SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Bridge amplification and fragment size BTS Illumina/Solexa 18 10-25-2016 07:27 AM
Insert size != Fragment size? Boel Bioinformatics 6 12-12-2013 08:28 AM
What fragment size do you use for your Illumina libraries? kmcarr Sample Prep / Library Generation 13 06-16-2013 04:24 PM
Fragment size limit Gina_P Sample Prep / Library Generation 11 01-06-2012 03:04 PM
Fragment Size shift on Egel bye using the new TruSeq protocol ? Claire1 Sample Prep / Library Generation 2 05-05-2011 12:37 PM

Reply
 
Thread Tools
Old 05-05-2011, 05:36 AM   #1
Claire1
Junior Member
 
Location: Spain

Join Date: Apr 2011
Posts: 6
Default Fragment Size shift on Egel bye using the new TrueSeq protocol ?

I am using the TruSeq protocols for RNA and DNA and I follow exactly the protocol, except Iím using an Egel (Egel: Invitrogen ĎSize Selectí 2%, Ladder: Invitrogen 100bp, 20ng/ul) before PCR instead of an Agarose Gel.
If I take my fragment size I want to use e.g. 600bp for my PCR and I run a Bioanalyzer after I see fragments around 500bp. So I have always to repeat my PCR's taking fragments around 700bp or higher to get fragments around 600bp.
This phenomenon I can see for DNA and RNA.

Anybody knows this problem?
Should I use a different Ladder?
Is the new adapter changing the migrating in the Egel?

thanks a lot for your help!
Claire1 is offline   Reply With Quote
Old 05-05-2011, 08:01 AM   #2
JHU-ChIPmaniac
Member
 
Location: Baltimore

Join Date: Jul 2010
Posts: 21
Default

Are you using the Egel specific ladders from Invitrogen? I use the 50 bp Egel ladder for my size selection of ChIP products and the isolated fragments correlate well with what we see on the bioanalyzer. I'ts possible that the regular size ladders don't run true to size on the Egels.
JHU-ChIPmaniac is offline   Reply With Quote
Old 05-05-2011, 11:44 PM   #3
Claire1
Junior Member
 
Location: Spain

Join Date: Apr 2011
Posts: 6
Default Fragment Size shift on Egel bye using the new TrueSeq protocol ?

I'm using the 100 bp DNA Ladder from Invitrogen (Cat. 15628-050), 1ug/ul diluted to 20ng/ul.
And until now this Ladder was working fine with the old Sample Prep protocol from Illumina.
Since I switched to the new TruSeq protocol from Illumina I have problems.
Are you using the new TruSeq protocol?

Thanks for your help!
Claire1 is offline   Reply With Quote
Old 05-07-2011, 06:07 AM   #4
ECO
--Site Admin--
 
Location: SF Bay Area, CA, USA

Join Date: Oct 2007
Posts: 1,358
Default

Quote:
Originally Posted by Claire1 View Post
Is the new adapter changing the migrating in the Egel?
Almost certainly, the bioanalyzer is very sensitive to ssDNA regions. Are these anomalies pre- or post-PCR ?
ECO is offline   Reply With Quote
Old 05-09-2011, 03:51 AM   #5
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

ECO,
The E-gel would be run prior to PCR, the bioanalyzer after. So your take, if I understand you, is that the Y-adapters are causing migration artifacts on the E-gels. Making the fragments appear 100 bp longer than they actually are. PCR converts the Y-ends to normal double-stranded adapter ends.

I guess if the size shift is consistent Claire1 could just always extract fragments 100 bp longer than the intended size range.

We have only just started making Illumina libraries. We did see some fairly crazy size changes at various steps in the procedure during the first round of library constructions using the TruSeq kits.

For the second set of libraries we tried doing the size selection after PCR, instead of before. This was mainly because we wanted to use our new Pippin prep system and the input amount called for by its protocols could not be met without doing the PCR step first. This worked, but the yields were much lower (approaching 10x lower).

Actually, when I saw the initial result--size prior to PCR substantially larger than after PCR--I was worried that PCR was selectively amplifying a small component of the total library and that we would end up bottle-necking the library. But I don't see lots of PCR duplicates in the resulting alignments, so I think this is not what happened.

Maybe it is the E-gels that are very sensitive to the single stranded regions in pre-PCR library amplicons?

--
Phillip
pmiguel is offline   Reply With Quote
Old 05-09-2011, 05:54 AM   #6
JHU-ChIPmaniac
Member
 
Location: Baltimore

Join Date: Jul 2010
Posts: 21
Default

Quote:
Originally Posted by Claire1 View Post
I'm using the 100 bp DNA Ladder from Invitrogen (Cat. 15628-050), 1ug/ul diluted to 20ng/ul.
And until now this Ladder was working fine with the old Sample Prep protocol from Illumina.
Since I switched to the new TruSeq protocol from Illumina I have problems.
Are you using the new TruSeq protocol?

Thanks for your help!
I'm not yet using the TruSeq protocol but plan on it in the future. I'll have to keep all this in mind.
JHU-ChIPmaniac is offline   Reply With Quote
Old 05-11-2011, 06:48 AM   #7
Claire1
Junior Member
 
Location: Spain

Join Date: Apr 2011
Posts: 6
Default

Even if I extract fragments 100bp higher on the Egel, I don't find the right fragment size immediately, as the ladder runs differently than the Samples.

thank you for all your answers!


Claire
Claire1 is offline   Reply With Quote
Old 05-11-2011, 07:01 AM   #8
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 508
Default

The TruSeq library prep protocol indicates that the new adapters cause anomalous size migration during electrophoresis either with or without ethidium bromide in the gel. It recommends using Sybr Gold staining to correct this problem.
HESmith 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 03:59 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO