Amplicon Sequence Variant-Based Oral Microbiome Analysis Using QIIME 2

Abstract

The bacterial composition of oral samples has traditionally been determined by PCR amplicon sequencing of 16S rRNA genes. Recent amplicon sequence variant (ASV)-based analyses of 16S rRNA genes differ from that based on operational taxonomic unit (OTU) clustering in the way it deals with sequences having potential errors. However, little information is available on its application in oral microbiome studies. Here, we conducted ASV-based analysis of oral microbiome samples using QIIME 2. We investigated the optimal parameters for sequence denoising, using DADA2, and found the trimming of the first 20 nucleotides from 5′-end of both paired reads avoided excessive sequence loss during chimera removal. Truncating reads at positions 240–245 allowed the removal of low-quality sequences while maintaining sufficient length to merge matching paired ends. Taxonomic assignment, using the naïve Bayes classifier trained with the V3-V4 region of reference 16S rRNA sequences in the extended human oral microbiome database (eHOMD), resulted in bacterial compositions similar to those of OTU-based analyses. Contrary to OTU-based clustering, ASV-based analysis showed taxonomic abundance at the genus or species level to not differ significantly in tongue microbiomes, regardless of brushing. QIIME 2 can, therefore, be a standard pipeline for ASV-based analysis of oral microbiomes.

A mouthwash formulated with o-cymen-5-ol and zinc chloride specifically targets potential pathogens without impairing the native oral microbiome in healthy individuals

“…The mean number of ASVs in the placebo and mouthwash groups was 197.26 ± 9.74 and 200.67 ± 9.68 at T0, and 201.98 ± 9.66 and 190.42 ± 10.31 at T14, respectively. The number of ASVs observed in the two groups was comparable to those observed in other studies of healthy oral microbiomes [ 41 , 42 ]. Although no significant differences were detected, Shannon’s and Simpson’s indices showed a tendency to decrease after 14 days in both groups.…”

Inter-site and interpersonal diversity of salivary and tongue microbiomes, and the effect of oral care tablets

“…Demultiplexed paired-end sequences were processed using QIIME 2 (v.2020.2) and its associated plugins 19 in a Docker container. Sequences obtained from independent Miseq runs were denoised separately using DADA2 (via q2-dada2) 20 applying previously-optimized parameters 14 (trim-left-f = 20; trim-left-r = 20; trunc-len-f and trunc-len-r were set between 241 and 248 depending on the sequence quality; other parameters followed the default settings, including chimera-method = “consensus”). The resulting exact amplicon sequence variants (ASVs) were merged (via q2-feature-table).…”

“…Therefore, methods that could alter the tongue microbiota to a healthy microbiota type could contribute to oral health. We have previously reported that tongue brushing does not alter the alpha or beta diversity of oral microbiota in healthy adults 14,15 . By contrast, according to a recent study, the use of oral care tablets decreases the amount of volatile sulfur compounds (VSCs) produced by bacteria 16 .…”

Cross-border comparison of mycobiomes in Parmelia and Peltigera lichens from Turkey and South Korea

“…All sequencing data were deposited in the National Center for Biotechnology Information (NCBI), Sequence Read Archive, under the accession number PRJNA1035977. The amplicon sequence variant (ASV) 34 table was imported from QIIME2 to R using the 'qiime2R' package (https://github.com/jbisanz/qiime2R). Host lichen sequences were ltered out, and the ASV table was rare ed to 1,800 sequences per lichen host for comparison in equal standard using the 'phyloseq' package 35 .…”