Indexed libraries best pooling strategy

[spdinesh]

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]

Quantitate your libraries by qPCR, not BioA.

[HESmith]

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]

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