Next-generation sequencing in the analysis of human microbiota: essential considerations for clinical application

The development of next-generation sequencing (NGS) presents an unprecedented opportunity to investigate the complex microbial communities that are associated with the human body. It offers for the first time a basis for detailed temporal and spatial analysis, with the potential to revolutionize our understanding of many clinically important systems. However, while advances continue to be made in areas such as PCR amplification for NGS, sequencing protocols, and data analysis, in many cases the quality of the data generated is undermined by a failure to address fundamental aspects of experimental design. While little is added in terms of time or cost by the analysis of repeat samples, the exclusion of DNA from dead bacterial cells and the extracellular matrix, the use of efficient nucleic acid extraction methodologies, and the implementation of safeguards to minimize the introduction of contaminating nucleic acids, such considerations are essential in achieving an accurate representation of the system being studied. In this review, the chronic bacterial infections that characterize lower respiratory tract infections in cystic fibrosis patients are used as an example system to examine the implications of a failure to address these issues when designing NGS-based analysis of human-associated microbiota. Further, ways in which the impact of these factors can be minimized are discussed.