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Old 03-03-2019, 09:26 PM   #1
depcone
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Location: New Jersey

Join Date: Jul 2010
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Default Miseq failure of Targeted Bisulfite multiplex amplicons from fluidigm Juno library

I made recently Fluidigm Juno library through targeted bisulfite multiplexing PCR using Juno machine. Juno is an upgraded version of Fluidigm Access array.

I performed Miseq to sequencing Juno library. But the Miseq run was failed and aborted itself an hour in with no data produced. For this run, 20pM library was loaded with 30% phiX library.

There are some images that looks like super high clustering, but illumina technical support said that is not actually over clustering, it is over contrast by camera. We've has a couple of other runs fail in similar ways. We used miseq reagent version3 600 cycles. To test miseq machine, we ran PhiX only with Miseq reagent v2 50 cycles, that looks ok.

Then, to check my library, I checked bioanalyser, qPCR to validate library using illumina primers and sanger sequencing through the cloning. All tests were passed, especially I confirmed the Juno library from sanger sequencing that the adapters and barcods were perfectly attached in target.

So, I really don't know why miseq was failed. Does anyone have similar problem, or knows the reason?

Now, I am planing to run Miseq for this library from other place where is running Miseq routinely.
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