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Old 02-07-2012, 10:13 AM   #1
spdinesh
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Default 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
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Old 02-07-2012, 01:27 PM   #2
VanessaS
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Quantitate your libraries by qPCR, not BioA.
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Old 02-07-2012, 04:05 PM   #3
HESmith
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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
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Old 02-08-2012, 12:21 AM   #4
Cofactor Genomics
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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
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