Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19

Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19

Introduction

The current pandemic of SARS-CoV-2 (or 2019-nCoV) is perhaps the greatest global health crisis faced in a generation and the efforts to mitigate it’s impact have been unprecedented. The disease that it causes, Corona Virus Disease 2019 (COVID-19) has a spectrum of presentations, from asymptomatic and sub-clinical presentations to mild flu-like symptoms, up to devastating and life-threatening pulmonary illness (Singhal, 2020)⁠. While many host genetic and lifestyle-based factors are thought to contribute to the severity of the illness, other factors likely contribute as well (Singhal, 2020)⁠.

For many viral infections, co-infection is often a major compounding factor that increases the morbidity and mortality of the original disease. Two examples are co-infections between influenza and Staphyloccoccus aureus, Streptococcus pneumoniae and Haemophilus influenzae, alongside rotavirus and Vibrio cholerae (Moore et al., 2020)⁠. In fact, a study in late January identified co-infections of SARS-CoV-2 and a range of fungal and bacterial pathogens (Chen et al., 2020)⁠. Despite this study, and given the extremely short timescale since SARS-CoV-2 emerged as a global pandemic, knowledge about the contribution of co-infections to the presentation and severity of COVID-19 is lacking. Thus rapid and efficient technologies are required in order to better understand and combat the threat posed by SARS-CoV-2019.

The Oxford Nanopore MinION (and associated technologies) is revolutionising various large-scale sequencing methodologies as it allows for the cheap and effectively real-time direct sequencing of both DNA and RNA on a highly portable device. The first major example of the benefits of such a technology in understanding rapidly progressing outbreaks was made evident during the Ebola outbreak in West Africa, where it was used to trace the course of the outbreak and characterise the infection (Hoenen et al., 2016)⁠. Additional technologies can reduce the cost and improve the speed of sequencing even more. One such method is amplicon-based sequencing, where primers are designed to amplify a portion of a genome or chromosome via PCR and the resultant amplicons generated are sequenced. Such a method used in tandem with the MinION represents a powerful way to rapidly identify and quantify the presence of viral pathogens in clinical samples.

As such, the authors of this preprint set out to identify SARS-CoV-2 and characterise the associated background microbiome in nasopharyngeal swab samples from two patients suffering from COVID-19. They performed a dual approach; amplicon-based sequencing using SARS-CoV-2-specific primers and a metagenomic approach to asses the microbiota present.

Key findings

• Amplicon-based sequencing of the patient samples successfully identified SARS-CoV-2 and confirms this approach as a proof-of-concept.

• The microbiomes of the two patients varied, with Fusobacterium periodonticum and human cytomegalovirus being identified.

Why I chose this paper

With the rapid and pervasive spread of SARS-CoV-2 throughout the globe, an equally rapid method for identifying and characterising the virus in clinical samples will be of huge benefit in understanding and mitigating the spread of the virus. Given the proven track record of MinION-sequencing in tracing outbreaks it is a good candidate for such a method, particularly when combined with amplicon-based sequencing to further reduce complexity, cost and time. More generally, this paper indicates at the importance of understanding viral infection from the perspective of the host-pathogen-microbiota axis and that real-world infectious disease can often be polymicrobial.

Future perspectives

• Multiplexed PCR primers could be used in order to expand the number of viruses or agents detected in a sample, or to allow for the detection of possible SARS-CoV-2 variants.

• The primer locations could be altered to flank more variable amplicons, that could be used to determine heterogeneity between SARS-CoV-2 strains, enabling phenotypical analysis and understanding of the evolution of the virus.

• The number of patients sampled could be expanded to detect any patterns between the microbiomes or co-infections of patients and these correlated or cross-referenced with clinical presentations of COVID-19.

References

Tags: amplicon, covid-19, minion, nanopore, ncov-2019, sars-cov-2

doi: https://doi.org/10.1242/prelights.18069

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