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discrepancy in miseq runs due to sample prep protocol?

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  • 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 Files

  • #2
    The simplest way to isolate the problem is to have new facility sequence the old (high quality) sample - if the data are good, it's not the facility. Alternatively, prepare your next sample with both the original and modified protocols (with indices to discriminate the two) and compare the sequence data.

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    • #3
      Originally posted by samd View Post
      ...
      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).
      ...
      Don't over interpret the differences between these two traces since they were obviously performed using different instruments. The one on the left appears to be an Advanced Analytical Fragment Analyzer and on the right an Agilent TapeStation. Due to differences in the electrophoretic matrix and run conditions the same library may appear to migrate differently on different instruments. Also note that the X axis (size scale) is much more compressed on the Fragment Analyzer relative to the TapeStation so naturally peaks will appear more compressed.

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