A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification

Metagenomic next-generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing-based mNGS method was first developed and evaluated using a set of samples with known aetiology. Its sensitivity for RNA viruses (influenza A, H1N1) was < 6.4 × 10 2 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then, the rapid turnaround time of Nanopore sequencing was tested by sequencing influenza A virus and adenoviruses. Furthermore, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analysed, and the pathogens identified by Illumina sequencing showed 81.8% concordance with qPCR results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1-positive patients with meningitis/encephalitis detected HIV-1 RNA and Toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol that realizes efficient metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.

Abstract licence: CC BY-NC-ND

1. 8.

   Performance of genetic programming optimised Bowtie2 on genome comparison and analytic testing (GCAT) benchmarks · 2015

2. 10.

   Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis · 2015

3. 13.

   Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples · 2014

4. 25.

   Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation · 2019

5. 31.

   Centrifuge: rapid and sensitive classification of metagenomic sequences · 2016

6. 37.

   Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples · 2019

34 more references, whose wording the paper’s licence keeps private.

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.