I'm working on the microbiome of a wild nectar feeding bat. This samples may contain a lot of plant material including Pollen. I was using the V4 region (515-806 primers) and extractation from fecal pellets.
Before I ran all my samples, I decided to run 15 samples and one control jsut to check if everything went ok.
The output of the run was quite particular and I found that a large percentage of the microbiome in 3 samples matches the following string:
I know that it is normal to get some mitochondria in the MB because the primers link this too. The problem is that this didn't went away with the filtering step:
"qiime taxa filter-table
There is one specific ASV that account for 10% of all the reads in my run!! and this is mainly concentrated in 3 samples. This seams wrong, so I explored different options to solve this, first I evaluated if this was a mistake in the bioinformatic processing:
In trying to correct this I:
+Re-ran the QIIME2 pipeline again with a less conservative cut 215 instead of 200 in the DADA2 step
+re assigned the taxonomy using Silva (the first try was with greengenes)
Conclusion: it's not something in the pipeline as far as I can tell (or is it?)
The second though was that this could be DNA from the Host, I Aligned the problem ASV, a "normal" ASV from my MB, a 16S sequence from my host and the sequence of the 2 top hits after blasting the problem ASV. The results, the problem ASV and the 16s sequence from the host do not aligned at all. Also the ASV and the sequence of the top hits are perfect matches while the "normal" ASV is quite different from the others.
When I blasted the problem ASV, the result the top hits are Mitochodrial DNA from plants that are NOT present in my study area. I'm starting to think that this sequence may come from undigested pollen that just when through the bat directly.
I was wondering if anyone was ever had a problem like this one... Im curious if this is a failure in the filtering step or if there may be some problem with the samples.
Any input would be greatly appreciated!