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Old 04-12-2011, 02:15 PM   #1
chadn737
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Default Multiplexing experimental design question.

We are doing an RNA-seq experiment comparing a wild type sample to a mutant. There are three biological reps of each, each tagged with a unique barcode.

These will be run on two lanes of an Illumina Hi-Seq. Our core averages around 65 million reads per lane.

I'm thinking that the ideal setup here is to multiplex all 6 samples on both lanes and combine the reads for each sample afterwards. In theory, this should eliminate any lane-lane variation between the samples as this will be spread across all 6 samples.

Or am I wrong in this? Is it better to multiplex 3 of all our wild type in one lane and 3 of our mutant in another. Or maybe a design of 2 WT 1 mutant in one lane and 2 mutant 1 WT in the other. I would think in either case we should average ~20 million reads per sample, but that the first is the ideal setup.

Any thoughts on this? I know many claim that the technical variation observed is minimal, but I like to eliminate as many confounding factors as possible.

Last edited by chadn737; 04-12-2011 at 02:18 PM.
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Old 04-12-2011, 04:18 PM   #2
Camg
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Default

I think your first idea is right, you should barcode each sample and run them all in each lane.

Check out this reference: http://www.genetics.org/cgi/reprint/185/2/405
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