using appropriate index, I think they can be part of the same run.

[Simone78]

If you prepare your library with Nextera sample prep kit+ Nextera index kit (or the XT sample prep kit + XT index kit) and THEN you pool the samples there is absolutely no problem, at least on a HiSeq 2000.

[exo]

Alright perfect, and that would not interfere with the adaptor trimming option on the MiSeq right? Assuming that i select "nextera" in my sample sheet.

[Sergioo]

Alright perfect, and that would not interfere with the adaptor trimming option on the MiSeq right? Assuming that i select "nextera" in my sample sheet.

I would recommend you then to choose "Generate FASTQ" workflow and analyze data using a third-party software by yourself.
I have a small experience with CLC Genomic Workbench and on it I manually enter adapter sequences to be used later on when trimming my sequences. I want to point out that I have never pooled both libraries together, so if you try it, please share your experience with us.