Hey guys,
We want to sequence P. falciparum (23 Mb) on a MiSeq with Nextera XT library prep. I have not worked with falciparum before but literature (see link) says that its extremely AT-biased genome makes library prep difficult because the Illumina polymerase does not effectively amplify AT-rich regions which results in uneven coverage. They suggest adding TMAC to the PCR and using Kapa Hifi as polymerase. However the paper is from 2012 and they did not use the Nextera Kit.
So my question is, has anyone experience with sequencing falciparum and/or has experience with tweaking the Nextera Kit.
Thanks a lot for your help.
Hi Martin,
Kapa HiFi polymerase should help. We had tried TMAC few years back (I dont remember the exact concentration) and it did improve the yields post enrichment PCR. We also used Kapa HiFi polymerase later and it did give better yields. We were using TruSeq LT DNA kits for the prep at that time. We didn't test both Kapa HiFi and TMAC in the same prep. For sequencing we tried to keep enough base diversity (have samples with normal GC content multiplexed with it) else P.Falciparum sequences used to decay in quality much before end of read.
Regards,
Rushiraj
Dear Rushiraj,
thanks a lot for your detailed answer. I will give the Kapa Hyper Plus Kit a go and try to spike in ~10% PhiX as well. I'll post the result here.
Kind regards,
Martin
Dear Martin,
I am very interested in the Kapa Hyper Plus Kit. DId you try it? Did it worked?
Also I normally used Kapa HIFI and I wonder whether adding TMAC makes any substantial difference in terms of reducing AT bias in Plasmodium.
Thanks!!
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