Massively scaled-up testing for SARS-CoV-2 RNA via next-generation sequencing of pooled and barcoded nasal and saliva samples

Abstract

Frequent and widespread testing of members of the population who are asymptomatic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the mitigation of the transmission of the virus. Despite the recent increases in testing capacity, tests based on quantitative polymerase chain reaction (qPCR) assays cannot be easily deployed at the scale required for population-wide screening. Here, we show that next-generation sequencing of pooled samples tagged with sample-specific molecular barcodes enables the testing of thousands of nasal or saliva samples for SARS-CoV-2 RNA in a single run without the need for RNA extraction. The assay, which we named SwabSeq, incorporates a synthetic RNA standard that facilitates end-point quantification and the calling of true negatives, and that reduces the requirements for automation, purification and sample-to-sample normalization. We used SwabSeq to perform 80,000 tests, with an analytical sensitivity and specificity comparable to or better than traditional qPCR tests, in less than two months with turnaround times of less than 24 h. SwabSeq could be rapidly adapted for the detection of other pathogens.

Fig. 1 |. 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 S 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 S standard that is key to allowing the method to work at scale. c, The in vitro S standard (abbreviated as S-Spike) differs from the virus S 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 S standard and the human RPP30. The RPP30 amplicon serves as a control for specimen collection. The in vitro S standard is critical to SwabSeq’s ability to distinguish true negatives.

Fig. 2.. Validation in clinical specimens demonstrate a limit of detection equivalent to sensitive RT-qPCR reactions.

a, Limit of Detection in nasal swab samples with no SARS-CoV2 were pooled and ATCC inactivated virus was added at different concentrations. Nasal Swab sample was RNA purified and using SwabSeq showed a limit of detection of 250 genome copy equivalents (GCE) per mL. b, RNA-purified clinical nasal swab specimens obtained through the UCLA Health Clinical Microbiology Laboratory were tested based on clinical protocols using FDA authorized platforms and then also tested using SwabSeq. This represents a subset of the total purified RNA samples used in our validation. We show 100% agreement with samples that tested positive for SARS-CoV-2 (n=63) and negative for SARS-CoV-2 (n=159). c, We also tested RNA purified samples from extraction-free nasopharyngeal swab and showed a limit of detection of 558 GCE/mL. d, Relationship between Ct from RT-qPCR targeting the S gene (x-axis) and SwabSeq ratio for extract-free swabs into normal saline or Tris-EDTA (y-axis) for patient samples classified as testing positive or negative for SARS-CoV-2 by the UCLA Clinical Microbiology Laboratory. Samples with no virus detected were assigned a Ct of 0 for this visualization. e, Extraction- free processing of saliva specimens show a limit or detection down to 1000 GCE per mL. f, Extraction-free processing of saliva clinical specimens using swabseq (y-axis) compared to classification of SARS-CoV-2 status from RNA-purified clinical nasal swab specimens for matched samples (x-axis).

Fig. 3.. Deployment of Clinical Testing at the UCLA COVID-19 SwabSeq Testing Laboratory.

a, Since November 2020, we have used SwabSeq for large-scale screening in conjunction with our saliva and nasal swab collection processes. We have scaled up to nearly 10,000 samples per week and are continuing to increase our capacity. Week number refers to the week of the year spanning 2020-2021. Our weekly percent positivity rate ranges between 0.3%-2.4% over this period. b, Our clinical deployment streamlined the pre-analytical testing process such that we receive tubes that are ready to be processed through our testing protocol.