Multiplexing using Nextera chemistry

[exo]

Hey Guys,

Just let me start by saying that this forum has been great support for me, been crawling here after midnight for some tips and tricks

I have a concern though, and i would really appreciate your input! I am planning a Nextera run using 10 samples on a miseq. I already have the library ready for each of the sample, which have been uniquely tagged. My concern is, I am now at the pooling part of my protocol, and i am not really sure when do i pool my samples or at what concentration! I mean, I performed a run before but i had only 3 samples so i actually was able to treat every library as a monoplex and use sufficient volumes of HT1 for diluting it down, but now with 10 samples this seems impossible - at least for a dummy like me. My thought was to actually pool the samples once i dilute them down to 4nM or 2nM would that be an option? Keeping in mind that i would like to load my samples using different "inputs" (loading 25% of the plate with one sample, and dividing the remaining between the others).

Another thing, once the library is frozen down directly after resuspension using the RSB buffer / how long is it stable for?

Looking forward for your input,
E

[barrmur]

Easiest thing to do would be to dilute all your samples to a standard concentration and then pool equal amount accordingly. I have done this with 96 sample plenty of times. It takes time to do it but the results are worth it. QPCR is best for quantification a but there are lots of other ways. You will never get the pools exactly right but you should be fine.

As for library storage, we are on 5 years and counting for some, and they sequence fine.

[exo]

Thank you for your reply!
I ran 1:3 dilution aliquotes of my libraries on a HS DNA Chip, before and after fragmentation. Seems like i have fragments ranging from 350bp to 1500bp, with a peak at 1kb (see attached). In their technical note, they mention that this kind of library is sequence-able but if we use 12pM library?

Has anyone tried sequencing such quality libraries and was happy with the result? I am only performing resequencing, so reads will be re-aligned to the reference - so i wouldnt really care about overlapping reads.

Best,
E