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-   -   Sample Prep for NGS Library (http://seqanswers.com/forums/showthread.php?t=22932)

addyblanch 08-30-2012 05:02 AM

Sample Prep for NGS Library
 
Hi Guys,

I'm new to all this and still struggling with some aspects and terminologies.

I preparing a library from Streptococcus with multi random inserts.

My plan was to fragment (Hydroshear to ~1.2kb), T4 ligate and inverse PCR amplify the inserts before preparing for Illumina (Miseq) library prep. I've now read something about GC bias that can be incorporated during PCR. As far as I understand this is normally down to the short PCR during Library prep, my question is could this also be a problem during my enrichment stage before library prep?

The GC content of my bug is ~36%.

I look forward to any input

Many thanks in advance
Adam

TonyBrooks 08-30-2012 05:07 AM

The Kapa Library Amplification is apparently better for GC/AT rich sequences when compared to the enzyme included in the Illumina/NEB kits (Phusion?). It's not something we looked at though, so you'll have to take their word.

http://www.kapabiosystems.com/public..._2011_Misc.pdf

addyblanch 08-30-2012 06:08 AM

do you think it would be worth using Kappa for the pre library enrichment?
Just to avoid any bias...


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