SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
How to get the median of read distribution in a genomic region? jajclement Bioinformatics 2 05-17-2016 12:53 PM
Read distribution at high sequence depth ForeignMan General 10 05-26-2011 03:50 AM
RNA-seq read distribution wenhuang RNA Sequencing 9 11-08-2010 05:00 PM
rna-seq read distribution wenhuang Bioinformatics 1 06-17-2010 09:07 AM
(a) Background Read Distribution (b) Spike-ins ANJAN PURKAYASTHA Bioinformatics 0 03-05-2009 07:02 AM

Reply
 
Thread Tools
Old 09-27-2017, 06:01 AM   #1
RickC7
Member
 
Location: Baton Rouge, Louisiana

Join Date: Feb 2010
Posts: 30
Default Index Read distribution

Hello,

I'm looking for advice, help, thoughts, etc on how to achieve more balanced read output when multiplexing. Typically I run all libraries on Agilent Bioanalyzer and pool according to the library peak in equimolar amounts. My last TruSeq mRNA seq project had between 2% and 8% reads for each sample, which varied read output 12 to 48million reads per sample. I see variations also when doing 16s(Schloss barcodes). I just wondering if variation is typical, some barcodes may amplify better than others, etc?? Do most labs see more even read distribution or is it more likely to "do the best you can and hope the outcome is acceptable"?

I'd be interested in any success stories using normalization protocols, plates, beads etc for library pooling.

Thanks for any help!

Last edited by RickC7; 09-27-2017 at 07:06 AM.
RickC7 is offline   Reply With Quote
Old 09-27-2017, 01:25 PM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,172
Default

The most accurate method for library quantification is qPCR.
nucacidhunter is offline   Reply With Quote
Old 09-27-2017, 01:47 PM   #3
RickC7
Member
 
Location: Baton Rouge, Louisiana

Join Date: Feb 2010
Posts: 30
Default

Thanks, I get that and have no problem when loading a sequencer for optimal cluster density. I'm also not going to quantify 192 16s libraries via qPCR for multiple projects. I more looking to get better/ more even read distribution for multiplexed library pools.
RickC7 is offline   Reply With Quote
Old 09-27-2017, 02:09 PM   #4
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,172
Default

For amplicons such as 16S that has been prepped using the same protocol, pooling based on dsDNA specific reagents such as PicoGreen results in up to 3x variation in output which is similar to bead or plate based normalisation methods.
nucacidhunter is offline   Reply With Quote
Old 06-11-2018, 01:18 PM   #5
DNA_Dan
Member
 
Location: Montana

Join Date: Nov 2008
Posts: 21
Default

Old post I know, sorry to be late to the party, but if you're still around RickC7, I'd like to see if you made any inroads on the normalization front. It seems like labs doing a lot of samples use the Nextera normalization method using beads and denaturing the library to get fragments off the bead. I am trying to create something similar for Truseq without denaturing.
DNA_Dan is offline   Reply With Quote
Old 06-13-2018, 10:14 AM   #6
RickC7
Member
 
Location: Baton Rouge, Louisiana

Join Date: Feb 2010
Posts: 30
Default

I haven't yet tried bead based normalization, but I have tried some plate normalization methods like SequalPrep. It did not work well in our hands. For larger sample numbers, like when we are doing 16s, we now use fluoro-plate reader, then create a normalization plate template for our EPMotion. This works best, still get a few outliers but not bad. I randomly check a few samples to make sure unincorporated primers and such are removed.

For smaller projects <24 samples, I just use qubit/bioanalyzer. Calculate and dilute all libraries down to 4nM then pool. I get better read distribution and cluster density using qubit/bioanalyzer than qPCR. I can easliy squeeze out 600million PF reads on our Next-Seq at 85% PF and 85% Q30. My %Phix loading is usually close to being spot-on, so that tells me my quantification is accurate.
RickC7 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 05:22 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO