SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
double 18S peak on Bioanalyzer crimor Sample Prep / Library Generation 3 02-13-2015 11:54 AM
RNA-seq libraries - double peak after size selection MLog Sample Prep / Library Generation 23 03-08-2012 10:22 AM
Truseq RNA library has 2 peak on bioanalyzer houda Sample Prep / Library Generation 7 07-06-2011 10:39 AM
Truseq library has dimer/trimer peak on bioanalyzer after amplfication sehrrot Sample Prep / Library Generation 4 06-15-2011 03:38 PM
TruSeq libraries - double sizing? jlove Illumina/Solexa 2 04-22-2011 12:34 PM

Reply
 
Thread Tools
Old 11-02-2011, 04:18 PM   #1
ssing
Member
 
Location: usa

Join Date: Jan 2009
Posts: 20
Default double peak in mRNA truseq kit

Hi all,

I have been doing some mRNA TruSeq sample prep, using the protocol *exactly* as advised by the Illumina folk (using PolyT beads to get mRNA, fragmenting for 7 minutes chemically, 15 PCR cycles), and I am getting an odd and consistent pattern where I have my desired peak ~300 bp and a second peak ~900 bp. See an exemplar trace attached.

Reading through the other posts, seems like most people think this due to overamplification... could that be my issue here? My biggest concern is (because the double peak is similar in size ratio to the rRNA size ratio) that this reflects rRNA contamination. Does anyone have experience with that?

Thanks!
Attached Files
File Type: pdf bioaz_double.pdf (136.7 KB, 892 views)
ssing is offline   Reply With Quote
Old 11-02-2011, 07:17 PM   #2
Prosuite
Junior Member
 
Location: Canada

Join Date: Feb 2011
Posts: 3
Default

The extra peak is due to SPRI bead carryover from post-PCR clean-up step. Use a
strong magnet for bead separation and pipette carefully during
elution to avoid disturbing beads. It should not affect the sequencing run.
Attached Files
File Type: pdf Bead_carryover.pdf (32.1 KB, 644 views)
Prosuite is offline   Reply With Quote
Old 11-07-2011, 08:38 AM   #3
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default

Hi Prosuite,
That is very interesting. Where did you find that document from Agilent? I was just looking at the instrument troubleshooting guide this morning and there was no mention of SPRI/AMPure beads.

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-07-2011, 09:39 AM   #4
Prosuite
Junior Member
 
Location: Canada

Join Date: Feb 2011
Posts: 3
Default

It is from Agilent eSeminar "BioAnalyzer Applications for Next Gen Sequencing : Updates and Tips-20110301". Here is the link: https://agilenteseminar.webex.com/ag...114f10d5514079
Prosuite is offline   Reply With Quote
Old 11-07-2011, 10:15 AM   #5
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default

Never seen this before. Interesting. Anyone buy the "ligase remaining attached to library molecule" issue presented on page 26?

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-07-2011, 10:19 AM   #6
ETHANol
Senior Member
 
Location: Western Australia

Join Date: Feb 2010
Posts: 308
Default

Delete what I wrote. I didn't read yours carefully enough. I doubt it is an over amplification issue because that peak usually is 2x the size of the correct peak.
__________________
--------------
Ethan

Last edited by ETHANol; 11-07-2011 at 10:24 AM.
ETHANol is offline   Reply With Quote
Old 11-07-2011, 12:30 PM   #7
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default

Quote:
Originally Posted by ETHANol View Post
Delete what I wrote. I didn't read yours carefully enough. I doubt it is an over amplification issue because that peak usually is 2x the size of the correct peak.
Did you check out the Prosuite attachment? Some info from Agilent claiming that SPRI beads can cause that result. (I have see it -- a peak much larger than one would expect from multimers/heterodimers/bubble amplicons, etc.)

If so had you ever seen that info? Be nice if Agilent provided that as part of their normal troubleshooting manual...

