Ultra-high-throughput sequencing approach enabling whole-community COVID-19 screening
T2020-337 REMBRANDT: a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 COVID-19 samples per day.
The SARS-CoV-2 virus, which causes COVID-19 syndrome, has rapidly spread throughout the world and was recently classified by the WHO as a pandemic pathogen. Severe disease that requires hospital admission occurs in 20% of cases, and a subset of these patients require ICU care; the death rate from COVID-19 increases proportionately with older age, being highest among older patients, and also those who are immunocompromised or have other co-morbidities. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. Large numbers of people will need to be tested and contact-tracing of positive patients will be required in order to successfully control reoccurring waves of infection. However, there are presently no testing platforms that enable more than 10,000 tests per day, severely limiting the ability to implement whole-community COVID-19 screening.
A team of researchers at The Ohio State University have developed the REMBRANDT (REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool) protocol, a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 COVID-19 samples per day. The REMBRANDT protocol contains a number of key advances that maximize output and sensitivity while retaining speed and efficiency. The protocol involves direct two-barcoded amplification of SARS-CoV-2 RNA and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. The two-barcode approach bypasses the need for RNA extraction or purification and rapidly generates stable DNA products; the approach also reduces the number of barcodes 100-fold from conventional barcoding methods. Once barcoded, the patient samples from multiple 96-well plates can be pooled and purified, ultimately minimizing reagent usage, time and sample-to-sample variation. Further sample multiplexing may be performed such that twelve different 9,216 combined samples may be further mixed together to perform Next Generation sequencing analysis of 110,592 patient samples on a number of Illumina-based platforms in a single run. The smaller numbers of barcodes require less time-consuming computational processing, as trimmed sequences can be quickly divided based on barcode. Overall, the protocol is designed to maximize SARS-CoV-2 detection efficiency while minimizing reagent usage, processing and turn-around time, thus enabling large scale screening of entire communities.
- COVID-19 screening
- Approach could be adapted to large-scale screening of other pathogens
- Extremely high-throughput sequencing
- Same degree of accuracy as existing methods
- US Provisional Patent Application Filed