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Old 01-04-2017, 01:42 PM   #21
RickC7
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Thanks for the info. I am not using a custom primer, but using custom SAGE libraries. I've modified a SuperSAGE protocol from Matsumura which incorporates Illumina adapters, so just load the library pool as a standard Illumina library, no custom sequencing primer. Even more concerning is the fact that the run QC looks great and the poly-G reads were only discovered after detecting a high number of unaligned reads when mapping to refseq. I plan to load again this week with a lower cluster density to see how that looks. I was at around 420million reads, but as stated, run metrics were great. This was even our first run with the FAS there, he saw the run metrics, we high-fived, then we found the Poly-G reads when going into the analysis...
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Old 01-05-2017, 08:17 AM   #22
Johnwang
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Default same issue with our run

Hi Yepler,
Great point!! It will be great if you provide more details on your custom primers ? such as length and Tm. By the way, did you use IDT web based tool to calculate your Tm ?

Thank you.
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Old 01-05-2017, 11:12 AM   #23
Yepler
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Quote:
Originally Posted by Johnwang View Post
Hi Yepler,
Great point!! It will be great if you provide more details on your custom primers ? such as length and Tm. By the way, did you use IDT web based tool to calculate your Tm ?

Thank you.
http://seqanswers.com/forums/showthread.php?t=71254 has some details.

I'm not familiar with SuperSAGE, but I took a quick look at the M&M from a paper, and I think we may have the same issue.

I don't technically have a "custom" primer, either - I'm using the Small RNA Sequencing Primer, which is Illumina's and is even in the NextSeq kit. It just doesn't work very well on the NextSeq! As far as I can tell from the Matsumura protocol, you are also using this primer. If that's the case, you can use the trick in the post I linked to to correct the issue.

Cheers-
Yepler

Last edited by Yepler; 01-05-2017 at 11:20 AM.
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