Benchmarking next- versus third-generation sequencing in metagenomics: performance metrics and diagnostic efficacy

Area of Science:

• Metagenomics and Microbial Diagnostics
• Next-Generation Sequencing (NGS) and Third-Generation Sequencing (TGS) Technologies

Background:

• Current pneumonia diagnostics are slow and may miss key pathogens, hindering timely and effective treatment.
• Metagenomic sequencing, using short-read (NGS) and long-read (TGS) platforms, offers potential for rapid and comprehensive microbial identification.

Purpose of the Study:

• To compare the analytical characteristics and diagnostic performance of NGS and TGS for metagenomic pathogen detection.
• To evaluate the impact of host DNA depletion on TGS performance and turnaround time.
• To provide practical guidance for implementing sequencing-based diagnostics in clinical settings.

Main Methods:

• Comparative analysis of Illumina (NGS), MGI (NGS), and Oxford Nanopore Technologies (ONT, TGS) platforms.
• Utilized defined mock microbial communities and 62 clinical bronchoalveolar lavage fluid (BALF) samples.
• Assessed diagnostic performance against culture, clinical microbiological tests (CMT), and a composite reference standard (CRS).

Main Results:

• ONT TGS achieved a significantly faster turnaround time (4-6 h) compared to NGS platforms (14-20 h).
• Host DNA depletion reduced ONT's false-negative rate for low-abundance microbes from 43.3% to 6.7%.
• Both NGS and TGS demonstrated high sensitivity and positive percent agreement (93.3% and 90.7%) against CRS, outperforming culture in polymicrobial infections.

Conclusions:

• Short-read sequencing offers high sensitivity and stability for pathogen detection.
• Host-depleted long-read nanopore sequencing provides a substantial turnaround-time advantage and is a valuable complementary approach for time-sensitive diagnostics.
• Sequencing-based methods significantly improve pathogen recovery, especially in complex polymicrobial infections, compared to traditional culture methods.