Abstract
Next-generation sequencing (NGS) technologies have been applied in bacterial flora analysis. However, there is no standardized protocol, and the optimal clustering threshold for estimating bacterial species in respiratory infection specimens is unknown. This study was conducted to investigate the optimal threshold for clustering 16S ribosomal RNA gene sequences into operational taxonomic units (OTUs) by comparing the results of NGS technology with those of the Sanger method, which has a higher accuracy of sequence per single read than NGS technology. This study included 45 patients with pneumonia with aspiration risks and 35 patients with lung abscess. Compared to Sanger method, the concordance rates of NGS technology (clustered at 100%, 99%, and 97% homology) with the predominant phylotype were 78.8%, 71.3%, and 65.0%, respectively. With respect to the specimens dominated by the Streptococcus mitis group, containing several important causative agents of pneumonia, Bray Curtis dissimilarity revealed that the OTUs obtained at 100% clustering threshold (versus those obtained at 99% and 97% thresholds; medians of 0.35, 0.69, and 0.71, respectively) were more similar to those obtained by the Sanger method, with statistical significance (p < 0.05). Clustering with 100% sequence identity is necessary when analyzing the microbiota of respiratory infections using NGS technology.
Highlights
Next-generation sequencing (NGS) technologies have been applied in bacterial flora analysis
We investigated the optimal threshold for clustering 16S ribosomal RNA (rRNA) gene sequences into operational taxonomic units (OTUs) using NGS technology (MiSeq sequencer; Illumina) by comparing the results with those obtained using the Sanger method
We demonstrated the difficulty in distinguishing the bacterial phylotype of the S. mitis group, which is the most common bacteria in respiratory infections, when the clustering threshold was set to low (< 99% OTUs)
Summary
Next-generation sequencing (NGS) technologies have been applied in bacterial flora analysis. This study was conducted to investigate the optimal threshold for clustering 16S ribosomal RNA gene sequences into operational taxonomic units (OTUs) by comparing the results of NGS technology with those of the Sanger method, which has a higher accuracy of sequence per single read than NGS technology. Targeted sequencing, including sequencing part of the 16S ribosomal RNA (rRNA) gene, is a superior approach for detecting and estimating bacterial species compared to metagenomic NGS methods because of their low complexity, low cost, and practical clinical application. Clustering of 16S rRNA sequences into operational taxonomic units (OTUs) was generally analyzed in the gastrointestinal and urological areas with thresholds of 99% and 97%, respectively[14,15,16,17], but it is unclear whether this approach is applicable to the microbiota in respiratory specimens. We have reported the usefulness and significance of this approach in evaluating respiratory infections[28,29,30,31,32,33]
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