Indexed libraries best pooling strategy

spdinesh · 02-07-2012, 10:13 AM

Hi
We recently sequenced 20 indexed libraries on one lane of Hi-Seq. Unfortunately, the pooling strategy we used based on bioanalyzer data was not even.

Any suggestion on the best pooling strategy to get uniform pool?

Dinesh

VanessaS · 02-07-2012, 01:27 PM

Quantitate your libraries by qPCR, not BioA.

HESmith · 02-07-2012, 04:05 PM

Search the forum for threads addressing accurate library quantification. There are advantages and limits to each technique. For example, adapter dimer contamination can wreak havoc with qPCR measurements.

Harold

Cofactor Genomics · 02-08-2012, 12:21 AM

Hello spdinesh,
In our experience, it is best to use multiple quantification platforms and go from there (bioanalyzer, Qbit, nanodrop), while knowing that +/- 20% is expected from pooling. Even pipette error can make a difference when pooling at n=20 with dilutions.

What was the standard dev. seen from your libraries?

jon

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Pooling multiplexed libraries after ligation, before PCR	ScottC	Illumina/Solexa	5	05-07-2016 02:10 PM
ELAND with indexed reads	wingtec	Introductions	0	12-27-2011 07:53 AM
PubMed: Genomic DNA pooling strategy for next-generation sequencing-based rare varian	Newsbot!	Literature Watch	0	09-18-2011 09:30 AM
Genomic DNA Pooling Strategy for Next-Generation Sequencing-Based Rare Variant Discov	Asashoryu	Literature Watch	0	03-11-2011 01:18 PM
Indexed Sure Select Capture	Bruce E	Illumina/Solexa	1	02-09-2011 07:54 AM

02-07-2012, 10:13 AM	#1
spdinesh Junior Member Location: Davis Join Date: Feb 2012 Posts: 1	Indexed libraries best pooling strategy Hi We recently sequenced 20 indexed libraries on one lane of Hi-Seq. Unfortunately, the pooling strategy we used based on bioanalyzer data was not even. Any suggestion on the best pooling strategy to get uniform pool? Dinesh

02-07-2012, 01:27 PM	#2
VanessaS Member Location: Dallass Join Date: Nov 2009 Posts: 49	Quantitate your libraries by qPCR, not BioA.

02-07-2012, 04:05 PM	#3
HESmith Senior Member Location: Bethesda MD Join Date: Oct 2009 Posts: 509	Search the forum for threads addressing accurate library quantification. There are advantages and limits to each technique. For example, adapter dimer contamination can wreak havoc with qPCR measurements. Harold

02-08-2012, 12:21 AM	#4
Cofactor Genomics Registered Vendor Location: St. Louis Join Date: Jan 2010 Posts: 52	Hello spdinesh, In our experience, it is best to use multiple quantification platforms and go from there (bioanalyzer, Qbit, nanodrop), while knowing that +/- 20% is expected from pooling. Even pipette error can make a difference when pooling at n=20 with dilutions. What was the standard dev. seen from your libraries? jon