Exploring a novel amplicon-based mpox virus sequencing strategy

In a recent study posted to the medRxiv* preprint server, researchers described developing a sequencing strategy based on amplicons for mpox detection.

Background

The multi-country epidemic of mpox in 2022, which coincided with the continuing coronavirus disease 2019 (COVID-19) pandemic, emphasized the necessity for genomic monitoring and quick pathogen whole genome sequencing. Numerous early mpox infections have been sequenced using metagenomic sequencing techniques. However, these methods tend to be resource-intensive and need samples comprising high viral deoxyribonucleic acid (DNA) levels. Given the unique clinical presentation of individuals associated with the epidemic and the ambiguity surrounding viral load throughout the infection course as well as anatomical body regions, there exists an urgent demand for a more sensitive and widely applicable sequencing technique.

About the study

In the present study, researchers employed PrimalScheme to develop primers for the human mpox virus in amplicon-based sequencing techniques.

Since early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron B.1 clade genomes had limited coverage, the team utilized the pre-outbreak genome of the closely associated A.1 clade as a reference for the primer design. The PrimalScheme-designed primer scheme consisted of 163 primer pairs with amplicon lengths ranging from 1597 to 2440 bp. At the Massachusetts State Public Health Laboratory (MASPHL), the team sequenced a total of 10 clinical samples with a variety of polymerase chain reaction (PCR) Ct values using amplicon-based as well as metagenomic sequencing methods.

To further evaluate the primer strategy, 145 additional clinical samples were sequenced in two separate laboratories. A total of  115 lesion swabs and eight oropharyngeal swabs were processed at the MASPHL, while 22 lesion swabs were processed for sequencing at the Yale School of Public Health (YSPH).

To gain a better understanding of the sequencing threshold, the team determined the PCR Ct value required to obtain 80% genome coverage at approximately 10X read depth per degree of downsampling using a logistic function analysis. To examine the variations in coverage depth for the downsampled data, the coverage depth was estimated at each nucleotide location across the genome. Furthermore, to discover which particular amplicons had low coverage, the depth of coverage was calculated for non-overlapping sections of amplicons that were randomly downsampled to one million reads per sample and for samples having Ct less than 31.

Results

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The study findings found equivalent genome coverage for amplicon and metagenomic sequencing in samples with low Ct (less than 18), but significantly greater genome coverage using amplicon sequencing in those with higher Ct. These preliminary results demonstrated that amplicon-based sequencing methods could enhance genome coverage of human mpox genomes having higher Ct levels extracted from clinical specimens, particularly at higher Ct values. The team noted that near-complete genomes could be effectively sequenced from oropharyngeal swabs with or without the presence of lesions. This showed that lesion swabs as well as oropharyngeal swabs may serve as sequencing samples.

The study also showed that reducing the total amount of sequencing reads to 0.5 million from two million reduced the 80% coverage Ct threshold, resulting in a 7.2% loss in genome coverage at 10X. If resources permit, the researchers suggested the production of at least one million sequencing reads in each sample to obtain an overall high genome coverage of over 80% at 10X read depth. Increasing the proportion of sequencing reads to two million facilitated the creation of higher coverage for specimens having comparatively high Ct values of over 31. However, for samples with Ct values less than 31, only slight variations of average 2.1% in coverage at 10X read depth were detected, averaging 2.1%. To optimize genome coverage, The team recommends choosing sequencing samples having a Ct value below 31 and producing at least one million reads per sample, especially in the case of limited resources.

This research also demonstrated that elevating Ct values and lowering sequencing reads per sample lead to a greater number of genomic areas with coverage of less than 10X depth. In addition, the depth of coverage varied across the genome. Amplicon-based sequencing resulted in rather consistent high genome coverage, leading to reducing coverage with increasing Ct values.

Overall, the study findings showed that amplicon-based sequencing could improve the sensitivity, depth, and breadth of the genomic coverage of the human mpox virus even when low DNA concentration samples were employed. Through the ability to "plug" the human mpox viral primer scheme into the current sequencing infrastructure, the present method can enable public health laboratories worldwide to rapidly adjust their existing procedures in response to the global mpox epidemic.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Bhavana Kunkalikar

Bhavana Kunkalikar is a medical writer based in Goa, India. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. Her educational background allowed her to foster an interest in anatomical and physiological sciences. Her college project work based on ‘The manifestations and causes of sickle cell anemia’ formed the stepping stone to a life-long fascination with human pathophysiology.