In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin–angiotensin–aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

This is the first FDA emergency-use-authorized hybridization capture-based next-generation sequencing (NGS) assay to detect the SARS-CoV-2 genome. Viral metagenomics and the novel hybrid capture NGS-based assay, along with its research-use-only analysis, can provide important genetic insights into SARS-CoV-2 and other emerging pathogens and improve surveillance and early detection, potentially preventing or mitigating new outbreaks.

We present two Delta (B.1.617.2) vaccine breakthrough individuals, a father and son living in separate households. The older, 63-year-old patient's symptoms were severe enough to require hospitalization. Despite having a high titer of anti-spike IgG in his serum, his symptoms resolved within 24 hours following monoclonal antibody (bamlanivimab/etesevimab) therapy.

Background As the SARS-CoV-2 (SCV-2) virus evolves, diagnostics and vaccines against novel strains rely on viral genome sequencing. Researchers have gravitated towards the cost-effective and highly sensitive amplicon-based (e.g. ARTIC) and hybrid capture sequencing (e.g. SARS-CoV-2 NGS Assay) to selectively target the SCV-2 genome. We provide an in silico model to compare these 2 technologies and present data on the high scalability of the Research Use Only (RUO) workflow of the SARS-CoV-2 NGS Assay. Methods In silico work included alignments of 383,656 high-quality genome sequences belonging to variant of concern (VOC) or variant of interest (VOI) isolates (GISAID). We profiled mismatches and sequencing dropouts using the ARTIC V3 primers, SARS-CoV-2 NGS Assay probes (Twist Bioscience) and 11 synthesized viral sequences containing mutations and compared the performance of these assays using clinical samples. Further, the miniaturized hybrid capture workflow was optimized and evaluated to support high-throughput (384-plex). The sequencing data was processed by COVID-DX software. Results We detected 101,432 viruses (27%) with > = 1 mismatch in the last 6 base pairs of the 3’ end of ARTIC primers; of these, 413 had > = 2 mismatches in one primer. In contrast, only 38 viruses (0.01%) had enough mutations ( > = 10) in a hybrid capture probe to have a similar effect on coverage. We observed that mutations in ARTIC primers led to complete dropout of the amplicon for 4/11 isolates and diminished coverage in additional 4. Twist probes showed uniform coverage throughout with little to no dropouts. Both assays detected a wide range of variants (~99.9% coverage at 5X depth) in clinical samples (CT value < 30) collected in NY (Spring 2020-Spring 2021). The distribution of the number of reads and on target rates were more uniform among specimens within amplicon-based sequencing. However, uneven genome coverage and primer dropouts, some in the spike protein, were observed on VOC/VOI and other isolates highlighting limitations of an amplicon-based approach. Conclusion The RUO workflow of the SARS-CoV-2 NGS Assay is a comprehensive and scalable sequencing tool for variant profiling, yields more consistent coverage and smaller dropout rate compared to ARTIC (0.05% vs. 7.7%). Disclosures Danny Antaki, PhD, Twist Bioscience (Employee, Shareholder) Mara Couto-Rodriguez, MS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee, Shareholder) Esteban Toro, PhD, Twist Bioscience (Employee) Bryan Höglund, BS, Twist Bioscience (Employee, Shareholder) Xavier O. Jirau Serrano, B.S., Biotia (Employee) Joseph Barrows, MS, Biotia (Employee) Christopher Mason, PhD, Biotia (Board Member, Advisor or Review Panel member, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder)

The emergence of novel SARS-CoV-2 genetic variants that may alter viral fitness highlights the urgency of widespread next-generation sequencing (NGS) surveillance. To profile genetic variants, we developed and clinically validated a hybridization capture SARS-CoV-2 NGS assay, integrating novel methods for panel design using dsDNA biotin-labeled probes, and built accompanying software. The positive and negative percent agreement were defined in comparison to an orthogonal RT-PCR assay (PPA and NPA: both 96.7%). The limit of detection was established to be 800 copies/ml with an average fold-enrichment of 46,791x. We identified novel 107 mutations, including 24 in the functionally-important spike protein. Further, we profiled the full nasopharyngeal microbiome using metagenomics and found overrepresentation of 7 taxa and macrolide resistance in SARS-CoV-2-positive patients. This 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.TEASERThis is the first target hybridization capture-based NGS assay to detect SARS-CoV-2 genetic variants for tracking viral evolution.