DNA synthesis technologies to close the gene writing gap.

Synthetic DNA is of increasing demand across many sectors of research and commercial activities. Engineering biology, therapy, data storage and nanotechnology are set for rapid developments if DNA can be provided at scale and low cost. Stimulated by successes in next generation sequencing and gene editing technologies, DNA synthesis is already a burgeoning industry. However, the synthesis of >200 bp sequences remains unaffordable. To overcome these limitations and start writing DNA as effectively as it is read, alternative technologies have been developed including molecular assembly and cloning methods, template-independent enzymatic synthesis, microarray and rolling circle amplification techniques. Here, we review the progress in developing and commercializing these technologies, which are exemplified by innovations from leading companies. We discuss pros and cons of each technology, the need for oversight and regulatory policies for DNA synthesis as a whole and give an overview of DNA synthesis business models.

Transmission of SARS-CoV-2 by children to contacts in schools and households: a prospective cohort and environmental sampling study in London.

BACKGROUND: Assessing transmission of SARS-CoV-2 by children in schools is of crucial importance to inform public health action. We assessed frequency of acquisition of SARS-CoV-2 by contacts of pupils with COVID-19 in schools and households, and quantified SARS-CoV-2 shedding into air and onto fomites in both settings. METHODS: We did a prospective cohort and environmental sampling study in London, UK in eight schools. Schools reporting new cases of SARS-CoV-2 infection to local health protection teams were invited to take part if a child index case had been attending school in the 48 h before a positive SARS-CoV-2 PCR test. At the time of the study, PCR testing was available to symptomatic individuals only. Children aged 2-14 years (extended to <18 years in November, 2020) with a new nose or throat swab SARS-CoV-2 positive PCR from an accredited laboratory were included. Incidents involving exposure to at least one index pupil with COVID-19 were identified (the prevailing variants were original, α, and δ). Weekly PCR testing for SARS-CoV-2 was done on immediate classroom contacts (the so-called bubble), non-bubble school contacts, and household contacts of index pupils. Testing was supported by genome sequencing and on-surface and air samples from school and home environments. FINDINGS: Between October, 2020, and July, 2021 from the eight schools included, secondary transmission of SARS-CoV-2 was not detected in 28 bubble contacts, representing ten bubble classes (participation rate 8·8% [IQR 4·6-15·3]). Across eight non-bubble classes, 3 (2%) of 62 pupils tested positive, but these were unrelated to the original index case (participation rate 22·5% [9·7-32·3]). All three were asymptomatic and tested positive in one setting on the same day. In contrast, secondary transmission to previously negative household contacts from infected index pupils was found in six (17%) of 35 household contacts rising to 13 (28%) of 47 household contacts when considering all potential infections in household contacts. Environmental contamination with SARS-CoV-2 was rare in schools: fomite SARS-CoV-2 was identified in four (2%) of 189 samples in bubble classrooms, two (2%) of 127 samples in non-bubble classrooms, and five (4%) of 130 samples in washrooms. This contrasted with fomites in households, where SARS-CoV-2 was identified in 60 (24%) of 248 bedroom samples, 66 (27%) of 241 communal room samples, and 21 (11%) 188 bathroom samples. Air sampling identified SARS-CoV-2 RNA in just one (2%) of 68 of school air samples, compared with 21 (25%) of 85 air samples taken in homes. INTERPRETATION: There was no evidence of large-scale SARS-CoV-2 transmission in schools with precautions in place. Low levels of environmental contamination in schools are consistent with low transmission frequency and suggest adequate cleaning and ventilation in schools during the period of study. The high frequency of secondary transmission in households associated with evident viral shedding throughout the home suggests a need to improve advice to households with infection in children to prevent onward community spread. The data suggest that SARS-CoV-2 transmission from children in any setting is very likely to occur when precautions are reduced. FUNDING: UK Research and Innovation and UK Department of Health and Social Care, National Institute for Health and Care Research.

A high-throughput pipeline for scalable kit-free RNA extraction.

An overreliance on commercial, kit-based RNA extraction in the molecular diagnoses of infectious disease presents a challenge in the event of supply chain disruptions and can potentially hinder testing capacity in times of need. In this study, we adapted a well-established, robust TRIzol-based RNA extraction protocol into a high-throughput format through miniaturization and automation. The workflow was validated by RT-qPCR assay for SARS-CoV-2 detection to illustrate its scalability without interference to downstream diagnostic sensitivity and accuracy. This semi-automated, kit-free approach offers a versatile alternative to prevailing integrated solid-phase RNA extraction proprietary systems, with the added advantage of improved cost-effectiveness for high volume acquisition of quality RNA whether for use in clinical diagnoses or for diverse molecular applications.

SARS-CoV-2 whole-genome sequencing using reverse complement PCR: For easy, fast and accurate outbreak and variant analysis.

During the course of the SARS-CoV-2 pandemic reports of mutations with effects on spreading and vaccine effectiveness emerged. Large scale mutation analysis using rapid SARS-CoV-2 Whole Genome Sequencing (WGS) is often unavailable but could support public health organizations and hospitals in monitoring transmission and rising levels of mutant strains. Here we report a novel WGS technique for SARS-CoV-2, the EasySeq™ RC-PCR SARS-CoV-2 WGS kit. By applying a reverse complement polymerase chain reaction (RC-PCR), an Illumina library preparation is obtained in a single PCR, thereby saving time, resources and facilitating high-throughput screening. Using this WGS technique, we evaluated SARS-CoV-2 diversity and possible transmission within a group of 173 patients and healthcare workers (HCW) of the Radboud university medical center during 2020. Due to the emergence of variants of concern, we screened SARS-CoV-2 positive samples in 2021 for identification of mutations and lineages. With use of EasySeq™ RC-PCR SARS-CoV-2 WGS kit we were able to obtain reliable results to confirm outbreak clusters and additionally identify new previously unassociated links in a considerably easier workaround compared to current methods. Furthermore, various SARS-CoV-2 variants of interest were detected among samples and validated against an Oxford Nanopore sequencing amplicon strategy which illustrates this technique is suitable for surveillance and monitoring current circulating variants.

Author Correction: A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.

There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control. Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results. We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples. The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.

A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics.

The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.