Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping

Abstract

The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq ("cRASL-seq"), which enables analysis of any targeted pathogen- (and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

Figure 1.

The cRASL-seq method. A. A ligation probe set is composed of a chimeric DNA-RNA 3’ acceptor probe and a phosphorylated 5’ donor probe. 20 nt target recognition sequences bring these probes adjacent to one another on a target RNA, enabling their enzymatic ligation. A biotinylated capture probe is included to separate the target sequence from irrelevant materials and excess ligation probes. B. cRASL/RASL-seq complementary assays, which can be performed in a single reaction. C. Sample (e.g. NP swab specimen) is added to lysis buffer containing cRASL probes. After lysis and annealing, targets are captured for subsequent ligation and sample-barcoding amplification, followed by amplicon pooling and NGS. D. Amount of ligation product formed on transcribed GAPDH RNA as a function of input amount; analysis by qPCR of ligation product. E. cRASL-seq test on a set of 9 blinded NP swabs (unextracted) from 6 patients with influenza A and 3 negative controls. F. Assay performed as in E, with influenza capture probe doped into a background of irrelevant capture probe at the ratio shown. For D-F, Molecular Equivalents are calculated by normalizing to a PCR spike-in sequence of defined copy number input.

Figure 2.

Universal cRASL-seq assay for pathogen-associated RNA analysis. Each reference organism was serially diluted into PBS and added directly to the lysis buffer and probe pool. NLC, No Ligase Control; NTC, No Template Control. The extraction free protocol of Fig. 1C was performed with all 116 probe sets in a single pool. Molecular Equivalents are calculated by normalizing read counts to a PCR spike-in sequence of known copy number. Detection at a signal >10x the NTC was used to calculate the assay’s limit of detection for each organism.

Figure 3.

Multiplexed SNP genotyping of SARS-CoV-2 gRNA directly from unextracted NP swabs. A. A probe pair is designed with SNP position in the middle of the 5’ phospho donor probe. A base-calling algorithm is applied to the reads from each alternative probe. B. 14 of 20 positions had a base call for the reference Washington isolate, which matched perfectly against the known genotype. C. The 3 samples from the set of 40 PCR+ samples analyzed, which had 5 or more base calls, and the reference isolate(). Red indicates mutant and blue indicates wildtype versus the reference Wuhan seafood market isolate. D. Network graph depicts each observed genotype (each individual node), two of which are linked if they do not have conflicting SNPs at any position. The blue nodes indicate a maximal vertex set of 9 independent genotypes detected among the 35 patient samples that passed QC. E. Comparison between reads from a SNP typing cRASL-seq probe set in the N gene, versus the Ct value from the RT-qPCR (n=37, 3 samples missing Ct values).