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Old 11-21-2012, 09:59 AM   #1
thdybwf
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Location: USA

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Question How much PhiX in MiSeq run?

Hello all -

Our lab is about to run a RADseq library on an Illumina MiSeq. It consists of multiplexed individuals with semi-random fragments from across the genome.

We're not sure how much PhiX control DNA we should use to spike the library. In our indexed forward adapter, after the Solexa adapter, there's a 5bp barcode, which should have balanced nucleotide composition. But then, there are 4bp of a restriction site overhang which is the same for all of our fragments. After that, the fragments should all be variable and balanced. So the only bit that is really a concern is that 4bp overhang common to all fragments.

Could you please offer any advice on amount of PhiX we should add? Thank you very much for any information you can give.
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Old 11-26-2012, 04:01 AM   #2
pmiguel
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If you are unsure, you probably want 50%. Then you can titrate down in subsequent runs, should it prove possible.

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Phillip
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