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Old 11-06-2019, 04:13 PM   #1
samd
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Location: California

Join Date: Nov 2019
Posts: 10
Default discrepancy in miseq runs due to sample prep protocol?

Hi all,

I have had an issue where my first ever Miseq run was great quality: 100k reads per sample with a nice distribution and then after switching sequencing facilities I get lower outputs and also a highly variable spread where many samples are under 10k reads per sample and some are as high as 300k. See original post here to learn more about the parameters if intersted: here

The libraries are all similar 16S libraries but the three minor things that did change were:
1. I added a heating step to the extractions using the PowerSoil Extractions Kits (65C for 10 mins and then 95C for 10 mins before the Power bead step)
2. I switched from AMPure Bead cleans after both PCR 1 and PCR 2 to only 1 bead clean with OMEGA Beads after the 2nd PCR.
3. I switched from 35 cycles in the 1st PCR to 28 cycles.

Nothing seemed to change in the "quality" of the DNA as I got similar gel results and quantifications. However I did get different QC results with the first good run having a nice skinny peak, and the bad runs having more of a lumpy hill around the fragment size (attached a picture below).

Any ideas if I should be worried about my lab protocol? Or should I rethink which sequencing facility I use (The new place is very very cheap which is why we switched)?

Thanks for the feedback!
Sam
Attached Images
File Type: png Run comparison.png (71.8 KB, 12 views)
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