Amplicon Deep Sequencing Elucidates the Complexity of Infection in Pathogens of Global Health Importance

Abstract

Next-generation sequencing (NGS) technologies have revolutionized the field of genomics, diagnostics, and surveillance. NGS enables rapid, cost-effective, and high-throughput analysis of nucleic acids. Many clinical applications of NGS can be used for detecting genetic variants associated with a disease, treatment failure, or surveillance purposes. Amplicon deep sequencing (ADS) is a powerful and highly sensitive molecular tool that allows for the characterization of an amplified product from a genomic region of interest. ADS can be used to accurately detect single nucleotide polymorphisms (SNPs), perform small genome assembly, identify the components within a microbial community, and act as a surveillance tool for pathogens of interest. In this thesis, I have described the applicability of ADS to three different microorganisms belonging to three taxonomic systems: Clostridioides difficile (a Gram-positive bacteria), SARS-CoV-2 (positive-sense single-stranded RNA virus), and Plasmodium falciparum (protozoan parasite). The findings of ADS applied to patients with C. difficile infection (CDI) have shown that the gut microbial diversity of CDI individuals is significantly lower when compared to a healthy cohort or to symptomatic patients with no CDI. ADS was also employed for the detection of SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI) by amplifying and sequencing the S gene of the virus, followed by a haplotype assessment. The results of this experiment indicate that ADS generates a higher depth of coverage which increases the likelihood of calling a VOC/VOI. The workflow employed here allowed for the appropriate mutant analysis of samples with a low viral load (Ct > 30). Finally, ADS was utilized for haplotype assessment and SNP calling in two longitudinal studies of patients diagnosed with P. falciparum malaria under constant treatment. The results of the study have demonstrated the superior power of ADS over traditional genotyping techniques. Overall, the findings in this thesis contribute to the literature on the epidemiological and transmission variables revolving around SARS-CoV-2, Clostridioides difficile, and Plasmodium falciparum. Amplicon deep sequencing can be easily scalable and adapted to any biological system to examine the behaviour of certain pathogens further.