The power of next-generation sequencing (NGS) has helped researchers and clinicians gain a better understanding of the genetic mechanisms behind the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the novel coronavirus disease (COVID-19), specifically in evaluating patient isolates. These are SARS-CoV-2 extracted from samples belonging to a single patient. There is currently only one strain of SARS-CoV-2, although there may be some changes in genome sequences.1
The significance of analyzing SARS-CoV-2 isolates include detection and identification of:
Phylogenetic relationships to follow viral spread. Using whole-genome sequencing (WGS) of SARS-CoV-2 in patients who were quarantined on a cruise ship, researchers were able to conduct network/phylogenetic analysis of the COVID-19 outbreak on board. It showed that the outbreak can be traced back to a single introduction, suggesting that using sequencing techniques can reveal transmission routes.2 A similar study used WGS to study the viral genome and determine epidemiological links and transmission events in Germany, being able to identify its origin.3
Nucleotide changes in patient isolates. Some mutations can increase infectivity such as in the spike protein D614G. Surveillance of these variants will impact how healthcare providers and regulatory agencies develop diagnostics and therapeutic interventions.4
Potential new strains. There is a possibility of changes in the biological properties of SARS-CoV-2, which could lead to a new strain. In this scenario, identification of this novel strain using methods such as NGS targeted capture could facilitate swift and effective intervention strategies.
Emerging resistance mutations to antiviral drugs. Remdesivir is an antiviral therapy that has shown to shorten hospitalization recovery time and lower respiratory tract infection in COVID-19 patients.5 If specific patients were to develop a resistance to remdesivir, genetic analysis of isolates using NGS could assist in determining which mutation caused it.
Scientists can use two workflows to sequence virus isolates. One method is to use whole viral genome sequencing, while the other is custom target enrichment panels. Using more advanced techniques, such as hybrid capture methods, can lead to improved uniform coverage and better detection of mutation variants.
With NGS RNA-seq, there is a large array of host responses that can be investigated including innate gene expression patterns or viral gene expression patterns in combination with host patterns. Researchers can use different sample types such as cell culture, animal models, patient blood, and tissue.
The graph shows an example of an RNA-seq workflow. Roche offers a product portfolio that provides workflow solutions at every step. The RNA enrichment stage involves either mRNA capture or the depletion of highly abundant ribosomal transcripts. The core library construction process includes RNA fragmentation, cDNA synthesis, A-tailing, and adapter ligation. In later stages, high-efficiency and low-bias library amplification and library quantitation before multiplexing, capture, or sequencing is incorporated into the workflow to improve NGS testing efficiency.
With a portfolio of NGS products that can help researchers gain insight into infectious disease, Roche remains committed to being at the forefront of the COVID-19 pandemic. Diving deeper into the genetic factors that follow transmission routes, new strains, and drug resistance will lead to improved diagnostics, vaccine development, and therapeutic strategies.
“NGS has provided insight in the type of virus, phylogeny, immunology and patient response, and ultimately has sped up the response to this pandemic significantly,” said Toumy Guettouche PhD, Director, Reagent and Assay Development at Roche Sequencing Solutions.