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

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.

Keywords: (Shotgun) metagenomics; 16S rRNA gene; Microbiota analysis; Next-generation sequencing; Pitfalls and biases; Routine clinical microbiological diagnostics.

Fig. 1

General overview of 16S rRNA gene NGS and shotgun metagenomics methods. Both methods start with the extraction of nucleic acids from a microbial sample. Next, the extracted DNA is either subjected to 16S rRNA gene PCR amplification (16S rRNA gene NGS) or sheared into small DNA fragments (shotgun metagenomics). The resultant 16S rRNA gene amplicons, or sheared DNA fragments, are sequenced using NGS techniques. Finally, all sequence data are processed using an extensive array of bioinformatics algorithms that allows the researcher to explore the taxonomic composition and/or the functional capacity of the sample tested. OTU operational taxonomic units—a group of very similar sequences.