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-07-2011, 04:54 PM   #8
ssing
Member
 
Location: usa

Join Date: Jan 2009
Posts: 20
Default

When I contacted Illumina, they gave me three possible explanations:
1. Incomplete fragmentation (which seems unlikely, given that I was using RNA within the range of what the recommended and a long fragmentation time)
2. Over-amplification (which I also doubted because I didn't do a ridiculous number of cycles)
3. Bead carryover (which made the most sense as this was a consistent pattern)

If it is bead carryover, when I run a normal gel of the cleaned product, I shouldn't see that smear anymore. I'm going to do that tomorrow. I'll report back what I see. Here's hoping that is what it is, because otherwise, they are recommending I do a size selection for all my samples...

Thanks for all the feedback.
ssing is offline   Reply With Quote
Old 11-07-2011, 05:03 PM   #9
ECO
--Site Admin--
 
Location: SF Bay Area, CA, USA

Join Date: Oct 2007
Posts: 1,358
Default

Quote:
Originally Posted by pmiguel View Post
Never seen this before. Interesting. Anyone buy the "ligase remaining attached to library molecule" issue presented on page 26?
Chiming in with two things from my experience that may be relevant...not commenting on "page 26" because I can't see where you're referring to that....

1. Intact ligase has a strong protective effect against any treatments with exonuclease. Denature the ligase or strip out the Mg++ and you're good to go.

2. The bioanalyzer (particularly the high sensitivity kit) can be used as a gel-shift assay to detect bound proteins (DNA polymerases in my experience)...it wouldn't surprise me in the slightest that active ligase causes a shift on the bioanalyzer....in fact that's probably why ILMN adds "stop solution" to their ligations.
ECO is offline   Reply With Quote
Old 11-08-2011, 04:27 AM   #10
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default Bound ligase shifts Illumina libraries sizes?

Quote:
Originally Posted by ECO View Post
Chiming in with two things from my experience that may be relevant...not commenting on "page 26" because I can't see where you're referring to that....
Sorry, Prosuite posted a link to the presentation in question above. You can skip to page 26 fairly easily. You do have to register so Agilent can spam you -- but I would say the information in the presentation is worth a little spam to anyone that uses the BioAnalyzer much.

The slide in question shows two electropherograms of an Illumina library after adapter ligation and PippinPrep size selection. One PippenPrepped after a proteinase K treatment, one without treatment. The peak with treatment is 368 bp. The one without is 291 bp.

Actually this addresses one of those perennial Illumina library prep questions that shows up on SeqAnswers. But I have never seen ligase binding suggested as the cause.

Quote:
Originally Posted by ECO View Post
1. Intact ligase has a strong protective effect against any treatments with exonuclease. Denature the ligase or strip out the Mg++ and you're good to go.

2. The bioanalyzer (particularly the high sensitivity kit) can be used as a gel-shift assay to detect bound proteins (DNA polymerases in my experience)...it wouldn't surprise me in the slightest that active ligase causes a shift on the bioanalyzer....in fact that's probably why ILMN adds "stop solution" to their ligations.
Good to know.

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-08-2011, 11:14 AM   #11
ssing
Member
 
Location: usa

Join Date: Jan 2009
Posts: 20
Default

In fact, this does not appear to be due to beads. I am not sure what the peak is, but I am going to size select ... trivial exercise and worth ensuring good cluster generation.
ssing is offline   Reply With Quote
Old 11-08-2011, 11:44 AM   #12
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default

So you saw the same phenomenon on your agarose gel? Any chance you could post that?

You might want to strand denature the sample (94 oC for 2-4 minutes followed by "snap cool" on ice) and run it on an RNA chip.

You can get double peak artifacts in TruSeq libraries that end up causing no real issues when clustered. Normally these peaks are closer in size to one another than the image you give above, though.

