SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Kapa library quantification martinjf Sample Prep / Library Generation 5 02-19-2015 12:08 AM
Library quantification suludana Illumina/Solexa 22 10-24-2013 03:52 PM
quantification of library maddalena Introductions 0 09-07-2012 01:00 AM
Problems with input DNA quantification skblazer Sample Prep / Library Generation 12 01-10-2012 07:49 AM
3'UTR library or random primed cDNA library for quantification? Rosanne82 Sample Prep / Library Generation 0 06-26-2009 05:27 AM

Reply
 
Thread Tools
Old 04-16-2019, 12:07 PM   #1
fishie
Junior Member
 
Location: Washington, DC

Join Date: Jul 2013
Posts: 3
Default problems with library quantification

Hi everyone,

I've been having issues with the quantification of my library pool, and I'm hoping you'll have some helpful suggestions on what is causing the problem.

My pool is 3.5 ng/uL based on HS Qubit. I ran a HS tapestation, and found an average fragment size of 364 bp with no adapter-dimer peak present. So my pool should be 14.6 nM. However, qPCR has consistently yielded results that are ~30 nM - double what I expected.

Since there's no adapter-dimer peak on the tapestation trace, any ideas what could be causing the problem?

The WGS libraries were KAPA Hyper preps with amplification, followed by a target enrichment with MyBaits. There was an additional amplification after the capture assay.

There is a double peak on my qPCR melt curve for this sample, but no evidence of adapter-dimer on the tapestation, so I'm not sure how much this relates to my quantification discrepancy.

Any input would be greatly appreciated! Thanks!
Attached Images
File Type: png meltcurve.png (94.8 KB, 11 views)
File Type: png tapestation.png (140.4 KB, 13 views)
fishie 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 05:46 PM.


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