Illumina BS-seq library prep query

matth431 · 04-27-2012, 08:16 AM

Apologies if this has been answered elsewhere, but I've had a glance through the threads and can't see anything obvious.

I've noted that Illumina's TruSeq kits are incompatible with BS-seq at present because the polymerase mix cannot tolerate uracil. My query is how much of an issue this is to sort out? Is it just a case of finding a different Taq for the library amplification (KAPA do one that seems suitable), or does this also affect the polymerases used to conduct end-repair? What about the polymerases in the SBS chemistry? We'd be wanting to prepare these for a run on a HiScanSQ.

Thanks,

Matt

frozenlyse · 04-29-2012, 11:43 PM

Publications eg http://www.ncbi.nlm.nih.gov/pubmed/19829295 generally use PhuTurboCx for the library prep PCR - as this replaces the uracils with thymidines the enzyme used in SBS, though I assume there are some tricks with basecalling etc bisulfite-seq due to the base composition imbalance - an experienced service provider should be fine with this though.

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04-27-2012, 08:16 AM	#1
matth431 Member Location: UK Join Date: Jan 2012 Posts: 22	Illumina BS-seq library prep query Apologies if this has been answered elsewhere, but I've had a glance through the threads and can't see anything obvious. I've noted that Illumina's TruSeq kits are incompatible with BS-seq at present because the polymerase mix cannot tolerate uracil. My query is how much of an issue this is to sort out? Is it just a case of finding a different Taq for the library amplification (KAPA do one that seems suitable), or does this also affect the polymerases used to conduct end-repair? What about the polymerases in the SBS chemistry? We'd be wanting to prepare these for a run on a HiScanSQ. Thanks, Matt

04-29-2012, 11:43 PM	#2
frozenlyse Senior Member Location: Australia Join Date: Sep 2008 Posts: 105	Publications eg http://www.ncbi.nlm.nih.gov/pubmed/19829295 generally use PhuTurboCx for the library prep PCR - as this replaces the uracils with thymidines the enzyme used in SBS, though I assume there are some tricks with basecalling etc bisulfite-seq due to the base composition imbalance - an experienced service provider should be fine with this though.