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Old 04-19-2016, 03:59 PM   #5
owik303
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Location: boulder, co

Join Date: Aug 2012
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mhgseq - we have resolved this issue greatly, I have demonstrated on the same library short reads cannibalizing up to 70%+ of the total reads form a 314, 316 and 318 chips even with deceased loading concentrations than recommended. The same library if heated to 95c for 5 min then slow cooled 0.1 degree / second to 37 (heat cooled as we call it) before being diluted and added to the ISP for emPCR dropped short reads to under 10% of the total. We believe this was due to aggeration of amplicons as our insert size is ~ 100 bases with ~25 bases of fixed primer region on both the 5' and 3' end. The reason this affects amplicon sequencing so much more is due to the majority of the library having identical fixed regions and limited diversity between them, which is not typically the case in genomic libraries. These aggerates carry through template dilution and emPCR seeding as its all done at 25c. This results in high polyclonal reads, high # of empty wells, high low quality reads and typically a larger # of what appear as truncated reads.
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