[nucacidhunter]

So the problem is in the library prep, somewhere. The only common reagent is the Omega Bio-tek beads from the PCR clean up. Is there any rationale that the beads could be causing this problem somehow?

I wonder if this is the first time that you have used this brand or batch of bead.

[djs150]

All of the reagents and procedures have been used in identical fashions for many months without issue. Regarding the beads specifically, we moved to this brand after having issues with Agencourt. It also didn't hurt that they were cheaper.

I agree with the idea that the adapters are not attaching to the reads, I'm going to run with that hypothesis and see where I end up.

[nucacidhunter]

To identify adapter ligation issues for RNA-Seq library you can do qPCR quantification and compare results to Qubit. If adapters have not been ligated then qPCR quantification should be substantially less than Qubit.

Although you have not run Nextera library for the second time but I doubt that they will have adapter issue because without addition of adapter sequences it will be impossible to PCR amplify those libraries.

[RickC7]

Similar but different issue on Next-Seq.

I am of course screwed b/c I'm using a custom Tag-sequencing protocol, no custom primers as we have Gex adapters. Lanes 1 and 3 look great, but lanes 2 and 4 contain a high number of poly-G reads. Do a phix run and everything looks good. Have not repeated the custom libraries a 2nd time, but a smaller sample subset did sequence fine on our Mi-Seq. I don't understand how the cluster is ID if there is no signal? The run is a short 51bp read and I have reads that are all G's, like 100 million of these reads.

Illumina actually mentioned they are aware of lane-lane variation when sequencing some custom libraries and that since the library is not validated and PhiX works, basically too bad. That the libraries are sequencing fine in lane 1 and 3 should be enough validation. I just feel something else is at work and not library issue in our case.

Anyone see similar when sequencing custom libraries on the Next-Seq?

EDIT: Quantified using Kapa qPCR for Illumina, got great cluster density using the concentration obtained.

[Yepler]

Similar but different issue on Next-Seq.

Lanes 1 and 3 look great, but lanes 2 and 4 contain a high number of poly-G reads. Do a phix run and everything looks good. Have not repeated the custom libraries a 2nd time, but a smaller sample subset did sequence fine on our Mi-Seq. I don't understand how the cluster is ID if there is no signal? The run is a short 51bp read and I have reads that are all G's, like 100 million of these reads.

Illumina actually mentioned they are aware of lane-lane variation when sequencing some custom libraries and that since the library is not validated and PhiX works, basically too bad. That the libraries are sequencing fine in lane 1 and 3 should be enough validation. I just feel something else is at work and not library issue in our case.

Anyone see similar when sequencing custom libraries on the Next-Seq?
.

Yep. I had the exact same issue (two lanes okay, two lanes pretty much all poly-G) when using a custom sequencing primer on the NextSeq. The NS is really sensitive to primer Tm. Are you using a custom sequencing primer? Do you know what the Tm of your primer is, and are you able to raise it? I ended up adding some LNA bases to mine (couldn't increase the length) and that fixed the problem. I'd be happy to talk in more detail if you like, just send a message.

[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]

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.

[Yepler]

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]

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]

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!