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Thread | Thread Starter | Forum | Replies | Last Post |
Failed NextSeq 500 v2 runs w/ custom sequencing primer | kakseq | Illumina/Solexa | 4 | 10-05-2016 05:25 PM |
Poly AT-stretches in ChIP-seq reads from NextSeq | Maflores11 | Sample Prep / Library Generation | 4 | 07-25-2016 12:45 AM |
poly-G in NextSeq | Asaf | Illumina/Solexa | 9 | 10-29-2015 01:08 AM |
MiSeq runs failed "focus out of spec" | thermophile | Illumina/Solexa | 6 | 09-23-2015 06:25 AM |
NextSeq Data | Brian Bushnell | Illumina/Solexa | 7 | 07-22-2015 01:48 PM |
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#21 |
Member
Location: Baton Rouge, Louisiana Join Date: Feb 2010
Posts: 28
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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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#22 |
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Location: NE, USA Join Date: Jun 2016
Posts: 14
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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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#23 | |
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Location: Tucson, AZ, USA Join Date: Oct 2010
Posts: 22
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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 10:20 AM. |
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#24 |
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Location: Baton Rouge, Louisiana Join Date: Feb 2010
Posts: 28
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Thanks, I will give it a try.
Where did you have your primer synthesized? Last edited by RickC7; 01-19-2017 at 09:34 AM. |
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#25 |
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Location: Tucson, AZ, USA Join Date: Oct 2010
Posts: 22
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#26 |
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Location: Baton Rouge, Louisiana Join Date: Feb 2010
Posts: 28
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So you did something like this? Thanks so much for your help!
ACAGGTTCAGAG+TTC+TAC+AGTCCGACGATC |
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#27 | |
Junior Member
Location: Cambridge Join Date: Sep 2016
Posts: 1
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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.? |
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#28 |
Member
Location: Baton Rouge, Louisiana Join Date: Feb 2010
Posts: 28
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The primer with LNA bases added to cartridge like custom primer fixed our issue. Thanks for the help!
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