High accuracy meets high throughput for near full-length 16S ribosomal RNA amplicon sequencing on the Nanopore platform

Xuan Lin, Katherine Waring, Hans Ghezzi, Carolina Tropini, John Tyson, Ryan Ziels (2024) High accuracy meets high throughput for near full-length 16S ribosomal RNA amplicon sequencing on the Nanopore platform PNAS Nexus (IF: 3.8) 3(10) pgae411

Abstract

Small subunit (SSU) ribosomal RNA (rRNA) gene amplicon sequencing is a foundational method in microbial ecology. Currently, short-read platforms are commonly employed for high-throughput applications of SSU rRNA amplicon sequencing, but at the cost of poor taxonomic classification due to limited fragment lengths. The Oxford Nanopore Technologies (ONT) platform can sequence full-length SSU rRNA genes, but its lower raw-read accuracy has so-far limited accurate taxonomic classification and de novo feature generation. Here, we present a sequencing workflow, termed ssUMI, that combines unique molecular identifier (UMI)-based error correction with newer (R10.4+) ONT chemistry and sample barcoding to enable high throughput near full-length SSU rRNA (e.g. 16S rRNA) amplicon sequencing. The ssUMI workflow generated near full-length 16S rRNA consensus sequences with 99.99% mean accuracy using a minimum subread coverage of 3×, surpassing the accuracy of Illumina short reads. The consensus sequences generated with ssUMI were used to produce error-free de novo sequence features with no false positives with two microbial community standards. In contrast, Nanopore raw reads produced erroneous de novo sequence features, indicating that UMI-based error correction is currently necessary for high-accuracy microbial profiling with R10.4+ ONT sequencing chemistries. We showcase the cost-competitive scalability of the ssUMI workflow by sequencing 87 time-series wastewater samples and 27 human gut samples, obtaining quantitative ecological insights that were missed by short-read amplicon sequencing. ssUMI, therefore, enables accurate and low-cost full-length 16S rRNA amplicon sequencing on Nanopore, improving accessibility to high-resolution microbiome science.© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.

Links

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11462149
http://www.ncbi.nlm.nih.gov/pubmed/39386005
http://dx.doi.org/10.1093/pnasnexus/pgae411

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