Detecting SARS-CoV-2 and its variant strains with a full genome tiling array

Abstract

Coronavirus disease 2019 pandemic is the most damaging pandemic in recent human history. Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and variant strains is paramount for recovery from this pandemic. Conventional SARS-CoV-2 tests interrogate only limited regions of the whole SARS-CoV-2 genome, which are subjected to low specificity and miss the opportunity of detecting variant strains. In this work, we developed the first SARS-CoV-2 tiling array that captures the entire SARS-CoV-2 genome at single nucleotide resolution and offers the opportunity to detect point mutations. A thorough bioinformatics protocol of two base calling methods has been developed to accompany this array. To demonstrate the effectiveness of the tiling array, we genotyped all genomic positions of eight SARS-CoV-2 samples. Using high-throughput sequencing as the benchmark, we show that the tiling array had a genome-wide accuracy of at least 99.5%. From the tiling array analysis results, we identified the D614G mutation in the spike protein in four of the eight samples, suggesting the widespread distribution of this variant at the early stage of the outbreak in the United States. Two additional nonsynonymous mutations were identified in one sample in the nucleocapsid protein (P13L and S197L), which may complicate future vaccine development. With around $5 per array, supreme accuracy, and an ultrafast bioinformatics protocol, the SARS-CoV-2 tiling array makes an invaluable toolkit for combating current and future pandemics. Our SARS-CoV-2 tilting array is currently utilized by Molecular Vision, a CLIA-certified lab for SARS-CoV-2 diagnosis.

Keywords: COVID-19; SARS-CoV-2; tiling array.

Figure 1

The overall tiling array design and bioinformatics analysis strategy. (A) The array design overview. Each position of the SARS-CoV-2 genome is covered by two probesets (sense and antisense). Each probeset contains four probes; each probe is a 25mer synthetic oligonucleotide, with middle nucleotide trying to capture the actual allele from the sample. Three exposure times were used to scan the array. (B) Two bioinformatics approaches were developed for inferring the allele: weighted voting method and ranked credibility score method.

Figure 2

Noise level and consistency of base calls. (A) Distribution of noise level for the unanimous and nonunanimous base calls from titling array data of eight samples. The background noise is lower with the unanimous calls than with the nonunanimous calls. (B) Heatmap of replicate consistency values for binned segments of SARS-Cov-2 genome across eight samples. For visualization clarity, the whole genome was segmented into 30 bins each of 1000 bp long. (C) Replicate consistency and its relatedness with sequencing depth and GC content. Both tiling array and sequencing suffer deficiency around roughly same genomic regions.

Figure 3

Accuracy measure of two base calling methods using sequencing result as the gold standard. Both bioinformatics methods performed excellently in relation to sequencing.

Figure 4

Tiling array detected point mutations in the SARS-CoV-2 genome. (A) Point mutations at 35 genomic positions across eight samples. Summing up mutations from individual samples; sequencing results presented 65 mutations in total, of which 61 were captured by tiling array. S1–S8: sample IDs. (B) Two mutations were identified in spike protein, one synonymous and one nonsynonymous. (C) Four mutations were identified in nucleocapsid protein, including two nonsynonymous ones.