Prospective Comparison Between Shotgun Metagenomics and Sanger Sequencing of the 16S rRNA Gene for the Etiological Diagnosis of Infections

Abstract

Bacteriological diagnosis is traditionally based on culture. However, this method may be limited by the difficulty of cultivating certain species or by prior exposure to antibiotics, which justifies the resort to molecular methods, such as Sanger sequencing of the 16S rRNA gene (Sanger 16S). Recently, shotgun metagenomics (SMg) has emerged as a powerful tool to identify a wide range of pathogenic microorganisms in numerous clinical contexts. In this study, we compared the performance of SMg to Sanger 16S for bacterial detection and identification. All patients’ samples for which Sanger 16S was requested between November 2019 and April 2020 in our institution were prospectively included. The corresponding samples were tested with a commercial 16S semi-automated method and a semi-quantitative pan-microorganism DNA- and RNA-based SMg method. Sixty-seven samples from 64 patients were analyzed. Overall, SMg was able to identify a bacterial etiology in 46.3% of cases (31/67) vs. 38.8% (26/67) with Sanger 16S. This difference reached significance when only the results obtained at the species level were compared (28/67 vs. 13/67). This study provides one of the first evidence of a significantly better performance of SMg than Sanger 16S for bacterial detection at the species level in patients with infectious diseases for whom culture-based methods have failed. This technology has the potential to replace Sanger 16S in routine practice for infectious disease diagnosis.

Metagenomic analysis unravels novel taxonomic differences in the uterine microbiome between healthy mares and mares with endometritis

“…The use of an Excel randomization function in the present study was intended to reduce any bias while assigning individual samples to the various pools within each group. A limitation of the 16S rRNA sequencing approach used in the present study is its inability to detect less abundant taxa in comparison to the relatively advanced and more sensitive metagenomic next generation sequencing (NGS) approach (Durazzi et al., 2021 ; Lamoureux et al., 2022 ). Future studies involving metagenomic NGS of individual samples are required to further our understanding of the uterine microbiome in healthy mares and mares with endometritis.…”

Comparison of Blood-Based Shotgun and Targeted Metagenomic Sequencing for Microbiological Diagnosis of Infective Endocarditis

“…Hong et al investigated the diagnostic accuracy of sMGS vs tMGS in sonicate fluid and found similar positive percent agreements: 72% for tMGS and 73% ( P = .83) for sMGS. Interestingly, another study found no significant difference between 16S rRNA PCR followed by Sanger sequencing and sMGS evaluated on 67 diverse tissue and fluid samples (including heart valves) [ 24 ]. However, all clinical metagenomics methods should be compared with caution.…”

Combination of Whole Genome Sequencing and Metagenomics for Microbiological Diagnostics

“…However, in primary non-sterile body sites, this may introduce a critical diagnostic bias. The performance in bacterial detection and identification was compared between shotgun metagenomics and 16S amplicon-based sequencing, where shotgun metagenomics showed a slightly higher sensitivity (46.3% vs. 38.8%) than 16S [ 119 ]. Similarly, Gu et al compared shotgun metagenomic sequencing using Illumina (short-read) and Nanopore sequencing (long-read) platforms for pathogen identification and validated the results with traditional culture-based methods and also with 16S and 28S-ITS PCRs for bacterial and fungal species.…”