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Old 09-01-2015, 12:37 AM   #1
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Location: Korea

Join Date: Jul 2015
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Default Data I can't understand

We ran 100 samples on Illumina Miseq following 16S rRNA-seq protocol.
All of the samples showed only Pseudomonas (>99%).

We QC'd via electrophoresis, Bioanalyzer during library prep and they looked normal.

What could cause this kind of weird data?
ewhameta2015 is offline   Reply With Quote
Old 09-01-2015, 02:21 AM   #2
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What kind of samples did you sequence?

Couldn't it be that the Pseudomonas is in fact the vastly predominant species?
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Old 09-01-2015, 06:32 AM   #3
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Was this a two-step PCR library prep? Did you run negative controls for the first round of PCR?

Also, how did you analyze your data? I've found MiSeq reporter to give some very strange results sometimes.
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Old 09-01-2015, 12:45 PM   #4
Brian Bushnell
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Pseudomonas is one of our most common contaminants in all datasets. Not sure if it is related to our lab, reagent suppliers, or the conditions in the sequencing machines, but I would be very wary of any data indicating a high level of Pseudomonas.

I suggest you try sequencing a blank (water or buffer with no DNA added) using the exact same protocol and see what you get, to determine your contamination background.
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