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Old 05-11-2018, 02:56 AM   #1
filipeuit
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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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Old 05-11-2018, 04:47 AM   #2
gabrieltw
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Check out

doi:10.1038/nprot.2018.019
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Old 05-13-2018, 11:04 PM   #3
filipeuit
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Quote:
Originally Posted by gabrieltw View Post
Check out

doi:10.1038/nprot.2018.019
Thank you very much, Gabriel. That is a very thorough protocol, that I was not aware existed.

EDIT: Do you think molarity differences between libraries will be an issue? If you run a TruSeq library prep protocol, and you start with e.g. 1 ug of sheared DNA per sample, you will end up with libraries that have different molarities. Before pooling libraries prior to sequencing, we usually QC through Picogreen/Qubit and qPCR so we can dilute and pool samples with the same (or similar) molarity. Would it be important to qPCR the samples prior to pooling before BP size selection?

Last edited by filipeuit; 05-14-2018 at 01:24 AM. Reason: Question pertinent to pooling of libraries
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Old 05-14-2018, 03:39 AM   #4
gabrieltw
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Look up the part that use limited primers to normalize libraries in the paper. After the limited primer normalization, most of the libraries should be around the same concentration. Basically steps are :

Library prep -> QC -> PCR enrichment with limited primer -> pooling -> size select -> pooled libraries loading.
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