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Old 08-26-2013, 02:04 PM   #1
days369
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Location: US

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Question High proportion of PolyT reads

Hi everyone,

I recently sequenced several cDNA libraries on HiSeq platform. But all the libraries ended up with more than 50% of polyT reads and not a lot of polyA reads. This phenomena made me very confused. Given that my libraries were supposed to be double-stranded, shouldn't I expect an equal proportion of polyT and polyA reads in one lane? On the other hand, although those samples's RNAs were expected to have some polyA sequences, it should not have such a high proportion of polyA or T based on my previous sequencing results.

I am wondering if anyone had similar experience before or have any insight to share with me.

For your information, I started with fragmented cDNAs and constructed libraries using TruSeq Nano DNA kit. One thing I did not exactly follow the protocol was that I amplified the libraries by 14 PCR cycles instead of 8 cycles. I determined that 14 cycles were necessary based on a PCR titration. Other than that, I did the same as the protocol suggested.

Dezhi
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