The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to the high rates of transmission by individuals who are asymptomatic at the time of transmission. Frequent, widespread testing of the asymptomatic population for SARS-CoV-2 is essential to suppress viral transmission and is a key element in safely reopening society. Despite increases in testing capacity, multiple challenges remain in deploying traditional reverse transcription and quantitative PCR (RT-qPCR) tests at the scale required for population screening of asymptomatic individuals.
Researchers at the David Geffen School of Medicine have developed SwabSeq, a high-throughput testing platform for SARS-CoV-2 that uses next-generation sequencing as a readout. SwabSeq employs sample-specific molecular barcodes to enable thousands of samples to be combined and simultaneously analyzed for the presence or absence of SARS-CoV-2 in a single run. Importantly, SwabSeq incorporates an in vitro RNA standard that mimics the viral amplicon, but can be distinguished by sequencing. This standard allows for end-point rather than quantitative PCR, improves quantitation, reduces requirements for automation and sample-to-sample normalization, enables purification-free detection, and gives better ability to call true negatives. The researchers show that SwabSeq can test nasal and oral specimens for SARS-CoV-2 with or without RNA extraction while maintaining analytical sensitivity better than or comparable to that of fluorescence-based RT-qPCR tests. SwabSeq is simple, sensitive, flexible, rapidly scalable, inexpensive enough to test widely and frequently, and can provide a turn around time of 12 to 24 hours.
SwabSeq Diagnostic Testing Platform for COVID19
A) The workflow for SwabSeq is a five step process that takes approximately 12 hours from start to finish. B) In each well, we perform RT-PCR on clinical samples. Each well has two sets of indexed primers that generate cDNA and amplicons for SARS-CoV-2 S2 gene and the human RPP30 gene. Each primer is synthesized with the P5 and P7 adaptors for Illumina sequencing, a unique i7 and i5 molecular barcodes, and the unique primer pair. Importantly, every well has a synthetic in vitro S2 standard that is key to allowing the method to work at scale. C) The in vitro S2 standard (abbreviated as S2-Spike) differs from the virus S2 gene by 6 base pairs that are complemented (underlined). (D) Read count at various viral concentrations (E) Ratiometric normalization allow for in-well normalization for each amplicon (F) Every well has two internal well controls for amplification, the in vitro S2 standard and the human RPP30. The RPP30 amplicon serves as a control for specimen collection. The in vitro S2 standard is critical to SwabSeq’s ability to distinguish true negatives.