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Old 05-11-2018, 02:56 AM   #1
filipeuit
Junior Member
 
Location: Tromsų

Join Date: Apr 2018
Posts: 2
Lightbulb Multiplexing BluePippin size selection

First post, but a longtime lurker!

Currently doing Illumina library preps for MiSeq sequencing, and even though we have mostly used TruSeq kits, we are now trying to develop a cheaper "in-house" alternative.
To that effect, we want to start using our BluePippin to do size selection (aiming for 500-600 bp inserts).

Since we work with >48 sample libraries, we were considering if it would be possible to multiplex samples in a BP cassette (e.g. 5 multiplexes of 10 samples each). That would mean size selecting only after adapter ligation and PCR enrichment. However, this poses a lot of problems, as far as I can tell.

First and foremost, will it work? Will it yield enough ligated DNA?
And then, it would mean that we would have to multiplex the samples twice. Once to run the BP and another time to sequence. At which point would I be able to run a qPCR with the libraries?
I have more doubts regarding this method, and even though I am willing to try to optimise and validate it, I was hoping I could start a discussion here about the potential positive aspects of it and any pitfalls that I might encounter.
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