Amplicon Sequence Variants Artificially Split Bacterial Genomes into Separate Clusters

Abstract

Amplicon sequencing variants (ASVs) have been proposed as an alternative to operational taxonomic units (OTUs) for analyzing microbial communities. ASVs have grown in popularity, in part because of a desire to reflect a more refined level of taxonomy since they do not cluster sequences based on a distance-based threshold. However, ASVs and the use of overly narrow thresholds to identify OTUs increase the risk of splitting a single genome into separate clusters. To assess this risk, I analyzed the intragenomic variation of 16S rRNA genes from the bacterial genomes represented in an rrn copy number database, which contained 20,427 genomes from 5,972 species. As the number of copies of the 16S rRNA gene increased in a genome, the number of ASVs also increased. There was an average of 0.58 ASVs per copy of the 16S rRNA gene for full-length 16S rRNA genes. It was necessary to use a distance threshold of 5.25% to cluster full-length ASVs from the same genome into a single OTU with 95% confidence for genomes with 7 copies of the 16S rRNA, such as Escherichia coli. This research highlights the risk of splitting a single bacterial genome into separate clusters when ASVs are used to analyze 16S rRNA gene sequence data. Although there is also a risk of clustering ASVs from different species into the same OTU when using broad distance thresholds, these risks are of less concern than artificially splitting a genome into separate ASVs and OTUs. IMPORTANCE 16S rRNA gene sequencing has engendered significant interest in studying microbial communities. There has been tension between trying to classify 16S rRNA gene sequences to increasingly lower taxonomic levels and the reality that those levels were defined using more sequence and physiological information than is available from a fragment of the 16S rRNA gene. Furthermore, the naming of bacterial taxa reflects the biases of those who name them. One motivation for the recent push to adopt ASVs in place of OTUs in microbial community analyses is to allow researchers to perform their analyses at the finest possible level that reflects species-level taxonomy. The current research is significant because it quantifies the risk of artificially splitting bacterial genomes into separate clusters. Far from providing a better representation of bacterial taxonomy and biology, the ASV approach can lead to conflicting inferences about the ecology of different ASVs from the same genome.

Keywords: 16S rRNA gene; ASV; OTU; bioinformatics; microbial communities; microbial ecology; microbiome.

FIG 1

The distance threshold required to prevent the splitting of genomes into multiple OTUs increased as the number of rrn operons in the genome increased. Each line represents the median distance threshold for each region of the 16S rRNA gene that is required for 95% of the genomes with the indicated number of rrn operons to cluster their ASVs to a single OTU. The median distance threshold was calculated across 100 randomizations in which one genome was sampled from each species. Only those numbers of rrn operons that were found in more than 100 species are included.

FIG 2

As the distance threshold used to define an OTU increased, the percentages of ASVs and OTUs representing multiple species increased. These data represent the median fractions for both measurements across 100 randomizations. In each randomization, one genome was sampled from each species.