Detecting New SARS-CoV-2 Variants and Other Microbiota in COVID-19 Patients
In a new study, Twist Bioscience and Biotia teamed up to detect new SARS-CoV-2 variants and other COVID-19-associated microbiota. The partnership leveraged Twist’s expertise in target enrichment and Biotia’s software chops to make the first hybrid capture-based NGS assay for tracking viral evolution.
Since COVID-19 was declared a pandemic more than one year ago, RT-PCR has been the go-to method for detecting SARS-CoV-2 infection because it offers high sensitivity for a relatively low cost. However, the continued emergence of variants like those originating from the UK (B.1.1.7), South Africa (B.1.351), and Brazil (P.1) threatens to make diagnostic tests more prone to failure.
RT-PCR tests that form the backbone of the current global COVID-19 response rely on primer sequences that exactly match target SARS-CoV-2 sequences, which continue to change as the virus evolves.
Recognizing the potential issue with RT-PCR-based tests, Twist and Biotia came up with a joint solution: an end-to-end workflow capable of identifying the novel SARS-CoV-2 and any present or future novel variants.
Case numbers outpace surveillance efforts
Efforts to track the evolution of SARS-CoV-2 have lagged far behind case numbers, but are improving. At the time of writing, the US has hit its weekly goal of 7000 positive samples sequenced for seven straight weeks, accounting for around 11% of the weekly moving average case number.
Simply, detecting SARS-CoV-2 is cheap. But sequencing its genome? That’s much more expensive.
There are essentially three ways to sequence a SARS-CoV-2 genome. One uses “shotgun” metagenomics using Next Generation Sequencing (NGS) to sequence all the genetic material found in a clinical sample - viral and human. While comprehensive, the sequencing of the human genetic material is costly and unnecessary. It also requires that the sample is sequenced to a higher depth for accurate detection, given the viral genetic material is a very small proportion of the total sample.
Two additional ways take a more targeted approach. One relies on PCR amplification to enrich for target sequences (SARS-CoV-2 genomes, in this case) that are then sequenced by NGS. The other relies on biotinylated hybridization capture probes to accomplish the same goal.
While both targeted NGS sequencing approaches enable viral evolution tracking at lower depths of sequencing (and therefore lower cost), hybrid capture offers a clear advantage: hybrid capture probes can tolerate mismatches better than PCR primers. This allows the assay to remain robust in the face of continual viral evolution.
Arguably, hybrid capture probes offer the most robust genomic surveillance on the market. Where PCR-based methods require an exact match to the viral genome, hybrid capture-based methods are able to tolerate mismatches of up to 20% of the viral genome. In the case of Twist’s target capture tools for SARS-CoV-2 surveillance, mismatch tolerance comes from four probes targeting every region of the viral genome, and capture probes being 120-base double-stranded DNA instead of the standard 20 to 30-base single-stranded DNA for PCR primers.
Twist & Biotia partner for new SARS-CoV-2 NGS Assay
To devise robust hybrid capture reagents for targeted sequencing of SARS-CoV-2 genomes, Twist and Biotia played to their strengths: Twist employed its DNA synthesis platform and target enrichment prowess, and Biotia on its software expertise. The system’s clinical performance is described in a recently deposited medRxiv preprint titled “Targeted Hybridization Capture of SARS-CoV-2 and Metagenomics Enables Genetic Variant Discovery and Nasal Microbiome Insights”
In the study, Twist and Biotia first demonstrated the clinical performance of their end-to-end workflow using 120 clinical samples (60 positive, 60 negative). Among these samples, the assay achieved positive and negative predictive agreements of 95.2% and 98.3% with the RT-PCR detection method. This means the new NGS assay’s results — both positive and negative — matched the gold-standard diagnostic approach more than 95% of the time.
In addition, analytical validation showed that the NGS assay could detect SARS-CoV-2 down to 800 genome copies per milliliter, making it more sensitive than many other NGS assays authorized for emergency use by the FDA.
Subsequent SARS-CoV-2 lineage analysis using Biotia software identified 261 mutations across an additional 71 positive samples, including 107 mutations not described in the GISAID (Global Initiative on Sharing Avian Influenza Data) database. Of the newly discovered mutations, 24 were found in the Spike protein, the primary therapeutic target of most neutralizing antibody and vaccine development campaigns.
The assay was also able to detect mutations specific to different geographic locations. For example, one mutation (T6394C) was observed in samples collected from Tennessee, but not New York.
Overall, these data highlight the exceptional performance of the new NGS assay, especially its ability to identify new variants that originate in specific geographic locations. According to the preprint, the “hybrid capture NGS assay, coupled with optimized software, is a powerful approach to detect and comprehensively map SARS-CoV-2 genetic variants for tracking viral evolution and guiding vaccine updates.”
Interrogating the COVID-19 microbiome
The targeted sequencing approach developed by Twist and Biotia could bolster ongoing efforts to reduce the morbidity and mortality associated with COVID-19 by making it easier to discover and track newer and more dangerous variants. That said, broader sequencing strategies are still needed to better understand why some individuals are more susceptible to COVID-19. The answer may lie in the nasal microbiome — the community of commensal pathogens found inside your nose.
Even before the pandemic, researchers speculated that the nasal microbiome helps maintain the nasal mucosal barrier and modulates immune responses against upper respiratory pathogens. To determine whether this was the case for COVID-19 patients, Twist and Biotia compared the microbiome of COVID-19-positive and negative nasopharyngeal samples.
Combining shotgun metagenomics with Biotia analysis software, the team observed no significant differences in the overall microbiome richness or evenness between the two sample types. However, they did observe an overrepresentation of several bacterial taxa, including Prevotella salivae. An abundance P. salivae frequently coincides with other upper respiratory tract pathologies, including respiratory infection and asthma.
The metagenomic analysis also found an overrepresentation of macrolide drug resistance in the nasal microbiome of SARS-CoV-2 patients. This presumably reflects the increased use of antibiotics to treat pneumonia. Thus, metagenomics can offer insight into the impact the COVID-19 pandemic has had on the development of antibiotic resistance.
In summary, despite over a year of research, we as a global community still have much to learn about COVID-19. The tools developed by Twist and Biotia will help facilitate this learning process and inform the global coronavirus response.
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