Poly-G Failed Runs on NextSeq

[RickC7]

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...

[Johnwang]

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.

[Yepler]

Originally Posted by Johnwang

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

[RickC7]

Thanks, I will give it a try.

Where did you have your primer synthesized?

[Yepler]

Originally Posted by RickC7

Thanks, I will give it a try.

Where did you have your primer synthesized?

I went through IDT. There's a little bit of lead time, since the LNA bases go through Exiqon - if you have a direct account with Exiqon already, that may save you some time.

[RickC7]

So you did something like this? Thanks so much for your help!

ACAGGTTCAGAG+TTC+TAC+AGTCCGACGATC

[Lei Hua]

Originally Posted by djs150

Approximately a month ago, our lab performed an RNASeq using a TruSeq Stranded mRNA library prep and a v2 75cyc High Output flowcell. The data that came back was almost entirely G's and was severely underclustered. When we went to re-run the same library, the machine died and would soon thereafter require all of the boards to be replaced.

We assumed that the poly-G run was an early symptom of the board failure, until we tried to run a genomic library prepared with Nextera DNA reagents on v2 300cyc High Output flowcell and again got nearly all G's (also very underclustered). There were no shared reagents between the two library preps and our lab has performed many of each type of prep without failure.

Thinking that it was a hardware issues still, we did a phiX run on a 300cyc Mid Output flowcell... which of course came out perfect. Illumina is of the opinion that it is a transient issue and that we should just continue trying, but there's got to be more to it than that. Have any of you run into this problem before or have thoughts on what may be going on?

Thank you very much for putting up this thread.
I recently encounter similar problem on NextSeq platform, but in my situation, the beginning 20-bp of all my reads looks fine, but after that, it all G bases, in all lanes. I also have extremely low cluster PF (~0.3%), but the cluster density is ok (~220 K/mm2).

I used Nugen Ovation DR rapid system to prepare my libraries, used Qiagen PCR purification kits to clean up my libraries. I used standard seq primers, no custom seq primers were used.

Illumina technical support suggested to remake library, but I was following the standard protocol of Nugen, I am afraid the remade library would not be different with the original one.

Have you sorted out the problem? What could be wrong with lib. prep.?

[RickC7]

The primer with LNA bases added to cartridge like custom primer fixed our issue. Thanks for the help!