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Identification, characterisation, surveillance, and screening of novel pathogens on a single sequencing platform


Date: 3rd December 2020

The combination of portable sequencing devices and real-time analysis enables the rapid identification, genomic analysis and screening for infectious pathogens in decentralised settings

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Fig. 1 A possible transmission cycle of a novel virus with pandemic potential

Pandemics occur when new, highly pathogenic diseases emerge and spread

Novel viral diseases are potentially serious threats to public health, because tests, vaccines and effective treatments are not readily available, as typified by the ongoing COVID-19 pandemic. Many highly pathogenic viruses, including Ebola and influenza, as well as SARS-CoV-2 originated in other animal host species, and were transmitted to humans from the original host, possibly via an intermediate animal host (Fig. 1). Most viruses that transfer to new host species cause limited outbreaks, but when a virus both acquires the ability to transmit efficiently from human to human and throughout communities and countries, and also retains a high level of pathogenicity, then the toll on human populations can be significant.

Fig. 2 Identifying a novel infectious agent a) SISPA workflow b)-d) analysis workflow

Identifying unknown infections with unbiased metagenomic sequence analysis

When faced with a cluster of patients who share similar symptoms but who test negative for the presence of known candidate pathogens, the first challenge is to identify the infectious agent and to derive a genome sequence. This allows the subsequent development of molecular tests for the pathogen. Sequence-independent, single primer amplification (SISPA) is an effective way to achieve this, particularly when the titre of the pathogen is high (Fig. 2a). Because SISPA is a sequence independent method, it is capable of revealing all organisms present in the sample, including any bacteria (Fig. 2b) and viruses (Fig. 2c). Sequence reads that have homology and align to a potentially pathogenic agent can be used to generate consensus sequences (Fig. 2d).

Fig. 3 Whole-genome analysis a) ARTIC workflow b)-c) sequence variation as the virus evolves

Using the ARTIC whole-genome protocol to understand how the pathogen is changing

The availability of the first genome sequences means that molecular analyses can be designed. In the early stages of an outbreak, it may be that genome sequences of the pathogen which have been obtained from different patients do not vary appreciably, but over time, as the pathogen is transmitted more widely from person to person, more genomic polymorphism is likely to emerge. Whole-genome sequencing of strains from large numbers of patients, obtained, for instance, using the protocol from the ARTIC group (Fig. 3a), can be used to monitor the rate of the pathogen’s evolution (Fig. 3b) and can provide insight into patterns of its transmission. Whole-genome sequences can also be used to develop accurate, rapid and high-throughput analyses for wide-scale testing and screening (Fig. 3c; 148,476 sequences analysed Nov. 2020).

Fig. 4 LamPORE a) overall workflow b) dual barcoding scheme c) typical results

Rapid, highly scalable screening for the presence or absence of SARS-CoV-2 RNA

As an outbreak continues to spread from person to person, the availability of scalable and rapid tests becomes increasingly important. LamPORE™ achieves high throughput using combinatorial barcoding (Figs. 4a-c), and once optimised, results can be obtained from 96 RNA extracts in under 2 hours. Provided that extensive whole-genome sequencing data is available, designing and maintaining a LamPORE assay for new target genomes is straightforward. Widespread testing and screening is one of several strategies that can be employed as a way to limit the spread of a disease, and is particularly useful in situations where significant numbers of infected people do not display obvious symptoms. It is essential for track and trace efforts, for protecting the most vulnerable, and to help businesses and schools to stay open.

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