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High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

ABSTRACT: Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

SUBMITTER: Callahan BJ

PROVIDER: S-EPMC6765137 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Callahan Benjamin J BJ Wong Joan J Heiner Cheryl C Oh Steve S Theriot Casey M CM Gulati Ajay S AS McGill Sarah K SK Dougherty Michael K MK

Nucleic acids research 20191001 18

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16 ...[more]

PMID: 31269198

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Project description:BackgroundSequencing variable regions of the 16S rRNA gene (≃300 bp) with Illumina technology is commonly used to study the composition of human microbiota. Unfortunately, short reads are unable to differentiate between highly similar species. Considering that species from the same genus can be associated with health or disease it is important to identify them at the lowest possible taxonomic rank. Third-generation sequencing platforms such as PacBio SMRT, increase read lengths allowing to sequence the whole gene with the maximum taxonomic resolution. Despite its potential, full length 16S rRNA gene sequencing is not widely used yet. The aim of the current study was to compare the sequencing output and taxonomic annotation performance of the two approaches (Illumina short read sequencing and PacBio long read sequencing of 16S rRNA gene) in different human microbiome samples. DNA from saliva, oral biofilms (subgingival plaque) and faeces of 9 volunteers was isolated. Regions V3-V4 and V1-V9 were amplified and sequenced by Illumina Miseq and by PacBio Sequel II sequencers, respectively.ResultsWith both platforms, a similar percentage of reads was assigned to the genus level (94.79% and 95.06% respectively) but with PacBio a higher proportion of reads were further assigned to the species level (55.23% vs 74.14%). Regarding overall bacterial composition, samples clustered by niche and not by sequencing platform. In addition, all genera with > 0.1% abundance were detected in both platforms for all types of samples. Although some genera such as Streptococcus tended to be observed at higher frequency in PacBio than in Illumina (20.14% vs 14.12% in saliva, 10.63% vs 6.59% in subgingival plaque biofilm samples) none of the differences were statistically significant when correcting for multiple testing.ConclusionsThe results presented in the current manuscript suggest that samples sequenced using Illumina and PacBio are mostly comparable. Considering that PacBio reads were assigned at the species level with higher accuracy than Illumina, our data support the use of PacBio technology for future microbiome studies, although a higher cost is currently required to obtain an equivalent number of reads per sample.

Project description:There is a need to profile microorganisms which exist pre-and-post-production of umqombothi, to understand its microbial diversity and the interactions which subsequently influence the final product. Thus, this study sought to determine the relative microbial abundance in umqombothi and predict the functional pathways of bacterial and fungal microbiota present. Full-length bacterial 16S rRNA and internal transcribed spacer (ITS) gene sequencing using PacBio single-molecule, real-time (SMRT) technology was used to assess the microbial compositions. PICRUSt2 was adopted to infer microbial functional differences. A mixture of harmful and beneficial microorganisms was observed in all samples. The microbial diversity differed significantly between the mixed raw ingredients (MRI), customary beer brew (CB), and optimised beer brew (OPB). The highest bacterial species diversity was observed in the MRI, while the highest fungal species diversity was observed in the OPB. The dominant bacterial species in the MRI, CB, and OPB were Kosakonia cowanii, Apilactobacillus pseudoficulneus, and Vibrio alginolyticus, respectively, while the dominant fungal species was Apiotrichum laibachii. The predicted functional annotations revealed significant (p < 0.05) differences in the microbial pathways of the fermented and unfermented samples. The most abundant pathways in the MRI were the branched-chain amino acid biosynthesis super pathway and the pentose phosphate pathway. The CB sample was characterised by folate (vitamin B9) transformations III, and mixed acid fermentation. Biotin (vitamin B7) biosynthesis I and L-valine biosynthesis characterised the OPB sample. These findings can assist in identifying potential starter cultures for the commercial production of umqombothi. Specifically, A. pseudoficulneus can be used for controlled fermentation during the production of umqombothi. Likewise, the use of A. laibachii can allow for better control over the fermentation kinetics such as carbohydrate conversion and end-product characteristics, especially esters and aroma compounds.

Dataset Information

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Publications

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

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