Understanding and overcoming the pitfalls and biases of next-generation sequencing (NGS) methods for use in the routine clinical microbiological diagnostic laboratory

Stefan A Boers,Ruud Jansen,John P Hays

doi:10.1007/s10096-019-03520-3

Abstract

Recent advancements in next-generation sequencing (NGS) have provided the foundation for modern studies into the composition of microbial communities. The use of these NGS methods allows for the detection and identification of (‘difficult-to-culture’) microorganisms using a culture-independent strategy. In the field of routine clinical diagnostics however, the application of NGS is currently limited to microbial strain typing for epidemiological purposes only, even though the implementation of NGS for microbial community analysis may yield clinically important information. This lack of NGS implementation is due to many different factors, including issues relating to NGS method standardization and result reproducibility. In this review article, the authors provide a general introduction to the most widely used NGS methods currently available (i.e., targeted amplicon sequencing and shotgun metagenomics) and the strengths and weaknesses of each method is discussed. The focus of the publication then shifts toward 16S rRNA gene NGS methods, which are currently the most cost-effective and widely used NGS methods for research purposes, and are therefore more likely to be successfully implemented into routine clinical diagnostics in the short term. In this respect, the experimental pitfalls and biases created at each step of the 16S rRNA gene NGS workflow are explained, as well as their potential solutions. Finally, a novel diagnostic microbiota profiling platform (‘MYcrobiota’) is introduced, which was developed by the authors by taking into consideration the pitfalls, biases, and solutions explained in this article. The development of the MYcrobiota, and future NGS methodologies, will help pave the way toward the successful implementation of NGS methodologies into routine clinical diagnostics.

Highlights

The detection, identification, and characterization of pathogenic microorganisms is the major step in establishing appropriate treatment for infectious diseases
Obligate anaerobes are known to cause serious infections, yet their detection may be sub-optimal within routine clinical microbiological diagnostic laboratories using traditional specimen collection and detection techniques, as special precautions are required to help preserve the anaerobic environment during specimen collection and transport, and the laboratory needs to provide growth components for culture-based detection methods [17]
The adaptation of collection, transport, and culture-independent next-generation sequencing (NGS) methods could play a major role in the detection and identification of anaerobic infections, or any other infection caused by fastidious or viable but non-culturable (VBNC) microorganisms—examples of VBNCs include antibiotic ‘damaged’ microorganisms that may be present within patients during antimicrobial therapy [18]

Summary

Introduction

The detection, identification, and characterization of pathogenic microorganisms is the major step in establishing appropriate (antimicrobial) treatment for infectious diseases. Shotgun metagenomics is an alternative approach to characterizing microbial communities that, in contrast to targeted amplicon methods, sequences the DNA content of a clinical sample directly and produces relative abundance information for all genes detected (including for example 16S rRNA genes).

Results

Conclusion