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Old 05-31-2018, 07:40 AM   #1
seanw
Junior Member
 
Location: New Brunswick, Canada

Join Date: Nov 2014
Posts: 2
Default 16S Library Plant Tissue Troubles

Hi all I thought I would seek advice here as I have tried a number of tweaks and not getting the results I would expect. Here is the background

DNA was extracted from plant stems to allow for a survey of bacterial endophytes using 16S amplicons in the v4 region

Samples were extracted using a Qiagen DNeasy Plant extraction kit, samples were quantified and run on a gel to check for quality. Yes it will be mostly plant DNA but still, this was done.
Library preparation was done following the Schloss Wet Lab SOP as I have done numerous times before for soil, roots, and yes some plant tissue (mostly combo runs when plant tissue as involved).
Samples were pools and placed on MiSeq and the following stats were obtained on my latest v2 500 cycle (2 x 250bp) run.
@ cycle 244 I got
Density 722K/mm2
Passing Filter 72.2%
PhiX spiked at 9.2%
Qscore >=30 32.9% and this only went down to about 25% by the end of the run

This was not the first time to run this library as I did a nano flow cell run with it and had spike in PhiX and a much higher concentration to increase diversity and yet still ended up with a low pass filter and poor qscore.

I have, in the past run a library with these stem samples (a subset of 4 samples and two different extraction types Qiagen and MoBio on each of the 4 samples, to determine the best extraction method/bacterial yield) on a nano flow cell (2 x 250) and got
These stats:

Density 520k/mm2
Passing filter 88.5%
Qscore >= 93.6%
PhiX was at 13.6
Yes there was a fair bit of chloroplast/host DNA contamination but was getting about 12-20 % out after processing through Mothur.

I have also run other soil samples using the reagents and all are fine. I have also a large number of various library preps under my belt but am at a bit of loss on this one…

Any ideas?
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