Generation of comprehensive ecosystems-specific reference databases with species-level resolution by high-throughput full-length 16S rRNA gene sequencing and automated taxonomy assignment (AutoTax)

AbstractHigh-throughput 16S rRNA gene amplicon sequencing is an essential method for studying the diversity and dynamics of microbial communities. However, this method is presently hampered by the lack of high-identity reference sequences for many environmental microbes in the public 16S rRNA gene reference databases, and by the absence of a systematic and comprehensive taxonomy for the uncultured majority. Here we demonstrate how high-throughput synthetic long-read sequencing can be applied to create ecosystem-specific full-length 16S rRNA gene amplicon sequence variant (FL-ASV) reference databases that include high-identity references (>98.7% identity) for nearly all abundant bacteria (>0.01% relative abundance) using Danish wastewater treatment systems and anaerobic digesters as an example. In addition, we introduce a novel sequence identity-based approach for automated taxonomy assignment (AutoTax) that provides a complete seven-rank taxonomy for all reference sequences, using the SILVA taxonomy as a backbone, with stable placeholder names for unclassified taxa. The FL-ASVs are perfectly suited for the evaluation of taxonomic resolution and bias associated with primers commonly used for amplicon sequencing, allowing researchers to choose those that are ideal for their ecosystem. The AutoTax taxonomy greatly improves the classification of short-read 16S rRNA gene amplicon sequence variants (ASVs) at the genus- and species-level, compared to the commonly used universal reference databases. Importantly, the placeholder names provide a way to explore the unclassified environmental taxa at different taxonomic ranks, which in combination with in situ analyses can be used to uncover their ecological roles.

Download Full-text

Generation of Comprehensive Ecosystem-Specific Reference Databases with Species-Level Resolution by High-Throughput Full-Length 16S rRNA Gene Sequencing and Automated Taxonomy Assignment (AutoTax)

mBio ◽

10.1128/mbio.01557-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 2

Author(s):

Morten Simonsen Dueholm ◽

Kasper Skytte Andersen ◽

Simon Jon McIlroy ◽

Jannie Munk Kristensen ◽

Erika Yashiro ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

Rrna Gene ◽

High Identity ◽

Reference Databases ◽

Reference Sequences

ABSTRACT High-throughput 16S rRNA gene amplicon sequencing is an essential method for studying the diversity and dynamics of microbial communities. However, this method is presently hampered by the lack of high-identity reference sequences for many environmental microbes in the public 16S rRNA gene reference databases and by the absence of a systematic and comprehensive taxonomy for the uncultured majority. Here, we demonstrate how high-throughput synthetic long-read sequencing can be applied to create ecosystem-specific full-length 16S rRNA gene amplicon sequence variant (FL-ASV) resolved reference databases that include high-identity references (>98.7% identity) for nearly all abundant bacteria (>0.01% relative abundance) using Danish wastewater treatment systems and anaerobic digesters as an example. In addition, we introduce a novel sequence identity-based approach for automated taxonomy assignment (AutoTax) that provides a complete seven-rank taxonomy for all reference sequences, using the SILVA taxonomy as a backbone, with stable placeholder names for unclassified taxa. The FL-ASVs are perfectly suited for the evaluation of taxonomic resolution and bias associated with primers commonly used for amplicon sequencing, allowing researchers to choose those that are ideal for their ecosystem. Reference databases processed with AutoTax greatly improves the classification of short-read 16S rRNA ASVs at the genus- and species-level, compared with the commonly used universal reference databases. Importantly, the placeholder names provide a way to explore the unclassified environmental taxa at different taxonomic ranks, which in combination with in situ analyses can be used to uncover their ecological roles.

Download Full-text

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

Nucleic Acids Research ◽

10.1093/nar/gkz569 ◽

2019 ◽

Vol 47 (18) ◽

pp. e103-e103 ◽

Cited By ~ 58

Author(s):

Benjamin J Callahan ◽

Joan Wong ◽

Cheryl Heiner ◽

Steve Oh ◽

Casey M Theriot ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Full Length ◽

Rrna Gene ◽

Single Nucleotide ◽

Full Complement ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

AbstractTargeted 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.

Download Full-text

Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

BMC Microbiology ◽

10.1186/s12866-021-02094-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yoshiyuki Matsuo ◽

Shinnosuke Komiya ◽

Yoshiaki Yasumizu ◽

Yuki Yasuoka ◽

Katsura Mizushima ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

Rrna Gene ◽

Short Read ◽

Short Read Sequencing ◽

Long Read

Abstract Background Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. Results We modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. Conclusions Our present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.

Download Full-text

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

10.1101/392332 ◽

2018 ◽

Cited By ~ 5

Author(s):

Benjamin J Callahan ◽

Joan Wong ◽

Cheryl Heiner ◽

Steve Oh ◽

Casey M Theriot ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Full Length ◽

Rrna Gene ◽

Single Nucleotide ◽

Full Complement ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

AbstractTargeted 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 allowedE. colistrains 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 severalE. colistrains.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.

Download Full-text

Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

10.1101/2020.05.06.078147 ◽

2020 ◽

Cited By ~ 3

Author(s):

Yoshiyuki Matsuo ◽

Shinnosuke Komiya ◽

Yoshiaki Yasumizu ◽

Yuki Yasuoka ◽

Katsura Mizushima ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

Rrna Gene ◽

Short Read ◽

Short Read Sequencing ◽

16S Amplicon Sequencing ◽

Long Read

AbstractBackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples.ResultsWe modified our existing protocol for full-length 16S amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition.ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene, which provided the requisite species-level resolution and accuracy in clinical settings.

Download Full-text

A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

PeerJ ◽

10.7717/peerj.2492 ◽

2016 ◽

Vol 4 ◽

pp. e2492 ◽

Cited By ~ 29

Author(s):

Catherine M. Burke ◽

Aaron E. Darling

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Single Molecule ◽

Illumina Miseq ◽

Full Length ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Bacterial Taxonomy

BackgroundThe bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision.ResultsWe describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection.ConclusionsThis method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.

Download Full-text

Superior resolution characterisation of microbial diversity in anaerobic digesters using full-length 16S rRNA gene amplicon sequencing

Water Research ◽

10.1016/j.watres.2020.115815 ◽

2020 ◽

Vol 178 ◽

pp. 115815 ◽

Cited By ~ 2

Author(s):

Theo Y.C. Lam ◽

Ran Mei ◽

Zhuoying Wu ◽

Patrick K.H. Lee ◽

Wen-Tso Liu ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Diversity ◽

Amplicon Sequencing ◽

Full Length ◽

Rrna Gene ◽

Anaerobic Digesters

Download Full-text

Establishment and Assessment of An Amplicon Sequencing Method Targeting The 16S-ITS-23S rRNA Operon For Analysis of The Equine Gut Microbiome

10.21203/rs.3.rs-156589/v1 ◽

2021 ◽

Author(s):

Yuta Kinoshita ◽

Hidekazu NIWA ◽

Eri UCHIDA-FUJII ◽

Toshio NUKADA

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Full Length ◽

Rrna Operon ◽

Rrna Genes ◽

Taxonomic Resolution ◽

23S Rrna ◽

Rrna Gene ◽

Fecal Samples

Abstract Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had >90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a third to almost a half, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to reflect archaeal genomes, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of bacterial and archaeal microbiota.

Download Full-text