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Old 08-30-2012, 05:02 AM   #1
addyblanch
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Lightbulb 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
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Old 08-30-2012, 05:07 AM   #2
TonyBrooks
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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
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Old 08-30-2012, 06:08 AM   #3
addyblanch
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do you think it would be worth using Kappa for the pre library enrichment?
Just to avoid any bias...
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gc content, illumina, library prep, miseq

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