# Found three OTUs/ASV that are highly abundant in my sample. How likely are they to be true OTUs/ASVs?
**URL:** https://forum.qiime2.org/t/found-three-otus-asv-that-are-highly-abundant-in-my-sample-how-likely-are-they-to-be-true-otus-asvs/21290
**Category:** General Discussion
**Created:** [November 9, 2021, 8:20am UTC](https://forum.qiime2.org/t/found-three-otus-asv-that-are-highly-abundant-in-my-sample-how-likely-are-they-to-be-true-otus-asvs/21290 "2021-11-09T08:20:44Z")
**Posts on this page:** 5
**Page:** 1
### Author:  [@Diki](https://forum.qiime2.org/u/Diki)
#### Post date: [November 9, 2021, 8:20am UTC](https://forum.qiime2.org/t/found-three-otus-asv-that-are-highly-abundant-in-my-sample-how-likely-are-they-to-be-true-otus-asvs/21290/1 "2021-11-09T08:20:44Z")
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?
Details:
1. We measure V3-V4 16s region.
2. The OTUs taxa is d\_\_Bacteria;p\_\_Proteobacteria;c\_\_Gammaproteobacteria;o\_\_Enterobacterales;f\_\_Morganellaceae;g\_\_Proteus;\_\_
3. This is the frequency according to table.qzv,
Thank you very much.
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### Author:  [@Diki](https://forum.qiime2.org/u/Diki)
#### Post date: [December 27, 2021, 8:01am UTC](https://forum.qiime2.org/t/found-three-otus-asv-that-are-highly-abundant-in-my-sample-how-likely-are-they-to-be-true-otus-asvs/21290/2 "2021-12-27T08:01:55Z")
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_, [Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries | Genome Biology | Full Text](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](http://future-science.com) (Atypon)_, [https://doi.org/10.2144/000113809](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_, [Redirecting](https://doi.org/10.1016/S1389-1723(03)90130-7).
- Krehenwinkel, Henrik, et al. “Estimating and Mitigating Amplification Bias in Qualitative and Quantitative Arthropod Metabarcoding.” _Scientific Reports_, vol. 7, no. 1, Dec. 2017, p. 17668. _[www.nature.com](http://www.nature.com)_, [Estimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding | Scientific Reports](https://doi.org/10.1038/s41598-017-17333-x).
- _Library Preparation for Next-Generation Sequencing: Dealing with PCR Bias_. [https://sequencing.roche.com/content/rochesequence/en/blog/library-preparation-for-next-generation-sequencing-dealing-with-pcr-bias.html](https://sequencing.roche.com/content/rochesequence/en/blog/library-preparation-for-next-generation-sequencing-dealing-with-pcr-bias.html). Accessed 27 Dec. 2021.
- _PCR Amplification Bias_. [PCR amplification bias](https://www.drive5.com/usearch/manual7/amplification_bias.html). Accessed 27 Dec. 2021.
- Silverman, Justin D., et al. “Measuring and Mitigating PCR Bias in Microbiota Datasets.” _PLOS Computational Biology_, vol. 17, no. 7, July 2021, p. e1009113. _PLoS Journals_, [Measuring and mitigating PCR bias in microbiota datasets](https://doi.org/10.1371/journal.pcbi.1009113).
- 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](http://DOI.org) (Crossref)_, [https://doi.org/10.1093/nar/25.21.4422](https://doi.org/10.1093/nar/25.21.4422).
Thank you all.
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### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [December 30, 2021, 3:35am UTC](https://forum.qiime2.org/t/found-three-otus-asv-that-are-highly-abundant-in-my-sample-how-likely-are-they-to-be-true-otus-asvs/21290/3 "2021-12-30T03:35:14Z")
> [@Diki](#):
>
> how likely are these OTUs to be a true results and not an artifact caused by faulty PCR process?
Did you run any positive or negative controls on these runs? Were the positive controls axenic / monoculture, or multi-taxa communities?
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### Author:  [@Diki](https://forum.qiime2.org/u/Diki)
#### Post date: [January 5, 2022, 8:18am UTC](https://forum.qiime2.org/t/found-three-otus-asv-that-are-highly-abundant-in-my-sample-how-likely-are-they-to-be-true-otus-asvs/21290/4 "2022-01-05T08:18:15Z")
> [@colinbrislawn](#):
>
> Did you run any positive or negative controls on these runs? Were the positive controls axenic / monoculture, or multi-taxa communities?
Hi! Thank you for your reply.
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?
Thank you.
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### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [January 5, 2022, 8:57pm UTC](https://forum.qiime2.org/t/found-three-otus-asv-that-are-highly-abundant-in-my-sample-how-likely-are-they-to-be-true-otus-asvs/21290/5 "2022-01-05T20:57:49Z")
Good afternoon!
> [@Diki](#):
>
> Unfortunately, we did not run any positive or negative controls.
That's a shame. But, now you know for next time!
> [@Diki](#):
>
> 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:
> [@Tutorial for filtering controls](https://forum.qiime2.org/t/tutorial-for-filtering-controls/557):
>
> Hi, as different controls (sequencing controls, DNA extraction controls, sample blanks, field blanks etc.) become more and more common in amplicon studies I thought it might be helpful to provide a QIIME2 tutorial specifically dealing with filtering sequences/features of controls. Here, I would suggest additional filtering options: e.g. subtraction of control read counts from counts in feature tables, filtering of RSVs coming from DADA2 in a similar fashion as the exclude\_seqs\_by\_blast.py scrip…
> [@Discussion: methods for removing contaminants and cross-talk](https://forum.qiime2.org/t/discussion-methods-for-removing-contaminants-and-cross-talk/5626/):
>
> Just to add further to this discussion, I've previously used decontam prior to it being incorporated into QIIME 2, and whilst I'm a huge fan of the principles it's built around, it didn't work fantastically for my samples. I will caveat that as I did only attempt the prevalence method with 2 negative controls (one kit negative and one sequencing negative) for around 20 samples, which is likely too low a number of controls to have any real statistical power, I'm not sure what the ideal number of …