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

Similar Threads
Thread Thread Starter Forum Replies Last Post
Indexing with bowtie2 luana Bioinformatics 4 05-04-2016 10:30 AM
Indexing with bfast runnerBio88 Bioinformatics 1 12-11-2015 01:05 AM
TruSeq Indexing felvis56 Illumina/Solexa 3 04-05-2013 09:32 AM
BFAST indexing phatjoe Bioinformatics 1 09-08-2011 07:39 PM
tophat indexing madsaan Bioinformatics 0 01-17-2011 01:13 PM

Thread Tools
Old 09-19-2016, 10:28 AM   #1
Junior Member
Location: San Diego, CA

Join Date: Aug 2016
Posts: 3
Default Indexing QC distribution

Hi all,

First thread here! My question involves optimizing indexing QC using the Nextera XT library prep kit for DNAseq via Miseq. I'm wondering if anyone can suggest a method for tightening up my indexing QC distribution. I have seen up to 30% CV for samples normalized and run using equal load factors. My goal is to optimize indexing QC distribution to my calculated loading fraction to ensure sufficient coverage on my libraries.

For reference, I perform a picogreen quant for input material, prepare the libraries, and validate fragment size and sample concentration using LabChip. I then normalize to 4nM using those values and proceed with DAL prep. My first thought was that my Caliper reagents or chip were old and generating inaccurate quantification, but I am still experiencing the same issues with a new chip and reagents.

Any ideas are appreciated!
evan_seq24 is offline   Reply With Quote
Old 09-20-2016, 12:19 AM   #2
Location: Montpellier (France)

Join Date: May 2008
Posts: 93

In my opinion, 30% CV is not that bad.
The problem is that clustering efficiency is not only depending on library concentration. It also depends on library size as there is a clustering bias toward smaller fragments. So even if you quantify your libraries accurately and mix them in equimolar (or so) proportion, you will face some variations in your final sequence equilibrium as your library profiles are differents.
You could add a size selection step but even if it improves your sequence number equilibirum, it may also reduce you library diversity. This last point can be an issue if you are working on complex/large genomes.
huguesparri is offline   Reply With Quote
Old 09-20-2016, 02:08 PM   #3
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238

Two points:
1- your quantification method accuracy is low and variability high so you need to use qPCR if you want more accurate quantification
2- library fragment size distribution as was pointed
nucacidhunter is offline   Reply With Quote
Old 09-21-2016, 12:28 PM   #4
Junior Member
Location: San Diego, CA

Join Date: Aug 2016
Posts: 3

Thank you both for the thoughtful responses. Looking at my data, I do see a direct correlation between fragment size and clustering efficiency. I appreciate it!
evan_seq24 is offline   Reply With Quote

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 04:02 PM.

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