Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables easy, fast and accurate outbreak analysis in hospital and community settings
Authors: Wolters Femke , Coolen Jordy P.M. , Tostmann Alma , van Groningen Lenneke F.J. , Bleeker-Rovers Chantal P. , Tan Edward C.T.H. , van der Geest-Blankert Nannet , Hautvast Jeannine L.A. , Hopman Joost , Wertheim Heiman F.L. , Rahamat-Langendoen Janette C. , Storch Marko , Melchers Willem J.G. 
Published: October 2020

AbstractBackgroundCurrent transmission rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still increasing and many countries are facing second waves of infections. Rapid SARS-CoV-2 whole-genome sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring and determining transmission links. Here we report the use of reverse complement polymerase chain reaction (RC-PCR), a novel technology for WGS of SARS-CoV-2 enabling library preparation in a single PCR saving time, resources and enables high throughput screening. Additionally, we show SARS-CoV-2 diversity and possible transmission within the Radboud university medical center (Radboudumc) during September 2020 using RC-PCR WGS.MethodsA total of 173 samples tested positive for SARS-CoV-2 between March and September 2020 were selected for whole-genome sequencing. Ct values of the samples ranged from 16 to 42. They were collected from 83 healthcare workers and three patients at the Radboudumc, in addition to 64 people living in the area around the hospital and tested by the local health services. For validation purposes, nineteen of the included samples were previously sequenced using Oxford Nanopore Technologies and compared to RC-PCR WGS results. The applicability of RC-PCR WGS in outbreak analysis for public health service and hospitals was tested on six suspected clusters containing samples of healthcare workers and patients with an epidemiological link.FindingsRC-PCR resulted in sequencing data for 146 samples. It showed a genome coverage of up to 98,2% for samples with a maximum Ct value of 32. Comparison to Oxford Nanopore technologies gives a near-perfect agreement on 95% of the samples (18 out of 19). Three out of six clusters with a suspected epidemiological link were fully confirmed, in the others, four healthcare workers were not associated. In the public health service samples, a previously unknown chain of transmission was confirmed.Significance statementSAR-CoV-2 whole-genome sequencing using RC-PCR is a reliable technique and applicable for use in outbreak analysis and surveillance. Its ease of use, high-trough screening capacity and wide applicability makes it a valuable addition or replacement during this ongoing SARS-CoV-2 pandemic.FundingNoneResearch in contextEvidence before this studyAt present whole genome sequencing techniques for SARS-CoV-2 have a large turnover time and are not widely available. Only a few laboratories are currently able to perform large scale SARS-CoV-2 sequencing. This restricts the use of sequencing to aid hospital and community infection prevention.Added value of this studyHere we present clinical and technical data on a novel Whole Genome Sequencing technology, implementing reverse-complement PCR. It is able to obtain high genome coverage of SARS-CoV-2 and confirm and exclude epidemiological links in 173 healthcare workers and patients. The RC-PCR technology simplifies the workflow thereby reducing hands on time. It combines targeted PCR and sequence library construction in a single PCR, which normally takes several steps. Additionally, this technology can be used in concordance with the widely available range of Illumina sequencers.Implications of all the available evidenceRC-PCR whole genome sequencing technology enables rapid and targeted surveillance and response to an ongoing outbreak that has great impact on public health and society. Increased use of sequencing technologies in local laboratories can help prevent increase of SARS-CoV-2 spreading by better understanding modes of transmission.