HiSpike sequencing of SARS-COV2 from Israel: S-0025
Positive COVID-19 samples of either primary nasopharyngeal swabs were collected, and total nucleic acids were extracted. Qualitative PCR was used to determine the cycle thresholds (Cts) of SARS-CoV-2 positive samples, which is relative to the samples amount of SARS-COV2 genome. Nucleic acid samples were converted to spike gene libraries for sequencing on an Illumina MiSeq instrument, using the HiSpike method. HiSpike involves two simple consecutive reactions: RT-PCR1 and PCR2. RT-PCR1 converts viral spike RNA to cDNA which is amplified using tailed primers to produce spike amplicons with forward and reverse universal tails. The spike annealing sites of these primers were primarily derived from the established ARTIC V3 primers for tiling amplicons along the SARS-CoV-2 genome. Likewise, RT-PCR1 was conducted in two multiplexed reactions to produce two overlapping sets of ~400 bp spike gene amplicons enclosed by forward and reverse universal tails. These amplicons were combined and subjected to PCR2, which added unique dual indexes to each sample and the Illumina P5 and P7 flow-cell adaptors.