SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
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

Reply
 
Thread Tools
Old 01-04-2017, 12:42 PM   #21
RickC7
Member
 
Location: Baton Rouge, Louisiana

Join Date: Feb 2010
Posts: 20
Default

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...
RickC7 is offline   Reply With Quote
Old 01-05-2017, 07:17 AM   #22
Johnwang
Member
 
Location: NE, USA

Join Date: Jun 2016
Posts: 14
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.
Johnwang is offline   Reply With Quote
Old 01-05-2017, 10:12 AM   #23
Yepler
Member
 
Location: Tucson, AZ, USA

Join Date: Oct 2010
Posts: 22
Default

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 10:20 AM.
Yepler is offline   Reply With Quote
Old 01-19-2017, 09:16 AM   #24
RickC7
Member
 
Location: Baton Rouge, Louisiana

Join Date: Feb 2010
Posts: 20
Default

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.
RickC7 is offline   Reply With Quote
Old 01-19-2017, 11:05 AM   #25
Yepler
Member
 
Location: Tucson, AZ, USA

Join Date: Oct 2010
Posts: 22
Default

Quote:
Originally Posted by RickC7 View Post
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.
Yepler is offline   Reply With Quote
Old 01-19-2017, 11:36 AM   #26
RickC7
Member
 
Location: Baton Rouge, Louisiana

Join Date: Feb 2010
Posts: 20
Default

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

ACAGGTTCAGAG+TTC+TAC+AGTCCGACGATC
RickC7 is offline   Reply With Quote
Old 01-27-2017, 06:17 AM   #27
Lei Hua
Junior Member
 
Location: Cambridge

Join Date: Sep 2016
Posts: 1
Default

Quote:
Originally Posted by djs150 View Post
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.?
Lei Hua is offline   Reply With Quote
Old 02-17-2017, 07:07 AM   #28
RickC7
Member
 
Location: Baton Rouge, Louisiana

Join Date: Feb 2010
Posts: 20
Default

The primer with LNA bases added to cartridge like custom primer fixed our issue. Thanks for the help!
RickC7 is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 06:27 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO