Inexpensive, Efficient Approach Developed to Make Pathogen Sequencing More Accessible

Inexpensive, Efficient Approach Developed to Make Pathogen Sequencing More Accessible
Salmonella bacteria, a common cause of food poisoning, invade an immune cell. (Credit: National Institute of Allergy and Infectious Diseases, National Institutes of Health)

An international group of researchers has developed a cheap and efficient method for sequencing large numbers of bacterial pathogens for as little as $10USD per genome, something they hope will make this kind of analysis more accessible for low- and middle-income countries.

The COVID-19 pandemic has highlighted the value of whole genome sequencing (WGS) of microbes, for example, for identification of new variants of concern of the SARS-CoV-2 virus. Similarly, WGS of pathogenic bacteria has become increasingly important for similar reasons.

Despite this, until recently large-scale bacterial sequencing could only be performed in a few sequencing centers around the world.

As reported in the journal Genome Biology, scientists led by the Earlham Institute and the University of Liverpool in the UK have developed a new pipeline approach to solve this problem.

“It has been 26 years since the first bacterial genome was sequenced, and it is now possible to sequence bacterial isolates at scale. However, access to this game-changing technology for scientists in low- and middle-income countries has remained restricted,” said study author and director of the Earlham Institute Neil Hall.

“The need to ‘democratize’ the field of pathogen genomic analysis prompted us to develop a new strategy to sequence thousands of bacterial isolates with collaborators based in many economically-challenged countries.”

The project focused on the bacteria Salmonella enterica, a bacterial infection that causes life threatening disease around the world. Mortality rates from this infection are particularly high in sub-Saharan Africa. The 10,000 Salmonella genomes research consortium includes scientists from 25 organizations in 16 countries and aims to make bacterial genomic data easier to access for low- and middle-income countries for use in pathogen surveillance. Within a year the project had collected more than 10,000 samples from countries in these regions.

“Limited funding resources prompted us to design an approach that ensured accurate sample tracking and captured comprehensive metadata for individual bacterial isolates while minimizing costs for the consortium,” write the authors. “A key driver was to assemble a set of genomic data that would be as informative and robust as possible.”

One factor that increases cost for bacterial WGS is refrigerated transportation. As part of setting up the sequencing pipeline for the project, the researchers tested the stability of the samples in non-refrigerated conditions by keeping them at room temperature for 7 days and then testing the quality of the extracted DNA. They also kept the number of steps for sample collection and send out to a minimum to improve efficiency and costs.

The sequencing itself was also optimized. Carried out at the Earlham Institute in Norwich, a low cost, low input, automated method called ‘LITE’ was used to rapidly sequence all the bacterial genomes. Construction of a gene library and all the bioinformatic analysis needed was done at a total cost of less than $10 per genome, when accounting for reagents needed.

“We saw the need to simplify and expand genome-based surveillance of salmonellae from Africa and other parts of the world, involving isolates associated with invasive disease and gastroenteritis in humans, and extending to bacteria derived from animals and the environment,” said Darren Heavens, a postdoctoral scientist at the Earlham Institute, who developed the whole-genome sequencing pipeline.

“Our pipeline represents a cost-effective and robust tool for generating bacterial genomic data from low- and middle-income countries, to allow investigation of the epidemiology, drug resistance and virulence factors of isolates.”