OTU and ASV

What are OTUs and ASVs?

OTUs (operational taxonomic units) and ASVs (amplicon sequence variants) are two ways of turning millions of sequencing reads into a countable list of microbial “types”. Both belong mainly to amplicon studies such as 16S rRNA sequencing, where one region of a marker gene is amplified and sequenced.

For years the default was to cluster sequences into OTUs at 97% identity. From the mid-2010s, ASVs, which resolve exact sequences, became the more common choice.

How they work

• OTUs: sequences are clustered together when their similarity exceeds a threshold. This absorbs sequencing errors but also blurs real differences between closely related organisms. De novo OTUs depend on the dataset: the same sequence can land in different OTUs in two separate studies.
• ASVs: algorithms such as DADA2 model sequencing errors and infer which sequences are genuine, down to a single-nucleotide difference. Because an ASV is the DNA sequence itself, it acts as a stable label that can be compared between samples and studies.

In a 2017 perspective in The ISME Journal, Callahan, McMurdie and Holmes argued that exact sequence variants should replace OTUs in marker-gene analysis, on the grounds of reproducibility, reusability and resolution.

Why it matters

The choice changes what ends up in a report:

• It alters the number of taxa detected and therefore richness and alpha and beta diversity.
• It limits comparisons between laboratories: reports produced with different pipelines are not directly interchangeable.
• Neither approach on its own ensures species-level identification; that depends on the region sequenced, read length and the reference database.

Shotgun metagenomics does not use OTUs or ASVs at all. Reads from all the DNA in a sample are assigned directly to reference genomes.

Example

A clinician reads two papers on the gut microbiome in the same condition. One, from 2014, reports around 450 OTUs per sample; the other, using DADA2, reports around 600 ASVs. The gap does not mean the second cohort harbours more bacteria. Much of it reflects the method, and a fair comparison would require reprocessing the raw data through the same pipeline.

OTUs and ASVs at Xenogene

Xenogene’s Gut Microbiota test is a metagenomic analysis using Nanopore sequencing which, according to its product page, detects the microorganisms present in a stool sample and assesses parameters including bacterial diversity. Understanding the difference between OTUs, ASVs and metagenomic read assignment helps clinicians see why diversity figures or taxon counts should not be compared directly with reports from laboratories using different methods.