SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > 454 Pyrosequencing

Similar Threads
Thread Thread Starter Forum Replies Last Post
Lots of false positives with SAMtools SNP discovery mikael Genomic Resequencing 4 02-28-2011 07:38 AM
Did Solid change their primer system? SongLi Bioinformatics 1 12-20-2010 04:56 PM
Going from biotinylated primer to unlabeled primer in emPCR chunnan20451 454 Pyrosequencing 0 09-01-2010 06:22 PM
CPB correction with emPCR format change 454User 454 Pyrosequencing 4 07-20-2010 10:49 AM
Primer extension capture tiayyba Sample Prep / Library Generation 3 03-28-2010 07:34 AM

Reply
 
Thread Tools
Old 07-06-2012, 09:48 AM   #1
bradfish
Member
 
Location: Colorado

Join Date: Jul 2012
Posts: 20
Default Issue with recent change to capture beads and amplification primer in new emPCR lots

We just tried our first run with the new lot emPCR kits in which Roche has included a note that states the capture beads were changed and the amplification primer concentration was changed. This was supposed to increase uniformity in the emPCR amplification. These changes began with LOT# 93880860.

What we experienced: Increased number of short quality failures, basically cutting our passed filter wells in half. Nothing else seemed abnormal, aside from getting about 1,000,000 enriched beads rather than the 4-600k we usually get.

Has anyone else tried these new lots and experienced anything like this? I am assuming they increased primer concentration.'

Thanks!
bradfish is offline   Reply With Quote
Old 07-09-2012, 12:15 AM   #2
RCJK
Senior Member
 
Location: Australia

Join Date: May 2009
Posts: 155
Default

The changes are described briefly in Tech bulletin 2012-007. It does say that the changes can increase the chance of amplifying short fragments, such as primer dimers. For the FLX these changes start with lot 93880220. I assume I'll get one of these lots with my next order.
RCJK is offline   Reply With Quote
Old 07-09-2012, 07:39 AM   #3
bradfish
Member
 
Location: Colorado

Join Date: Jul 2012
Posts: 20
Default

Yes we read the tech bulletin and thought we should be okay as we use a Lonza flash gel system to purify amplicon of any primer dimers and short fragments. I suppose there could be some getting through but this seems more like an issue with the reagents, not my purification technique, we sequenced the day before this run using the old, unmodified lots and did not have nearly as many short quality failures. The histrogram indicates there were not short reads in the run using the new reagents as well.

Would like to hear experiences with these new reagents if uyou guys get them.
bradfish is offline   Reply With Quote
Old 07-17-2012, 08:31 AM   #4
Anthony.287
Member
 
Location: Ohio

Join Date: Dec 2010
Posts: 95
Default

Does anybody have any more info on these new reagents? How enrichment percentages compare, for example?
Anthony.287 is offline   Reply With Quote
Old 07-17-2012, 12:57 PM   #5
bradfish
Member
 
Location: Colorado

Join Date: Jul 2012
Posts: 20
Default

We just ran 2 more pools of amplicon last night for emPCR. Broke and enriched this morning, ended up with about 3x enriched beads that we usually get (up to 1.5 million enriched from our usual 500k).

Will let you know how sequencing turns out tomorrow. I would expect that we get a high number of short quality filter trims again.

J
bradfish is offline   Reply With Quote
Old 07-17-2012, 12:58 PM   #6
bradfish
Member
 
Location: Colorado

Join Date: Jul 2012
Posts: 20
Default

Quote:
Originally Posted by bradfish View Post
We just ran 2 more pools of amplicon last night for emPCR. Broke and enriched this morning, ended up with about 3x enriched beads that we usually get (up to 1.5 million enriched from our usual 500k).

Will let you know how sequencing turns out tomorrow. I would expect that we get a high number of short quality filter trims again.

J
These were both using the new lots, obviously.

ANYONE ELSE?! Somebody has to be using these new lots
bradfish is offline   Reply With Quote
Old 07-17-2012, 01:05 PM   #7
Anthony.287
Member
 
Location: Ohio

Join Date: Dec 2010
Posts: 95
Default

Would you be able to provide cpb ratios and enrichment percentages comparing the old lots to the new? I know that would help me out!
I had to go up to 32 cpb to get 7.7% last week, (with the older lots) and now I have the new lots and I have no idea where to start..
Anthony.287 is offline   Reply With Quote
Old 07-17-2012, 01:10 PM   #8
bradfish
Member
 
Location: Colorado

Join Date: Jul 2012
Posts: 20
Default

Right now I don't have time to post a lot of data, but I can tell you we are doing 1 cpb with new and old lots. Sequencing 400 bp amplicon, with 1/4 the amount of reccomended amp primer. We went from getting 400-600k enrichement consistenly for the last year to over 1 million on the last few runs with nothing changing except the lots.
bradfish is offline   Reply With Quote
Old 07-17-2012, 01:13 PM   #9
Anthony.287
Member
 
Location: Ohio

Join Date: Dec 2010
Posts: 95
Default

Which kits are you using?
Anthony.287 is offline   Reply With Quote
Old 07-17-2012, 01:15 PM   #10
bradfish
Member
 
