SARS-CoV-2 variant detection with ADSSpike

Abstract

The SARS-CoV-2 coronavirus pandemic has been an unprecedented challenge to global pandemic response and preparedness. With the continuous appearance of new SARS-CoV-2 variants, it is imperative to implement tools for genomic surveillance and diagnosis in order to decrease viral transmission and prevalence. The ADSSpike workflow was developed with the goal of identifying signature SNPs from the S gene associated with SARS-CoV-2 variants through amplicon deep sequencing. Seventy-two samples were sequenced, and 30 mutations were identified. Among those, signature SNPs were linked to 2 Zeta-VOI (P.2) samples and one to the Alpha-VOC (B.1.17). An average depth of 700 reads was found to properlycorrectly identify all SNPs and deletions pertinent to SARS-CoV-2 mutants. ADSSpike is the first workflow to provide a practical, cost-effective, and scalable solution to diagnose SARS-CoV-2 VOC/VOI in the clinical laboratory, adding a valuable tool to public health measures to fight the COVID-19 pandemic for approximately $41.85 USD/reaction.

Keywords: Amplicon deep sequencing; S gene; SARS-CoV-2; Variants of concern; Variants of interest.

Fig. 1

Spike gene amplicon deep sequencing (ADS) pipeline named ADSSpike. The presented workflow was divided among 4 steps: sample processing, PCR and library preparation, sequencing, and data analysis (read filtering, alignment, SNP calling, and variant assessment).

Fig. 2

Read depth and coverage assessment. (A) Plot displaying the mean read depth distribution by individual nucleotide position. Depth was assessed by SARS-CoV-2 positive iPCR samples, SARS-CoV-2 negative samples, and negative controls (NFWiPCR). The dashed purple line represents a 700 read depth. (B) Plot displaying the mean read depth distribution by individual nucleotide position amongst Ct value groups. Depth was assessed by SARS-CoV-2 positive iPCR samples. (C) Side by side box plot of coverage by Ct value groups assessed by a Kruskal-Wallis test and multiple-paired comparisons (*: adjusted P-value < 0.05; **: adjusted P-value < 0.01; ***: adjusted P-value < 0.001). (D) Dot plot displaying the position-specific depth of the identified SNPs for the VOC/VOI (left) vs missed SNPs. The blue dashed line represents the lowest depth for a called SNP, and the red dashed line represents the highest depth recorded for a missed SNP.

Fig. 3

Identified SNPs indels SARS-CoV-2 positive samples. (A) Non synonymous mutations, deletions, and nonsense mutants identified amongst the 64 iPCR SARS-CoV-2 positive samples in the S and flanking ORF3a genes region. Blue dashed lines represent the receptor binding domain (RBD) delimited by the nucleotides 318 and 510, and its receptor binding motif (RBM). Red mutants are flagged for their presence in VOC/VOI. (B) Pie chart of the SNP type distribution. (C) Side-by-side bar chart of the read depth by SARS-CoV-2 positive samples with no SNPs detected vs samples with at least one identified SNP (D) Box plot of the 5 most prevalent mutants identified across all positive samples (n = 72).