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

Abstract

33Species-level genetic characterization of complex bacterial communities has important 34 clinical applications. In the present study, we assessed the performance of full-length 35 16S rRNA gene analysis of human gut microbiota using the nanopore long-read 36 sequencer MinION™. A new strategy for library construction with an optimized primer 37 set overcame PCR-associated bias and produced accurate taxonomic classifications of a 38 broad range of bacterial species. Our present microbiome study, comparing the 39 discriminatory power of full-length and short-read sequencing, clearly illustrated the 40 analytical advantage of sequencing the full-length 16S rRNA gene, which provided 41 higher species-level resolution and accuracy. 42 43 Keywords: 44 16S rRNA, gut microbiota, metagenome, MinION™, nanopore sequencing 45 46 4 Background 47 Recent advances in DNA sequencing technology have had a revolutionary impact on 48 clinical microbiology [1]. Next-generation sequencing (NGS) technology enables 49 parallel sequencing of DNA on a massive scale to generate vast quantities of accurate 50 data. NGS platforms are now increasingly used in the field of clinical research [2]. 51 Metagenomic sequencing offers numerous advantages over traditional culture-based 52 techniques that have long been the standard test for detecting pathogenic bacteria. This 53 method is particularly useful for characterizing uncultivable bacteria and novel 54 pathogens [3].55 Among the metagenomic sequencing strategies, amplicon sequencing of the 16S 56 ribosomal RNA (rRNA) gene has proven to be a reliable and efficient option for 57 taxonomic classification [4, 5]. The bacterial 16S rRNA gene contains nine variable 58 regions (V1 to V9) that are separated by highly conserved sequences across different 59 taxa. For bacterial identification, the 16S rRNA gene is first amplified by polymerase 60 chain reaction (PCR) with primers annealing to conserved regions and then sequenced.61 The sequencing data are subjected to bioinformatic analysis in which the variable 62 regions are used to discriminate between bacterial taxa [6]. 63 Since the conventional parallel-type short-read sequencer cannot yield reads covering 64 the full length of the 16S rRNA gene [7], several regions of it have been targeted for 65 sequencing, which often causes ambiguity in taxonomic classification [8]. New 66 sequencing platforms have overcome these technical restrictions, particularly those 67 affecting read length. A prime example is the MinION™ sequencer from Oxford 68Nanopore Technologies, which is capable of producing long sequences with no 69 theoretical read length limit [9][10][11]. MinION™ sequencing targets the entire 16S rRNA 70 gene, allowing the identification of bacteria with more accuracy and sensitivity [12, 13]. 71Furthermore, MinION™ produces sequencing data in real time, which reduces 72 turnaround time for data processing [14, 15]. 73 5Given these features of MinION™ sequencing, we had previously conducted full-length 74 16S amplicon sequencing analyse...

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“…Moreover, we could not detect Proprionibacterium in our nanopore dataset, even though it was detected as a NICU biomarker by Illumina sequencing. Approaches such as the design of custom primers have been proved to be effective in circumventing amplification limitations and providing more accurate community profiles with nanopore sequencing efforts (23).…”

“…Thus, most reports for microbial community analysis using 16S we found applied different Q Score thresholds (usually Q Score > 7) and variable cutoff values (e.g. 1300-1950bp, <1700bp, 1400-1700 bp, and 1350-1650 bp) (19,(22)(23)(24).…”

Homogeneous selection promotes microdiversity in the glacier-fed stream microbiome

“…Nevertheless, the short amplicon lengths used in most studies might not be adequate to properly resolve the topology at the tips of the phylogeny. While this should have no effect on the identification of phylogenetic clades with distinct z-scores compared to outgroups, it might affect the identification of specific SVs and thus full-length 16S rRNA gene amplicons [66][67][68] might be used to construct phylogenetic trees with highly supported topologies near the tips.…”

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“…Full-length 16S rRNA sequencing using Pac-Bio, MiniION or Nanopore and shotgun sequencing [37][38][39][40] offer advantages over shortread sequencing, including the low cost and rapidness as well as the higher resolution and accuracy of Nanopore sequencing. 38 However, short-read sequencing is cost effective, more sensitive for microbial genes, and more broadly distributed than full-length sequencing.…”