Emu: species-level microbial community profiling of full-length 16S rRNA Oxford Nanopore sequencing data.

16S ribosomal RNA-based analysis is the established standard for elucidating the composition of microbial communities. While short-read 16S rRNA analyses are largely confined to genus-level resolution at best, given that only a portion of the gene is sequenced, full-length 16S rRNA gene amplicon sequences have the potential to provide species-level accuracy. However, existing taxonomic identification algorithms are not optimized for the increased read length and error rate often observed in long-read data. Here we present Emu, an approach that uses an expectation-maximization algorithm to generate taxonomic abundance profiles from full-length 16S rRNA reads. Results produced from simulated datasets and mock communities show that Emu is capable of accurate microbial community profiling while obtaining fewer false positives and false negatives than alternative methods. Additionally, we illustrate a real-world application of Emu by comparing clinical sample composition estimates generated by an established whole-genome shotgun sequencing workflow with those returned by full-length 16S rRNA gene sequences processed with Emu.

Curry KD ，Wang Q ，Nute MG ，Tyshaieva A ，Reeves E ，Soriano S ，Wu Q ，Graeber E ，Finzer P ，Mendling W ，Savidge T ，Villapol S ，Dilthey A ，Treangen TJ ... -  《-》

Microbial Community Profiling Protocol with Full-length 16S rRNA Sequences and Emu.

16S rRNA targeted amplicon sequencing is an established standard for elucidating microbial community composition. While high-throughput short-read sequencing can elicit only a portion of the 16S rRNA gene due to their limited read length, third generation sequencing can read the 16S rRNA gene in its entirety and thus provide more precise taxonomic classification. Here, we present a protocol for generating full-length 16S rRNA sequences with Oxford Nanopore Technologies (ONT) and a microbial community profile with Emu. We select Emu for analyzing ONT sequences as it leverages information from the entire community to overcome errors due to incomplete reference databases and hardware limitations to ultimately obtain species-level resolution. This pipeline provides a low-cost solution for characterizing microbiome composition by exploiting real-time, long-read ONT sequencing and tailored software for accurate characterization of microbial communities. © 2024 Wiley Periodicals LLC. Basic Protocol: Microbial community profiling with Emu Support Protocol 1: Full-length 16S rRNA microbial sequences with Oxford Nanopore Technologies sequencing platform Support Protocol 2: Building a custom reference database for Emu.

Curry KD ，Soriano S ，Nute MG ，Villapol S ，Dilthey A ，Treangen TJ ... -  《-》

The newest Oxford Nanopore R10.4.1 full-length 16S rRNA sequencing enables the accurate resolution of species-level microbial community profiling.

Zhang T ，Li H ，Ma S ，Cao J ，Liao H ，Huang Q ，Chen W ... -  《-》

Comparative evaluation of analytical pipelines for illumina short- and nanopore long-read 16S rRNA gene amplicon sequencing with mock microbial communities.

Utility of a recently developed long-read pipeline, Emu, was assessed using an expectation-maximization algorithm for accurate read classification. We compared it to conventional short- and long-read pipelines, using well-characterized mock bacterial samples. Our findings highlight the necessity of appropriate data-processing for taxonomic descriptions, expanding our understanding of the precise microbiome.

Ota Y ，Yasunaga K ，Mahazu S ，Prah I ，Nagai S ，Hayashi T ，Suzuki M ，Yoshida M ，Hoshino Y ，Akeda Y ，Suzuki T ，Gu Y ，Saito R ... -  《-》

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

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. 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. 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.

Matsuo Y ，Komiya S ，Yasumizu Y ，Yasuoka Y ，Mizushima K ，Takagi T ，Kryukov K ，Fukuda A ，Morimoto Y ，Naito Y ，Okada H ，Bono H ，Nakagawa S ，Hirota K ... -  《BMC MICROBIOLOGY》