A workflow for high throughput SARS-CoV-2 variant analysis via NGS with molecular barcodes

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. Researchers from Barcode Diagnostics 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. This 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, the researchers 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, this 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.

A schematic showing the overall workflow, including viral RNA extraction, one-step reverse transcription-PCR using barcoded-specific primers, and next-generation sequencing to quantify the amounts of barcoded amplicons.

Cohen-Aharonov LA, Rebibo-Sabbah A, Yaacov A, Granit RZ, Strauss M, Colodner R, et al. (2022) High throughput SARS-CoV-2 variant analysis using molecular barcodes coupled with next generation sequencing. PLoS ONE 17(6): e0253404. [article]

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