Attention Ion Torrent 16S Metagenomics Kit Users: Feedback Wanted

Hello Qiime2 users,

My colleagues and I have been using the IonTorrent 16S Metagenomics kit to prepare libraries which are then sequenced on the IonTorrent Platform. We have found it difficult / near impossible to perform downstream analysis on our own because of multiple reasons. We have reached out to the company to request that they start providing clients with sequences that have been separated and identified both by forward and reverse reads, and by targeted gene region. However, in order to build our case, we are looking to provide a list of clients that are also having difficulty analyzing their data because of this lack of information. If you use the IonTorrent 16S Metagenomics Kit to prepare your libraries and you believe receiving data in this format would better serve your needs, please contact us with your first and last name, your email, and the approximate number of samples you have prepared using the 16S Metagenomics kit. Please be aware that this information will be sent to ThermoFisher and that you may or may not be contacted in the future.

Thank you for your time,


For some context, Lauren is talking about kits like these. From the website:

The kit includes 2 sets of primers that can be used to amplify the corresponding hypervariable regions of the 16S rDNA gene in bacteria:
• Primer set V2-4-8
• Primer set V3-6,7-9

These primers are proprietary, and Thermo Fishes has never published them.
This makes analysis hard. Some people have made progress: paper.

These secret primers might be based on this set from 2007 manuscript. Or maybe this paper from 2017?

Except… this other paper from 2017 mentions “short amplicon sequencing were designed for 16S rDNA variable regions (V2, V3, V4, V6–7, V8, and V9)”, all the same regions covered by the Ion 16S™ Metagenomics Kit, BUT they never mention the Ion 16S™ Metagenomics Kit by name, and they actually publish their full primers on page 10 of their supp document, which no-one else ever does when using this kit because maybe they legally can’t or they don’t know either.

Spoiler warning!

Back in 2011, four brothers from Detroit published a paper in which they introduce and test five primer pairs, and because they are not working for Thermo Fisher, they list the full sequences in Table 1. But then, just 5 pages later, they also state:

“The multiple-primer sequencing strategy described here may have wider applications… may be applicable to other short-read next generation sequencers, such as the Ion Torrent.”


It’s a fun little mystery! :female_detective: :male_detective:

Compare this to Illumina: “What sequences do I use for adapter trimming?”
Want more detail? Try this pdf that lists every single primer they sell
Or how about all their primers in a single .fasta file



I’d also like to offer that I’ve been doing a lot of work recently on scaffolding multiple regions. The best theoretical algorithm I can find for short reads is computationally feasible only because you have the primers and can therefore do alignment. Knowing the hypervariable region isn’t enough: you need to know the primers.

It’s also a big issue in terms of meta analysis. If Im looking for a specific ASV and I don’t have the primer sequences the ion torrent data becomes impossible to use/reuse. Which perhaps isn’t a concern for a company but would make me wary as a researcher.


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