Hello, I have an experiment with 20 over lapping long range PCR fragments that span a single gene. The average size is 5kb (max 10, min 1.5kb) with equimolar pooling the balance between fragments is poor, we have seen this with a number of samples and the pattern is consistent, some fragments have a few hundred fold coverage, others >100,000x (we're aiming for deep coverage over all amplicons)
Using our results from equimolar pooling we increased the proportion of template for weak fragments but the pattern of high/low coverage between fragments is exactly the same?
Is anyone having better results pooling PCR for NGS? and why would one PCR fragment be more successful than another when they are sheared together?! (GC content is not significantly different).
JPC
Using our results from equimolar pooling we increased the proportion of template for weak fragments but the pattern of high/low coverage between fragments is exactly the same?
Is anyone having better results pooling PCR for NGS? and why would one PCR fragment be more successful than another when they are sheared together?! (GC content is not significantly different).
JPC
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