Application of next-generation sequencing to identify different pathogens

Abstract

Early and precise detection and identification of various pathogens are essential for epidemiological monitoring, disease management, and reducing the prevalence of clinical infectious diseases. Traditional pathogen detection techniques, which include mass spectrometry, biochemical tests, molecular testing, and culture-based methods, are limited in application and are time-consuming. Next generation sequencing (NGS) has emerged as an essential technology for identifying pathogens. NGS is a cutting-edge sequencing method with high throughput that can create massive volumes of sequences with a broad application prospects in the field of pathogen identification and diagnosis. In this review, we introduce NGS technology in detail, summarizes the application of NGS in that identification of different pathogens, including bacteria, fungi, and viruses, and analyze the challenges and outlook for using NGS to identify clinical pathogens. Thus, this work provides a theoretical basis for NGS studies and provides evidence to support the application of NGS in distinguishing various clinical pathogens.

Keywords: Sanger; bacteria; fungi; next generation sequencing; pathogens.

Figure 1

The Sanger sequencing method in seven steps. Including denaturing dsDNA, forming multiple copies of a segment, attaching primer, addition of polymerase solutions, amplifying the chains, denaturing chains, and electrophoreses solution. Dideoxynucleotides are labeled with four fluorescent markers, and the extension of the DNA strand is terminated when ddNTP is incorporated into the DNA strand. The resulting mixture of DNA fragments of different lengths is separated in capillary electrophoresis, and the base type and accuracy are determined by detecting the fluorescence intensity of different colors after laser irradiation.

Figure 2

Comparing the different processes of the Sanger method and NGS in detecting different pathogens. Sanger method’s methodology comprises amplification, automated cycling, sequence analysis, and reconstruction. This technique generates several copies of the target DNA region. The workflow of NGS (take Illumina for example) in clinical setup, including sample separation and preparation, NGS based on the addressed request, Illumina process, related bioinformatics data processing, and retrieval of the final results. The different procedures of two methods result in different application in detecting various pathogens, including viruses, bacteria, and fungi.