Considerations in performing whole human genome bisulfite sequencing on the Illumina NovaSeq system

Today at the NGS workshop at WEHI, Melbourne, I presented some findings related a pilot study of 12 methylomes studied with whole genome bisulfite sequencing. Two of those libraries were also sequenced on the HiSeq4000 platform to similar depth so there were some subtle but interesting differences between the systems. What we found was that the actual sequence coverage obtained was substantially less than that projected due to 2 problems. Firstly that the insert size was too small - which looks like it could be due to the inner workings of the Illumina TruSeq methylation kit. And secondly that there was a high proportion of duplicate reads observed - that is same strand and coordinates which are likely not independent observations. I will need to look into further detail at whether these are PCR duplicates or "cluster" duplicates. Perhaps the library prep or clustering protocols need some tweaking for bisulfite sequencing.

So as promised, here is the link to the slides.


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