High throughput SARS-CoV-2 variant analysis using molecular barcodes coupled with next generation sequencing

PLoS One. 2022 Jun 21;17(6):e0253404. doi: 10.1371/journal.pone.0253404. eCollection 2022.

Abstract

The identification of SARS-CoV-2 variants across the globe and their implications on the outspread of the pandemic, infection potential and resistance to vaccination, requires modification of the current diagnostic methods to map out viral mutations rapidly and reliably. Here, we demonstrate that integrating DNA barcoding technology, sample pooling and Next Generation Sequencing (NGS) provide an applicable solution for large-population viral screening combined with specific variant analysis. Our solution allows high throughput testing by barcoding each sample, followed by pooling of test samples using a multi-step procedure. First, patient-specific barcodes are added to the primers used in a one-step RT-PCR reaction, amplifying three different viral genes and one human housekeeping gene (as internal control). Then, samples are pooled, purified and finally, the generated sequences are read using an Illumina NGS system to identify the positive samples with a sensitivity of 82.5% and a specificity of 97.3%. Using this solution, we were able to identify six known and one unknown SARS-CoV-2 variants in a screen of 960 samples out of which 258 (27%) were positive for the virus. Thus, our diagnostic solution integrates the benefits of large population and epidemiological screening together with sensitive and specific identification of positive samples including variant analysis at a single nucleotide resolution.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19* / diagnosis
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Pandemics
  • SARS-CoV-2* / genetics

Supplementary concepts

  • SARS-CoV-2 variants

Grants and funding

This work was partly supported by a grant from the Israel Innovation Authority. The funder provided support in the form of salaries for authors [L.C.A., A.R.S., R.G., A.Y and R.E.], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.