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-08-2011, 05:01 PM   #13
ssing
Member
 
Location: usa

Join Date: Jan 2009
Posts: 20
Default

Hi Philip,

See attached. The ladder is a 100 bp ladder (NEB).
Attached Images
File Type: jpg illuminaGel.jpg (16.7 KB, 262 views)
ssing is offline   Reply With Quote
Old 11-09-2011, 03:57 AM   #14
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default

(The upper loading dye is really distracting. Xylene cyanol?) Is this TBE EtBr? How many cycles of enrichment PCR did you do?

Anyway, yes there is a high molecular weight band. No harm in doing a size selection. Actually we nearly always size select (after pooling most or all of the indexes that will go in a lane). I doubt the high MW stuff will cause any issues, though.

Still, mysterious. Wonder if it could be the polymerase still bound to some of the molecules. (See ECO's comments above...)

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-09-2011, 03:55 PM   #15
ssing
Member
 
Location: usa

Join Date: Jan 2009
Posts: 20
Default

This is TAE EtBr and I did 15 cycles of enrichment PCR.

Not sure what this is, but I went ahead and size selected. I'll let you all know when I get my data...hopefully this is just a weird artifact and doesn't suggest anything went wrong with my library prep.
ssing is offline   Reply With Quote
Old 11-14-2011, 04:48 PM   #16
ssing
Member
 
Location: usa

Join Date: Jan 2009
Posts: 20
Default

So, I ended up size-selecting my libraries, and then I used Bioanalyzer to look at the curve -- and it was exactly the same. Again, the desired peak at 300 bp and the higher MW peak centered around 1400 bp. So, I took Philip's suggestion (seen on other posts) to heat up the library to 95 degrees, cool slowly and then ran on an agarose gel (we don't have a Bioanalyzer in house...), and finally, I only saw one band at exactly the desired size. Not sure why my bubble products/concatenamers/daisy chains (whatever they are) have such a different pattern from others seen on this forum, but there you go. I guess the denaturing during quantification via qPCR and during the actual sequencing should mean these products will have no downstream effects.

Submitting for sequencing tomorrow, so hopefully I'll have good results to report back to you in a month or so!
ssing is offline   Reply With Quote
Old 01-10-2012, 08:53 AM   #17
ssing
Member
 
Location: usa

Join Date: Jan 2009
Posts: 20
Default

Just wanted to follow up and say that I got good quality sequences at good yields on the HiSeq. Our sequencing facility head said he had seen similar bioanalyzer profiles with the TruSeq kit -- why is unclear -- but the results are fine, nonetheless.

Thanks for all your help!
ssing is offline   Reply With Quote
Old 03-16-2019, 01:00 AM   #18
KB*
Member
 
Location: UK

Join Date: May 2018
Posts: 20
Question also have HMW tail

Hi ssing,
Are you still around? I have samples looking similar to yours (check the image). I am preparing libraries on ChIPed DNA and all my IPs and IgG IPs have this tail. Only input does not have it. Input was amplified in a different PCR machine and had 3 less cycles. But the initial concentration of my input DNA was 2.5-5 times more than that of IP samples.

I did not experiment with the "stock" libraries, but I diluted them to ~100-300 pg/uL (100 ul total). I tried separation on the magnet; re-size-select; spin - nothing changed. Just like in your case..... well may be my libraries were too diluted for second run with Ampure beads??

You said you denatured the library at 95 oC and then cooled slowly. What do you mean "slowly"? I would like to try that as well. As I understand that was just a test. Or did you denatured all of your libraries before submission? I need to pool the libraries together before submission. If you did denaturation for your libraries, shall I denature them before pooling? I guess the concentration stays the same?

The sequencing facility proposed to do a trail on MiSeq first and then to sequence on NovaSeq. Huh.. that is the huge overpay They gave the quote for all the samples. I guess if I do miSeq trail I only shall do one sample although not sure it is possible...
Attached Images
File Type: jpg Qabtlib.jpg (73.1 KB, 12 views)
KB* 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 06:44 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO