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Old 11-17-2011, 11:23 AM   #1
Simcom
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Location: East Coast US

Join Date: Jun 2010
Posts: 8
Default Sequencing a Low diversity library on the HiSeq

I am preparing a custom multiplexed library that will fall into the "low diversity" category. Low diversity meaning the first 5 nucleotides of read 1 will be identical among all clusters. There is a well known and well documented problem with cluster identification for low diversity libraries (outlined here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030592/ ).

The above paper and many of the comments on these forums refer specifically to the GAII, and suggest that spiking the library with 40-50% phiX control resolves the cluster calling issue.

Now that the GAIIx has been all but phased out, I need to run my low diversity library on the HiSeq. The problem is that I don't know of anyone that has successfully run a low diversity library on a HiSeq, and my core informed me today that they have tried several times to run low diversity libraries but got awful results on the HiSeq, even after spiking with phiX %50.

My question is, has anyone had success running low a diversity library on the HiSeq? If so, how did you manage to get it to work. Because my study does not require a massive number of reads, I am considering spiking my sample with up to 90-95% gDNA, hopefully drastically increasing the diversity and resolving cluster identification problems. Does anyone have experience running low diversity libraries on the HiSeq that could give me some advice?

Thanks so much!
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