Location: Colorado

Join Date: Jul 2012
Posts: 20
Default

I'm using the rapid library Lib-A kit, on GS Junior. I know the concentrations changed similarly for FLX sequencing too, as this was included on the tech bulletin. See above post for the actual lot number.
bradfish is offline   Reply With Quote
Old 07-17-2012, 01:18 PM   #11
Anthony.287
Member
 
Location: Ohio

Join Date: Dec 2010
Posts: 95
Default

So you've gone from ~10% enrichment to 20-30%. Interesting.
Anthony.287 is offline   Reply With Quote
Old 07-17-2012, 01:39 PM   #12
bradfish
Member
 
Location: Colorado

Join Date: Jul 2012
Posts: 20
Default

Quote:
Originally Posted by Anthony.287 View Post
So you've gone from ~10% enrichment to 20-30%. Interesting.
Thats true, but its a low quality enrichment. My overall passed filter reads have been reduced in half from 100k to 50k, because of short quality failures.
bradfish is offline   Reply With Quote
Old 07-17-2012, 01:48 PM   #13
Anthony.287
Member
 
Location: Ohio

Join Date: Dec 2010
Posts: 95
Default

I wonder if there are actually short fragments, or if it's one of the other parameters that is included in the short quality filter..I'm not familiar with the Jr, but this was a common problem with the FLX.
Anthony.287 is offline   Reply With Quote
Old 07-18-2012, 08:06 AM   #14
bradfish
Member
 
Location: Colorado

Join Date: Jul 2012
Posts: 20
Default

Actually had a decent run yesterday, given the huge enrichment. 90k reads, only 88k short quality. Maybe it was a fluke? Running another tonight, this will sway me one way or another.
bradfish is offline   Reply With Quote
Old 07-18-2012, 01:01 PM   #15
bradfish
Member
 
Location: Colorado

Join Date: Jul 2012
Posts: 20
Default

Quote:
Originally Posted by A_Big_Dope View Post
My group actually figured out that if you don't add any of the emPCR enzyme mix, we would get excellent sequencing data. We dun' get it but we keep on keepin' on ya know?
I don't know whether to laugh or cry about this post. And yes, you certainly are a big dope.
bradfish is offline   Reply With Quote
Old 07-18-2012, 01:08 PM   #16
adaptivegenome
Super Moderator
 
Location: US

Join Date: Nov 2009
Posts: 437
Default

Quote:
Originally Posted by bradfish View Post
I don't know whether to laugh or cry about this post. And yes, you certainly are a big dope.
Posting garbage once is possibly funny, posting it more than once is spam.

Last edited by adaptivegenome; 07-18-2012 at 01:11 PM. Reason: typo
adaptivegenome is offline   Reply With Quote
Old 07-18-2012, 01:11 PM   #17
ECO
--Site Admin--
 
Location: SF Bay Area, CA, USA

Join Date: Oct 2007
Posts: 1,358
Default

Let's keep it relatively serious and professional, Dope. Thanks.
ECO is offline   Reply With Quote
Old 07-26-2012, 01:54 AM   #18
GraemeFox
Member
 
Location: Manchester

Join Date: Oct 2011
Posts: 14
Default

I enquired to Roche about just how the concentration of the primer in the emPCR kits had been changed and I have just had a response.

"The concentration of the primer in the kit was actually deceased (sic) by about 4 fold."
GraemeFox is offline   Reply With Quote
Old 07-26-2012, 07:00 AM   #19
Aniki
Member
 
Location: Toronto

Join Date: Mar 2011
Posts: 39
Default

so....

I am having one heck of a time with old emPCR kits. I am having an enrichment nightmare where I simply cannot get any freaking enriched beads out of this (extremely low %, regardless of CPB). I hope these new kits can do better for me but you guys aren't exactly giving me confidence.
Aniki is offline   Reply With Quote
Old 07-28-2012, 02:38 PM   #20
DNA_Dan
Member
 
Location: Montana

Join Date: Nov 2008
Posts: 21
Default

Quote:
Originally Posted by bradfish View Post
Right now I don't have time to post a lot of data, but I can tell you we are doing 1 cpb with new and old lots. Sequencing 400 bp amplicon, with 1/4 the amount of reccomended amp primer. We went from getting 400-600k enrichement consistenly for the last year to over 1 million on the last few runs with nothing changing except the lots.
We are doing the same except using Lib-L kits on a FLX in LV format. Key is reducing that primer amount. Also I think if you are sequencing all the same amplicon, you have higher signal inteference and this gets filtered out in post processing as a short or failed read. The passing filter rate drops significantly the less unique the sequencing pool is.

As for the increasing enrichments with the newer kits, we haven't really noticed this, however our enrichments are higher on plates that sit for a while after the final emulsion shake. We find that these "false" higher enrichments tend to sequence poorly. Same CPB, Same reagents, just the 1st cup sitting out longer while the second is being aliquoted = poorer sequence. As a result, we have gone to processing one LV cup at a time, putting them on the thermalcycler ASAP once aliquoted. Processing the cups this way is much more consistent, albeit more time consuming.
DNA_Dan 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 09:44 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO