Dataset Information

Multicenter assessment of shotgun metagenomics for pathogen detection.

ABSTRACT:

Background

Shotgun metagenomics has been used clinically for diagnosing infectious diseases. However, most technical assessments have been limited to individual sets of reference standards, experimental workflows, and laboratories.

Methods

A reference panel and performance metrics were designed and used to examine the performance of shotgun metagenomics at 17 laboratories in a coordinated collaborative study. We comprehensively assessed the reliability, key performance determinants, reproducibility, and quantitative potential.

Findings

Assay performance varied significantly across sites and microbial classes, with a read depth of 20 millions as a generally cost-efficient assay setting. Results of mapped reads by shotgun metagenomics could indicate relative and intra-site (but not absolute or inter-site) microbial abundance.

Interpretation

Assay performance was significantly impacted by the microbial type, the host context, and read depth, which emphasizes the importance of these factors when designing reference reagents and benchmarking studies. Across sites, workflows and platforms, false positive reporting and considerable site/library effects were common challenges to the assay's accuracy and quantifiability. Our study also suggested that laboratory-developed shotgun metagenomics tests for pathogen detection should aim to detect microbes at 500 CFU/mL (or copies/mL) in a clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with an efficient read depth of 20M.

Funding

This work was supported by National Science and Technology Major Project of China (2018ZX10102001).

SUBMITTER: Liu D

PROVIDER: S-EPMC8608867 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Publications

Multicenter assessment of shotgun metagenomics for pathogen detection.

Liu Donglai D Zhou Haiwei H Xu Teng T Yang Qiwen Q Mo Xi X Shi Dawei D Ai Jingwen J Zhang Jingjia J Tao Yue Y Wen Donghua D Tong Yigang Y Ren Lili L Zhang Wen W Xie Shumei S Chen Weijun W Xing Wanli W Zhao Jinyin J Wu Yilan Y Meng Xianfa X Ouyang Chuan C Jiang Zhi Z Liang Zhikun Z Tan Haiqin H Fang Yuan Y Qin Nan N Guan Yuanlin Y Gai Wei W Xu Sihong S Wu Wenjuan W Zhang Wenhong W Zhang Chuntao C Wang Youchun Y

EBioMedicine 20211120

<h4>Background</h4>Shotgun metagenomics has been used clinically for diagnosing infectious diseases. However, most technical assessments have been limited to individual sets of reference standards, experimental workflows, and laboratories.<h4>Methods</h4>A reference panel and performance metrics were designed and used to examine the performance of shotgun metagenomics at 17 laboratories in a coordinated collaborative study. We comprehensively assessed the reliability, key performance determinant ...[more]

PMID: 34814051

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Project description:Identification and phenotypic drug-susceptibility testing for mycobacteria are time-consuming and challenging but essential for managing mycobacterial infections. Next-generation sequencing (NGS) technologies can increase diagnostic speed and quality, but standardization is still lacking for many aspects (e.g., unbiased extraction, host depletion, bioinformatic analysis). Targeted PCR approaches directly on sample material are limited by the number of targets that can be included. Unbiased shotgun metagenomics on direct material is hampered by the massive amount of host DNA, which should be removed to improve the microbial detection sensitivity. For this reason, we developed a method for NGS-based diagnosis of mycobacteria directly from patient material. As a model, we used the non-tuberculous mycobacterium (NTM) Mycobacterium abscessus. We first compared the efficiency of three different DNA extraction kits for isolating DNA (quality and concentration). The two most efficient kits were then used in a follow-up study using artificial sputum. Finally, one extraction kit was selected and further evaluated for DNA isolation from a patients' sputum mixture spiked with M. abscessus at three concentrations (final concentrations 108, 107, 106 CFU/ml). The spiked sputum samples were processed with and without saponin treatment (ST) in combination with DNAse treatment prior to bacterial DNA extraction to evaluate the recovery of bacteria and depletion of host DNA by PCR and Illumina sequencing. While Ct values of the qPCR targeting mycobacterial ITS DNA remained rather stable, Ct values in the qPCR targeting the human β-actin gene increased by five Ct values in ST samples. In subsequent Illumina sequencing, a decrease of 89% of reads mapped to the human genome was observed in ST samples. The percentage of reads mapped to M. abscessus (108 CFU/ml) increased by 89%, and the sequencing depth increased two times when undergoing ST. In conclusion, the sensitivity of M. abscessus detection in artificial sputum was increased using a saponin pre-treatment step. The saponin followed by the DNase I treatment approach could be efficiently applied to detect and characterize mycobacterial infections, including tuberculosis, directly from sputum.

Dataset Information

Multicenter assessment of shotgun metagenomics for pathogen detection.

Background

Methods

Findings

Interpretation

Funding

Publications

Multicenter assessment of shotgun metagenomics for pathogen detection.

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