SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Library preparation Renatospl Sample Prep / Library Generation 1 09-19-2011 01:50 PM
Fragment library preparation for SOLiD 4.0 nedoluzhko Sample Prep / Library Generation 0 03-23-2011 04:05 AM
illumina library preparation and multiplexing jgibbons1 Illumina/Solexa 9 03-01-2010 12:29 AM
library preparation cost Tom Haltern Sample Prep / Library Generation 6 02-22-2010 07:22 AM
Problems with Globin genes in mRNAseq paired-end library preparation div1982 Illumina/Solexa 4 08-25-2009 03:46 AM

Reply
 
Thread Tools
Old 06-19-2012, 03:02 AM   #1
Xavi_
Junior Member
 
Location: Valencia

Join Date: Jun 2012
Posts: 1
Post Multiplexed amplicon library preparation: problems and considerations

Hello,

Iím about starting a sequencing experiment with a HiScan SQ.
The point is that we have to prepare the library from multiplexed fragments, varying from 200 to 800 bp, but we are trying to find out how to have a final DNA input where all the amplicons got an equimolecular representation in order to provide a good number of reads and coverage at the end.

Does anyone know how to normalize the effect of mixes with different number of amplicons and concentration? Or this isnít that important as far as you keep your samples in a good average amplified range?

We tried to get them at the same concentration but it has been impossible. We will have to amplify some of them even in uniplex.

Our aim is to identify SNPs and we are afraid we introduce a bias already before starting the library prep that could decrease the final quality during the analysis.
We usually work with NEBnext and TrueSeq DNA kits and fragment by Covaris method.

I would appreciate very much every help or experience you could provide.

Best regards,
Xavi
Xavi_ is offline   Reply With Quote
Old 06-21-2012, 06:17 AM   #2
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
Default

Ostensibly you can do qPCR titration of each individual library then pool based on your results. We use KAPA (SYBR-green based) kits for this as most do, so we have to adjust our molarities to account for the difference in signal produced by longer amplicons. So you would also need to verify the average size of your amplicons using a gel or Bioanalyzer run. Alternatively you could just design a Taqman assay instead and avoid the need to adjust for amplicon lengths.

As far as pooling amplicons of different lengths in the same lane -- it may not be possible to get even representation of these. From recent results we found when assessing the lengths of inserts of single library pools of a wide size range, it looks like there is heavy bias towards shorter amplicons in any given pool -- even after correcting estimates of molarity for length. Almost like the shorter amplicons reach the flowcell first and longer ones are left trying to find a "seat" among those few remaining. On molecular level, I don't see how this could be the case, though.

My gut feel is that if you had a pool that was 50% 200 bp molecules and 50% 800 bp molecules, >90% of your resulting reads would be from the 200 bp molecules. By far, your best bet would be to pool all the 200 bp molecules in one lane, and the 800 bp molecule in another.

--
Phillip
pmiguel is offline   Reply With Quote
Reply

Tags
equimolar ratios, illumina, library prep, multiplex

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 11:46 PM.


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