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Old 07-12-2016, 10:08 PM   #3
Location: college station

Join Date: Jan 2012
Posts: 22

Dear Adam, thank you for the suggestions.
Originally Posted by adam.geber View Post
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?
I always get a peak at around 303bp after PCR, regardless of what size selection I tried before PCR. The first figure shows the fragmented DNA (without adapters), while the second one includes adapters (~150bp in my case). So the size discrepancy is not explained by the sizing precision of the D5000 assay. Now I'm wondering if the primers have non-specific bindings to the genomic DNA, so it always amplifies the same peak. I will try to use raw gDNA for a PCR to find out.

Originally Posted by adam.geber View Post
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.
We are not using the KAPA kit, but only the KAPA Hifi premix for the PCR step. I have read through the guide, doesn't seem to talk much about size distribution bias during PCR or the ways to mitigate the effect. It has some guides on bead size selection, which is something I'm already doing. It suggests that getting the fragment size distribution correct from the beginning is important since the bead size selection doesn't produce a tight range.

Originally Posted by adam.geber View Post
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?
Yes I have tried the double cut SPRI selection. The post-PCR size selection isn't very helpful, it shifts the peak to the right by ~30bp (330bp), but due to the much higher peak in the small size range in the PCR product, it doesn't generate my desired the insert size (350-650bp fragment, 200-500bp insert). This is why I try to optimize the distribution after PCR to make sure that it's as close to the target size as possible.
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