New workflow developed by Brown University researchers compresses hours of manual lab labor into a single automated process, unlocking reliable genetic sequencing for local public health labs.
PROVIDENCE, R.I. [Brown University] - Biomedical engineers at Brown University have developed a fully automated workflow that simplifies and accelerates the preparation of bacterial samples for genetic sequencing.
The method, dubbed Pathogen2Read, streamlines a critical bottleneck in the genetic sequencing process and could enable small labs without high-throughput automation to more effectively participate in outbreak-monitoring networks operated by the U.S. Food and Drug Administration (FDA) and the Centers for Disease Control. That could make for faster responses to foodborne illnesses and other outbreaks.
"Next-generation sequencing has become a staple in outbreak detection and prevention," said Kathryn Whitehead, a graduate student in Brown's School of Engineering who led the work. "But sample preparation involves labor-intensive manual preparation and culture isolation, which can delay real-time outbreak responses. Our laboratory has developed what is, to our knowledge, the first fully automated scientific method that bypasses these limitations."
Research describing and testing the method, which was developed in collaboration with researchers at the FDA and with funding from the biotech firm Revvity, is published in BMC Genomics.
Next-generation sequencing has revolutionized modern epidemiology by allowing scientists to rapidly sequence the entire genomes of potential pathogens in just a few hours or days. That enables researchers and public health professionals to quickly identify pathogens involved in illness outbreaks or to pinpoint new genetic mutations that may make known pathogens harder to treat.
But the process involved in preparing samples for sequencing is complex and labor-intensive. It's a multistep process that involves isolating microbes, breaking cell membranes open (a process called lysis), extracting and purifying the DNA, and arranging it into readable segments. Current techniques require eight to 10 hours of hands-on work plus up to 16 hours of waiting time. Any mistakes along the way could require doing the entire process all over again.
The Pathogen2Read workflow includes assay preparation steps, custom software and a specially prepared enzyme cocktail that enables a desktop liquid-handling machine to handle all the steps of DNA sample prep - lysis, extraction and library preparation - entirely on its own. The researchers successfully compressed the hands-on preparation time from nearly a full day to under 45 minutes. Once an operator loads the raw samples and reagents onto a single plate, the system handles the rest automatically over a six-hour run, outputting pristine, sequencer-ready DNA libraries.
The quality of the DNA libraries is critical, particularly when looking for mutations in a bacterial strain.
"Because you're looking for small mutations that may be involved in drug resistance, for example, it's easy to miss them if you're not capturing all the sequences," said co-author Anubhav Tripathi, a professor of engineering and a faculty affiliate of Brown's Institute for Biology, Engineering and Medicine. "So the quality of the sample preparation is critically important."
At the crux of the new workflow, Whitehead says, is the enzyme cocktail, which quickly and effectively breaks open different types of bacteria. Bacteria come in two broad structural forms, gram-negative and gram-positive. The membrane structure of gram-positive bacteria makes them more difficult to crack open to extract DNA. That can cause the presence of gram-positive bacteria to be missed in a sample if the preparation process fails to crack them open. The researchers demonstrated an enzyme cocktail that had a nearly 2.5-fold improvement over standard methods in capturing gram-positive DNA, and reduced waiting time from 16 hours to 30 minutes.
The researchers are hopeful the method will improve outbreak monitoring and prevention by bringing smaller, local public health laboratories into the fold.
"The reason we're so excited about this is it was developed with real-world impact in mind," Whitehead said. "Having that collaboration with the FDA, being able to get their responses and their input on what they need to see, has allowed us to develop a method that actually can be used and doesn't have some of the limitations that you may sometimes see going from academic to translational research."
