A heritability-based comparison of methods used to cluster 16S rRNA gene sequences into operational taxonomic units

Abstract

A variety of methods are available to collapse 16S rRNA gene sequencing reads to the operational taxonomic units (OTUs) used in microbiome analyses. A number of studies have aimed to compare the quality of the resulting OTUs. However, in the absence of a standard method to define and enumerate the different taxa within a microbial community, existing comparisons have been unable to compare the ability of clustering methods to generate units that accurately represent functional taxonomic segregation. We have previously demonstrated heritability of the microbiome and we propose this as a measure of each methods’ ability to generate OTUs representing biologically relevant units. Our approach assumes that OTUs that best represent the functional units interacting with the hosts’ properties will produce the highest heritability estimates. Using 1,750 unselected individuals from the TwinsUK cohort, we compared 11 approaches to OTU clustering in heritability analyses. We find that de novo clustering methods produce more heritable OTUs than reference based approaches, with VSEARCH and SUMACLUST performing well. We also show that differences resulting from each clustering method are minimal once reads are collapsed by taxonomic assignment, although sample diversity estimates are clearly influenced by OTU clustering approach. These results should help the selection of sequence clustering methods in future microbiome studies, particularly for studies of human host-microbiome interactions.

Circulating levels of the anti-oxidant indoleproprionic acid are associated with higher gut microbiome diversity

“…21 De novo OTU clustering was then carried across all reads using Sumaclust within QIIME 1.9.0, grouping reads with a 97% identity threshold. 22,23 OTU counts were converted to log-transformed relative abundances, with zero counts handled by the addition of an arbitrary value (10 −6 ). The residuals of the OTU abundances were taken from linear models, accounting for technical covariates including sequencing depth, sequencing run, sequencing technician and sample collection method.…”

The Relationships Between the Free-Living and Particle-Attached Bacterial Communities in Response to Elevated Eutrophication

“…Chimeras were identified and were removed with the program usearch (version 10) (Edgar, 2010). OTUs were clustered with 97% similarity cutoff using vsearch (version 2.8.1) (Jackson et al, 2016). The representative sequence of each phylotype was selected and aligned by PDP Classifier (version 16) against the SILVA 16s rRNA database (release 132) with a confidence threshold of 70%.…”

The Xylella fastidiosa-Resistant Olive Cultivar “Leccino” Has Stable Endophytic Microbiota during the Olive Quick Decline Syndrome (OQDS)

“…Chimeric sequences were removed using Vsearch [92]. Operational taxonomic units (OTUs) were generated at 97% sequence similarity by using the sumaclust method, which was shown to be more accurate than other clustering methods [93], and were identified, after alignment, using the uclust method and the databases SILVA, release 132 [94], for prokaryotes and UNITE v.7.2, release_s_01.12.2017 [95], for fungi. Unidentified and contaminant (plastidic and mitochondrial for Prokaryotes, Metazoa for Fungi) OTUs were removed, using the QIIME script "filter_taxa_from_otu_table.py", as well as OTUs with less than 10 reads, to reduce noise.…”