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.
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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