# Processing Thermo Ion 16S Metagenomics Kit data in QIIME2: primer trimming, denoise-pyro, and SILVA classifier choice **URL:** https://forum.qiime2.org/t/processing-thermo-ion-16s-metagenomics-kit-data-in-qiime2-primer-trimming-denoise-pyro-and-silva-classifier-choice/34185 **Category:** General Discussion **Created:** [May 22, 2026, 11:30am UTC](https://forum.qiime2.org/t/processing-thermo-ion-16s-metagenomics-kit-data-in-qiime2-primer-trimming-denoise-pyro-and-silva-classifier-choice/34185 "2026-05-22T11:30:13Z") **Posts on this page:** 1 **Showing post:** 2
### Author: ![colinbrislawn](https://forum.qiime2.org/user_avatar/forum.qiime2.org/colinbrislawn/32/6221_2.png) [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn) #### Post date: [May 23, 2026, 1:47am UTC](https://forum.qiime2.org/t/processing-thermo-ion-16s-metagenomics-kit-data-in-qiime2-primer-trimming-denoise-pyro-and-silva-classifier-choice/34185/2 "2026-05-23T01:47:17Z")
> [@DHWANI\_DHOLAKIA1](#): > > I identified the following primer sequences in the data: You found the sequences! **We were looking for these 6 years ago.** At the time, some forums users were working with the kits and were having a hard time partly because the primers and regions were not published. > [@Possible Analysis Pipeline for Ion Torrent 16S Metagenomics Kit Data in QIIME2?](https://forum.qiime2.org/t/possible-analysis-pipeline-for-ion-torrent-16s-metagenomics-kit-data-in-qiime2/13476): > > Hi QIIME2 community, My colleague @Lauren I have been using the Ion torrent 16S Metagenomics Kit to sequence some clinical samples that we have. Our sample sets are both longitudinal. The kit consists of two primer sets, one for V2-4-8 and one for V3-6,7-9. The products of the reactions are pooled per sample, and then the rest of library prep is completed. Each sample gets its own barcode. Ion Torrent produces single end reads. The issue here is that the reads from each of the 6 variable regio… The fact that you got this all the way through DADA2 is remarkable. Good job! * * * To your questions: > [@DHWANI\_DHOLAKIA1](#): > > - Does this workflow look appropriate for **Thermo Ion 16S Metagenomics Kit** data? It's a multi-region kit, so any workflow is going to be non-standard. It has to be! The taxonomy assignment looks promising. Did you include any positive controls? > [@DHWANI\_DHOLAKIA1](#): > > `dada2 denoise-pyro` with `--p-trunc-len 0` reasonable here, Sure. Cutadapt has other settings you could use for extra trimming, if that was a problem. > [@DHWANI\_DHOLAKIA1](#): > > - For taxonomy, is using the full-length **SILVA 138 99% Naive Bayes classifier** appropriate for this mixed-region Thermo 16S kit? Yes! You need full length because you have all the regions. I don't know if SILVA has been benchmarked with multiple regions, but you could do this with the positive controls! > [@DHWANI\_DHOLAKIA1](#): > > - Since SILVA reference sequences are often much longer than my reads/ASVs, would it be better to train a custom classifier using extracted reads around the Thermo primer regions, or is the full-length SILVA classifier acceptable? Yes, per-region classifiers should perform better, but you would need to make quite a few of them! I think it's easier to use a full-length skl classifier or switch to a search+LCA classifier like vsearch. Speaking of which... > [@DHWANI\_DHOLAKIA1](#): > > - Would VSEARCH-based taxonomy assignment be preferable for this type of multi-region Thermo 16S data? I'm not sure any of the classifiers were optimized for multi-region classification. I'm a big fan of vsearch because it's conceptually simple and runs fast. Try it out and report back! * * * It's hard to comment more about alpha and beta diversity without know more about the study design. I'll wait to write more. 🦠 Thank you for sharing your pipeline with us. And thank you for posting those primers after 6 years. --- _[View the full topic](https://forum.qiime2.org/t/processing-thermo-ion-16s-metagenomics-kit-data-in-qiime2-primer-trimming-denoise-pyro-and-silva-classifier-choice/34185)._