I found three OTUs that are highly abundant in my sample. I have eight unique stool samples from eight laboratory mice. We want to look microbiome dynamics from three timepoints (three sequencing preparation batches of these mice).
We got results from second batch not long ago, and found above-mentioned three highly abundant OTUs in the second batch. We observe very small number of them in the previous first batch.
I noticed we used different thermal cycler between batches for PCR. We suspect that the second batch thermal cycler is in a very questionable condition, how likely are these OTUs to be a true results and not an artifact caused by faulty PCR process?
We measure V3-V4 16s region.
The OTUs taxa is d__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacterales;f__Morganellaceae;g__Proteus;__
Hi, please let me know if I am not allowed to put some PCR bias amplification reference for my question above. Supposedly considered as spam for example.
Aird, Daniel, et al. “Analyzing and Minimizing PCR Amplification Bias in Illumina Sequencing Libraries.” Genome Biology, vol. 12, no. 2, Feb. 2011, p. R18. BioMed Central, https://doi.org/10.1186/gb-2011-12-2-r18.
Dabney, Jesse, and Matthias Meyer. “Length and GC-Biases during Sequencing Library Amplification: A Comparison of Various Polymerase-Buffer Systems with Ancient and Modern DNA Sequencing Libraries.” BioTechniques, vol. 52, no. 2, Feb. 2012, pp. 87–94. future-science.com (Atypon), https://doi.org/10.2144/000113809.
Kanagawa, Takahiro. “Bias and Artifacts in Multitemplate Polymerase Chain Reactions (PCR).” Journal of Bioscience and Bioengineering, vol. 96, no. 4, Jan. 2003, pp. 317–23. ScienceDirect, https://doi.org/10.1016/S1389-1723(03)90130-7.
Warnecke, P. M., et al. “Detection and Measurement of PCR Bias in Quantitative Methylation Analysis of Bisulphite-Treated DNA.” Nucleic Acids Research, vol. 25, no. 21, Nov. 1997, pp. 4422–26. DOI.org (Crossref), https://doi.org/10.1093/nar/25.21.4422.
Unfortunately, we did not run any positive or negative controls.
However, I am curious with the follow up if we had run the controls. Suppose negative control was monoculture and it was these Proteus above. Can we quickly assume that Proteus is contaminant and therefore justifiable to be removed?
Yes. Because you know the composition of positive controls, you can use them to calibrate other parts of the process. Just like you said, if these three mystery OTUs appears in your second batch of samples, including your monoculture control sample you ran on both Illumina runs, you could argue it's artificial and remove it.
It's possible that the second batch really does have a highly abundant g__Proteus in it.
Here's some more discussions of positive controls and filtering out strange microbes, if you are interested: