Mass COVID-19 patient screening using UvsX and UvsY mediated DNA recombination and high throughput parallel sequencing

Abstract

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10–20% of symptomatic patients, particularly those with underlying medical conditions. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. A number of molecular assays are currently used to detect SARS-CoV-2. Many of them can accurately test hundreds or even thousands of patients every day. However, there are presently no testing platforms that enable more than 10,000 tests per day. Here, we describe the foundation for the REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool (REMBRANDT), a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 samples per day. The REMBRANDT protocol includes direct two-barcoded amplification of SARS-CoV-2 and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. This protocol represents a potentially powerful approach for community screening of COVID-19 that may be modified for application to any infectious or non-infectious genome.

A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

“…The barcoding step can be achieved either using commercial kits, like the kits from Illumina and Oxford Nanopore Technologies, or a customized design ( Table 1 ). In the latter case, a sequence of a barcode is often embedded in primers as an overhang at the step of reverse transcription of viral RNA or PCR amplification of the RT product ( Figure 1B ) ( Bhoyar et al, 2021 ; Bloom et al, 2021 ; Duan et al, 2021 ; Gauthier et al, 2021 ; Ludwig et al, 2021 ; Stüder et al, 2021 ; Wu et al, 2021 ; Cohen-Aharonov et al, 2022 ; Credle et al, 2022 ; Gallego-García et al, 2022 ; Palmieri et al, 2022 ; Warneford-Thomson et al, 2022 ; Yermanos et al, 2022 ). Barcoded primers used in SwabSeq ( Bloom et al, 2021 ) and by Cohen-Aharonoc et al (2022) ( Cohen-Aharonov et al, 2022 ) are compatible with one-step RT-PCR.…”

“… Gallego-García et al (2022) spiked in a string of 20 random nucleotide barcode sequences inserted in the forward- and reverse primer to minimize cross-sample contamination. Palmieri et al (2022) and Yermanos et al (2022) applied two-dimensional barcoding primers to specify samples pooled in wells and plates. Similarly, Duan et al (2021) directly included a known sequence of a barcode (8 bp) and a UMI with three random nucleotides in RT primers at the same time to multiplex samples and correct sequencing reads.…”