# Assessment of Microbiome-Based Pathogen Detection Using Illumina Short-Read and Nanopore Long-Read Sequencing in 144 Patients Undergoing Bronchoalveolar Lavage in a University Hospital in Germany

## TL;DR

This study compares Illumina and Nanopore sequencing for detecting pathogens in lung fluid samples from 144 patients, showing that NGS can identify more bacteria than traditional culturing.

## Contribution

The study evaluates the performance of short-read and long-read sequencing for pathogen detection in bronchoalveolar lavage samples, highlighting their potential in clinical diagnostics.

## Key findings

- Short-read sequencing detected cultured bacteria at the genus level in ~85% of cases.
- Long-read sequencing identified the uncommon pathogen Tropheryma whipplei in three cases missed by culturing.
- NGS revealed a broader spectrum of bacteria and fastidious potential pathogens compared to culture.

## Abstract

Lower respiratory tract infections (LRTIs) represent a significant global health concern, and the accurate identification of pathogens is crucial for patient care. Culture-based methods are the gold standard, but their detection abilities are limited. Next-generation sequencing (NGS) offers a promising method for comprehensive microbial detection, providing valuable information for clinical practice. In this study, 144 bronchoalveolar lavage fluid samples were collected, culture-based diagnostics were performed, and bacterial microbiome profiles were generated by short-read sequencing of the V4 region of the 16S rRNA gene using Illumina technologies and long-read sequencing with Oxford Nanopore Technologies (ONT) to determine the full-length 16S rRNA gene. The most common genera detected by NGS included Streptococcus, Staphylococcus, Veillonella, Prevotella, Rothia, Enterococcus, and Haemophilus. Short-read sequencing detected cultured bacteria at the genus level in ~85% of cases, while long-read sequencing demonstrated agreement with cultured species in ~62% of cases. In three cases, long-read sequencing identified the uncommon potential lung pathogen Tropheryma whipplei not detected with traditional culturing techniques. The NGS results showed a partial overlap with culture as the current diagnostic gold standard in LRTI. Additionally, NGS detected a broader spectrum of bacteria, revealed fastidious potential pathogens, and offered deeper insights into the complex microbial ecosystem of the lungs.