Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables fast and accurate outbreak analysis

Wolters, F.; Coolen, J. P. M.; Tostmann, A.; van Groningen, L. F. J.; Bleeker-Rovers, C. P.; Tan, E. C. T. H.; van der Geest-Blankert, N.; Hautvast, J. L. A.; Hopman, J.; Wertheim, H. F. L.; Rahamat-Langendoen, J. C.; Storch, M.; Melchers, W. J. G.

doi:10.1101/2020.10.29.360578

bioRxiv · 10.1101/2020.10.29.360578

Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables fast and accurate outbreak analysis

Wolters, F.·Coolen, J. P. M.·Tostmann, A.·van Groningen, L. F. J.·Bleeker-Rovers, C. P.·Tan, E. C. T. H.·van der Geest-Blankert, N.·Hautvast, J. L. A.·Hopman, J.·Wertheim, H. F. L.·Rahamat-Langendoen, J. C.·Storch, M.·Melchers, W. J. G.

View original source ↗Export metadata ↓

Abstract

BackgroundCurrent 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. FundingNone Research in contextO_ST_ABSEvidence before this studyC_ST_ABSAt 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.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Wolters, F., Coolen, J. P. M., Tostmann, A., van Groningen, L. F. J., Bleeker-Rovers, C. P., Tan, E. C. T. H., van der Geest-Blankert, N., Hautvast, J. L. A., Hopman, J., Wertheim, H. F. L., Rahamat-Langendoen, J. C., Storch, M., Melchers, W. J. G.. 2020-10-29. Novel SARS-CoV-2 Whole-genome sequencing technique using Reverse Complement PCR enables fast and accurate outbreak analysis. https://doi.org/10.1101/2020.10.29.360578

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Microbially conjugated bile acids differentially affect the lifecycle and pathogenesis of Clostridioides difficile

Clostridioides difficile is a Gram-positive bacterium that causes C. difficile infection (CDI). In healthy adults, it has been proposed that the gut microbiota provides colonization resistance against C. difficile by competition through nutrient limitation and antagonism through secondary bile acid metabolism, the latter of which is mediated by the deconjugation of host-derived bile acids with bacterial bile salt hydrolases (BSH). Recently, BSHs have also shown an ability to reconjugate non-canonical (i.e., not glycine or taurine) amino acids to sterol cores, resulting in novel microbially conjugated bile acids (MCBAs). MCBAs have been associated with the resolution of recurrent CDI, though the mechanism underlying these effects are not well understood. To address this gap, we synthesized 36 MCBAs with nine amino acids (cysteine, isoleucine, leucine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine) conjugated to the primary and secondary bile acids found in the human gut (CA, DCA, CDCA, and LCA) and tested their impact on various stages of C. difficiles lifecycle including bacterial outgrowth, membrane integrity, sporulation, and toxin activity. We show that BSH-mediated amidation of bile acid sterol cores can either increase or decrease inhibitory effects on C. difficile in an amino acid-dependent manner. For example, serine MCBAs enhanced C. difficiles ability to grow in otherwise inhibitory bile acid concentrations, increased spore production, and elevated toxin activity. Tryptophan MCBAs increased inhibition. By demonstrating these variable effects, our work clarifies the molecular determinants of MCBA activity and highlights specific MCBAs as promising targets for the development of C. difficile microbial-based therapeutics.

microbiology↗

bioRxiv2026

Magnetic organelle gene clusters enable bacterial growth and ATP homeostasis under oxidizing conditions

Biological organisms can produce magnetic nanoparticles made of magnetite to detect the Earth's magnetic field for orientation. The evolutionary mechanisms that led to the acquisition of this capacity remain unknown. The most ancient organisms known to perform magnetic orientation are magnetotactic bacteria. Under tight genetic control, they produce chains of magnetite nanoparticles in dedicated organelles to allow efficient navigation in aquatic environments containing molecular oxygen. Yet, these bacteria have been proposed to emerge before molecular oxygen became abundant on Earth, at a time when this navigation system could not have served its current function. Here, we show that gene clusters responsible of magnetite formation and organization enable resistance against hydrogen peroxide, a toxic oxygen-derived compound that was produced during the early stages of Earth's oxygenation when iron oxidation consumed molecular oxygen. Characterization of the nanoparticle's oxidation state supported the involvement of magnetite in resistance to hydrogen peroxide-induced oxidative stress. These findings are compatible with an early emergence of magnetotactic bacteria, before natural waters became oxygenated. We propose a conceptual framework to explain the origin of magnetic orientation in bacteria, and offer tools to identify the initiation of Earth's oxygenation in a time-resolved manner.

microbiology↗

bioRxiv2026

Identification and characterization of new P. aeruginosa phages using a surface receptor platform

Bacteriophages are of increasing interest for their potential use as alternatives to antibiotics for treating drug-resistant infections. Phage steering, specifically receptor-based steering, involves use of phages that bias the development of resistance in bacteria towards phenotypes such as reduced fitness. Phage therapy typically involves administering cocktails of phages with different targets to reduce the likelihood of resistance emerging. To ensure effective steering and cocktail design, there is a need to identify the receptors used by phages of interest, and to isolate phages that use diverse receptors. Here we describe a SURFAce Receptor (SURFAR) platform consisting of a set of defined Pseudomonas aeruginosa strains and mutants for rapid characterization of phage receptors. This platform can also be used to enrich for and isolate diverse phages from environmental samples, yielding novel phage genomes from wastewater. Using the SURFAR platform, two new phages were isolated and characterized: Dalewood, which targets both flagella and the conserved LPS outer core, and Bowman, a biofilm targeting phage capable of supressing growth of a subset of carbapenem-resistant clinical isolates. This tool helps to standardize the laborious but important task of phage receptor characterization, with the goal of expanding the known repertoire of P. aeruginosa phage receptors and phages.

microbiology↗