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vanillasky 03-22-2014 10:37 PM

Contaminant in dscDNA sample?
1 Attachment(s)
A recent illumina run analysis showed that the majority of our sequences were contaminated with 16S rRNA. Prior to dscDNA we used a rRNA depletion method using the RiboZero Bacteria Magnetic kit. We ran our samples on a Pico Chip and saw our sample had lost the peaks from 16S and 23S so the depletion step works. If contamination occurred it must have been either during dscDNA construction or prior to the illumina run. Could you help me determine which step might have introduced contaminants? In my attachment you will see the dscDNA run on a high sensitivity chip. Does this look fine? Please ignore the empty lanes.

buthercup_ch 06-19-2014 05:03 PM

Hi vanillasky,
In my experience, you should see a one and only smooth band in your dscDNA preparation when running a DNA HS chip… I can't see that in the file you attached. So I do not recommend you to proceed with sequencing for this sample.
I also think that after RIboZero treatment, running a NanoChip is enough and better than the Pico one.

vanillasky 06-19-2014 10:17 PM

Thank you for the reply. Unfortunately, I did end up sequencing and found that there were contaminants in there. I was never able to get this particular sample to work with the library preparation kit I used. I have talked to Agilent tech support in the past and they recommended the pico chip for mRNA analysis (which was my target). They told me the nanochip is good for quantitation vs the picochip. Also, the concentrations of my sample are so low that working with the picochip is better. Anyway, thank you for your post, I will keep this in mind if I ever work on this again.

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