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

Abstract

There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called "capture RASL-seq" (cRASL-seq), which 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 purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol 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 technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

Fig. 1

The cRASL-seq assay. A Ligation probe set is composed of a chimeric DNA-RNA 3′ acceptor probe (2 & 3) and a phosphorylated 5′ donor probe (4 & 5). Each probe contains common PCR primer binding sequences (2 & 5). Two 20 nt target recognition sequences (3 & 4) bring these probes adjacent to one another on a target RNA, enabling their enzymatic ligation. A biotinylated capture probe (1) is used to separate the target sequences from irrelevant materials and excess ligation probes. B Complementary oligonucleotide probe ligation 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 probe set 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 cRASL ligation product. E cRASL-seq test on a set of nine blinded NP swabs (unextracted) from six patients with influenza A and three negative controls. F Assay performed as in (E), with influenza capture probe doped into a background of irrelevant capture probe as indicated. For D–F molecular equivalents are calculated by normalization to a PCR spike-in sequence of defined copy number input.

Fig. 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 80 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 of aggregate probes' read counts at a signal >10× the NTC was used to calculate the assay's limit of detection for each organism.

Fig. 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′ phosphorylated 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 the known genotype without errors. C The 35 samples from the set of 40 PCR+ samples analyzed, which had 5 or more base calls, and the reference isolate (green diamond). Red indicates mutant, blue indicates wildtype versus the reference Wuhan seafood market isolate. Yellow indicates no call. 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 nine 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 values from the corresponding RT-qPCR.