Nanopore sequencing of the 16S rRNA (V1-V3) and rpoB genes directly from clinical samples.

Broad-range amplification and sequencing of the 16S rRNA gene enhances bacterial infection diagnosis. Recent improvements in sequencing accuracy of the Oxford Nanopore Technologies (ONT) sequencing platform, makes it a promising option for this approach in clinical microbiology. However, even full-length 16S rRNA sequencing may lack resolution to distinguish closely related species, a problem exacerbated by the remaining ONT error rate. The rpoB gene is an alternative bacterial phylogenetic marker providing better species-level discrimination. We investigate a strategy combining ONT sequencing of the 16S rRNA (V1-V3) gene with a segment of the rpoB gene. Our results on complex clinical samples showed that rpoB ONT sequencing provided species-level identification in 91.2% of detections, as compared to 62.8% with 16S rRNA ONT sequencing. However, 16S rRNA ONT sequencing provided a higher sensitivity of 93.0%. Combining 16S rRNA and rpoB ONT sequencing yielded a sensitivity of 98.8% with 89.0% species-level discrimination. For both gene targets, ONT sequencing paralleled Illumina sequencing. Combining ONT sequencing with high-sensitivity broad-range amplification of the 16S rRNA gene and amplification of the rpoB gene for improved taxonomic resolution, enable accurate bacterial detection and identification directly from clinical samples. This approach represents a promising tool for clinical microbiology diagnostics.

16S核糖体RNA（16S rRNA）基因的广谱扩增与测序技术可提升细菌感染诊断效能。牛津纳米孔科技（Oxford Nanopore Technologies, ONT）测序平台的测序准确率近年取得显著提升，使其成为临床微生物学领域应用该检测方案的极具潜力的备选平台之一。然而，即便采用全长16S rRNA测序，其分辨能力仍不足以区分亲缘关系相近的菌种，而ONT平台尚存的测序错误率进一步加剧了这一难题。rpoB基因是一种备选的细菌系统发育标记基因，可提供更优异的菌种水平分辨能力。本研究探讨了一种联合检测策略：对16S rRNA（V1-V3）基因与rpoB基因片段分别开展ONT测序。针对复杂临床样本的研究结果显示，rpoB基因ONT测序的菌种水平鉴定检出率可达91.2%，而16S rRNA基因ONT测序的对应检出率仅为62.8%。但16S rRNA基因ONT测序的检测灵敏度更高，可达93.0%。联合16S rRNA与rpoB基因ONT测序则可实现98.8%的检测灵敏度，以及89.0%的菌种水平鉴定准确率。针对这两类基因靶点，ONT测序的表现与因美纳（Illumina）测序平台相当。将ONT测序与高灵敏度16S rRNA基因广谱扩增技术、rpoB基因扩增技术相结合以提升分类学分辨能力，可直接从临床样本中实现精准的细菌检测与鉴定。该方案为临床微生物学诊断提供了一款极具潜力的工具。