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Old 07-12-2016, 04:38 PM   #2
adam.geber
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Join Date: May 2014
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1) The D5000 assay has 10% sizing accuracy, which is within the range of the fragment sizes you're seeing. Have you seen a size discrepancy for your libraries across multiple preps?

2) PCR bias towards smaller fragments is definitely a factor, although this can be partially mitigated by increasing your extension time. I'm assuming that you're using a KAPA prep kit -- have you looked in their technical guide for size distribution optimization? They have some good figures showing the the effects of different size selection methods and some recommendations that seem quite relevant to your question.

3) Finally, the libraries you show are post-PCR amplification and pre-cleanup, correct? Why not just do an additional SPRI-based size selection after your PCR enrichment?

Last edited by adam.geber; 07-12-2016 at 08:33 PM.
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