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Old 09-27-2017, 06:01 AM   #1
RickC7
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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.
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Old 09-27-2017, 01:25 PM   #2
nucacidhunter
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The most accurate method for library quantification is qPCR.
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Old 09-27-2017, 01:47 PM   #3
RickC7
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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.
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Old 09-27-2017, 02:09 PM   #4
nucacidhunter
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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.
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