Should OTU or ASV Clustering Be Used in 16S Sequencing?
In 16S rRNA sequencing, two main clustering approaches are commonly employed: OTU (Operational Taxonomic Units) clustering and ASV (Amplicon Sequence Variants) clustering. Each method offers distinct advantages and limitations.
OTU Clustering
1. Advantages
OTU clustering is a conventional approach that groups sequences into OTUs based on a predefined similarity threshold, typically set at 97%. This method effectively mitigates the impact of random sequencing errors and has been extensively applied in large-scale biodiversity research.
2. Limitations
A key limitation of OTU clustering is the potential overmerging of closely related species, reducing taxonomic resolution. Furthermore, the criteria for defining OTUs may vary across studies, which can compromise data comparability.
ASV Clustering
1. Advantages
ASV clustering, a more recent approach, distinguishes sequences at the single-nucleotide level, offering finer taxonomic resolution. This method captures subtle differences between microbial communities and is particularly suitable for studies requiring precise species-level differentiation.
2. Limitations
ASV clustering, however, is susceptible to misinterpreting technical artifacts (e.g., PCR errors) as true biological diversity, potentially leading to an overestimation of species richness. Additionally, ASV processing is computationally intensive, requiring significantly more resources compared to OTU clustering.
The selection between OTU and ASV clustering depends on the specific research objectives. OTU clustering remains a preferred choice for large-scale biodiversity studies, while ASV clustering is more advantageous for microbial community analyses requiring high taxonomic resolution.
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