HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene

Abstract

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

Fast and cost-effective SARS-CoV-2 variant detection using Oxford Nanopore full-length spike gene sequencing

“…The Illumina-based S gene tiling sequencing approach for the ectodomain of the SARS-CoV-2 S gene was used for 10-50 % of all COVID-19 cases in Austria from January to June 2021 [16]. Also, the HiSpike method was developed, using a three-step protocol with a turnaround time of ~30 h consisting of RT-PCR1 and PCR2, and a single cleanup step of the pooled MiSeq library [35]. Several other methods have been developed based on Sanger sequencing [36][37][38][39][40][41][42] and also for the Oxford Nanopore platform [43].…”

STArS (STrain-Amplicon-Seq), a targeted nanopore sequencing workflow for SARS-CoV-2 diagnostics and genotyping

“…Moreover, they have been shown to produce a higher percentage of reads that are unsuitable for bioinformatic analysis, likely due to both fragmentation of synthesized molecules and prematurely aborted molecules during sequencing [ 60 ]. Recently, novel protocols have been proposed which are based on the sequencing of the Spike gene [ 31 , 61 ]. Although these protocols share a similar underlying philosophy, STArS protocol benefits of sequencing additional genomic regions corresponding to RT–qPCR amplicons and builds upon the largely tested and iteratively improved ARTIC amplicons scheme.…”

Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

“…HiSpike, developed by Fass and colleagues, can identify variants within the S gene for the 20I/501Y.V1 and 20H/501Y.V2 variants with the help of a small Illumina MiSeq instrument. 477 Compared with the conventional sequencing method, HiSpike improves the detection throughput and significantly reduces the detection costs and time. In another study, Castañeda developed ADSSpike similar to HiSpike.…”