South San Francsico-based Twist Bioscience Corp. and Biotia Inc., of New York, received the first FDA emergency use authorization (EUA) for a capture-based next-generation sequencing (NGS) assay for the coronavirus that causes COVID-19. The technique reduces the likelihood of misdiagnosing or failing to identify mutations compared to standard sequencing.

The in vitro diagnostic test analyzes the entire RNA viral sequence to determine the presence or absence of SARS-CoV-2 in a process that enables identification of a much larger range of genetic variants than amplicon-based sequencing, reducing the risk of missing emerging variants. The World Health Organization’s Genome Sequencing for SARS-CoV-2 noted that “[o]ne advantage of using a capture-based approach over a PCR amplicon-based approach is that capture-based approaches can tolerate sequence differences from the probe sequences of 10% to 20%.”

The Twist/Biotia assay’s ability to tolerate greater divergence in sequences arises from use of 120-base double-stranded DNA probes, while amplicon-based approaches normally use 20 to 30 bases, Twist CEO and co-founder Emily Leproust told BioWorld. The greater length makes the probes less sensitive to mismatches and permits them to bind to the larger sequence and capture its variations, resulting in more information on the evolution of the virus and fewer false negatives.

In contrast, one mismatch in the primer region in the amplicon-based approach could derail or impair binding, as seen in some RT-qPCR tests which contained an S gene target in a region that had significant mutations in the B.1.1.7 variant.

“We have shown that in the Twist capture system, we are able to efficiently capture targets that contain multiple mismatches within the probe binding regions,” Leproust said. “Combined with 4x probe tiling density, we are confident that the panel can capture all the SARS-CoV-2 variants in the foreseeable future.” High-density tiling microarrays facilitate characterization of complete genomes.

Tracking mutations

The emergence and rapid dominance in many regions of variants with a large number of mutations, increased transmissibility and potentially greater lethality has led labs and countries around the world to rapidly ramp up sequencing efforts.

A research use only report available with the Twist/Biotia assay profiles the full sequence of the virus, allowing identification of genetic variants and analysis of the lineages of SARS-CoV-2 samples. The report is especially useful for labs taking a significant role in global surveillance, though smaller labs that have stepped up to conduct variant tracking would benefit as well.

“For our research use only report we list the genetic variants [including significant mutations] and clade for individual reports, and we also provide a summary spreadsheet for all variants and clades for a batched run of samples,” Leproust noted. “The individual reports are built to easily find genetic variants of concern and the summary spreadsheet facilitates downstream analysis for researchers, including tracking variants over time, and supporting global surveillance efforts.”

Diagnostic ability

Twist Bioscience and Biotia’s NGS SARS-CoV-2 assay

The SARS-CoV-2 NGS Assay is designed to use nasopharyngeal, oropharyngeal, anterior nasal and mid-turbinate nasal swabs, nasopharyngeal wash/aspirates, nasal wash/aspirates as well as from bronchoalveolar lavage specimens from individuals suspected of having COVID-19 by their health care provider. The test can identify strains of SARS-CoV-2 in samples with as few as 800 viral copies per milliliter.

The Biotia COVID-Dx software generates a clinical report from the sample indicating the presence or absence of the virus. Labs upload Fastq files with sequencing output to the cloud to obtain the results.

A non-peer reviewed study published on the preprint server Medrxiv found that the assay had a positive and negative percent agreement of 96.7% compared to an orthogonal RE-PCR assay and more accurately identified variants. Sample sequencing with the capture-based approach takes more time than an amplicon-based approach, the company noted.

“While there are many available high-throughput diagnostic tests available for COVID-19, our solution enables clinicians and researchers the ability to sequence and surveil the evolution of mutations in the virus over time and geography,” said Leproust. “Importantly, while many labs are conducting individual sequencing runs for each patient sample, this assay and the accompanying software provide a way to batch about 100 samples together, providing actionable information that can then be used to inform public health and clinical decisions.”