ABSTRACT
In this work, Oxford Nanopore sequencing is tested as an accessible method for quantifying heterogeneity of amplified DNA. This method enables rapid quantification of deletions, insertions, and substitutions, the probability of each mutation error, and their locations in the replicated sequences. Amplification techniques tested were conventional polymerase chain reaction (PCR) with varying levels of polymerase fidelity (OneTaq, Phusion, and Q5) as well as rolling circle amplification (RCA) with Phi29 polymerase. Plasmid amplification using bacteria was also assessed. By analyzing the distribution of errors in a large set of sequences for each sample, we examined the heterogeneity and mode of errors in each sample. This analysis revealed that Q5 and Phusion polymerases exhibited the lowest error rates observed in the amplified DNA. As a secondary validation, we analyzed the emission spectra of sfGFP fluorescent proteins synthesized with amplified DNA using cell free expression. Error-prone polymerase chain reactions confirmed the dependency of reporter protein emission spectra peak broadness to DNA error rates. The presented nanopore sequencing methods serve as a roadmap to quantify the accuracy of other gene amplification techniques, as they are discovered, enabling more homogenous cell-free expression of desired proteins.
ABSTRACT
Wearable health sensors for an expanding range of physiological parameters have experienced rapid development in recent years and are poised to disrupt the way healthcare is tracked and administered. The monitoring of environmental contaminants with wearable technologies is an additional layer of personal and public healthcare and is also receiving increased focus. Wearable sensors that detect exposure to airborne viruses could alert wearers of viral exposure and prompt proactive testing and minimization of viral spread, benefitting their own health and decreasing community risk. With the high levels of asymptomatic spread of COVID-19 observed during the pandemic, such devices could dramatically enhance our pandemic response capabilities in the future. To facilitate advancements in this area, this review summarizes recent research on airborne viral detection using wearable sensing devices as well as technologies suitable for wearables. Since the low concentration of viral particles in the air poses significant challenges to detection, methods for airborne viral particle collection and viral sensing are discussed in detail. A special focus is placed on nucleic acid-based viral sensing mechanisms due to their enhanced ability to discriminate between viral subtypes. Important considerations for integrating airborne viral collection and sensing on a single wearable device are also discussed.
