An international group of researchers from the University of Gothenburg in Sweden, the Ontario Institute for Cancer Research, and Boston University School of Medicine have recently published the specifics about their newly developed technique to avoid a major problem with common laboratory methods and improve the sensitivity of important cancer tests. The investigator's findings were published recently in Nature Protocols through an article entitled “Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing.”
The new technique is a process by which the sensitivity of DNA sequencing can be improved. The technology, called SiMSen-Seq, could aid in detecting the recurrence of cancers, catching possible disease relapses faster than current methods, and improving patient outcomes.
When sequencing DNA, researchers often use polymerase chain reaction (PCR) to increase the amount of DNA available from a sample. Yet, PCR can introduce mistakes that can limit many researchers' ability to detect real mutations in the original DNA molecules. To track the original molecules in a sample, molecular tags called DNA barcodes are added. This technique is essential for sensitive detection of mutations but can lead to other errors, as components of the tags can interfere with each other and affect the final results.
"We created a DNA barcode with a hairpin structure that opens up to be read when heated and contracts when cooled,” explained study co-author Paul Krzyzanowski, Ph.D., program manager of OICR's Genome Technologies Program. “This allows us to 'hide' the barcode and analyze more patient DNA fragments in a single reaction.”
Current genome sequencing technologies return results with error rates of approximately one percent, meaning that for researchers to be certain that a mutation exists it has to be detected in a sample at a rate of greater than one percent. SimSen-Seq technology has lowered this error rate 100-fold, meaning that the recurrence of cancers could be detected at lower levels—and earlier—than before, allowing patients to receive additional treatment sooner.
“Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (~4 h) library construction protocol,” the authors wrote. “The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory.”
The team has patented their technique, and while it can conceivably be performed in any molecular biology lab, the group also hopes to make their expertise in using the method available to the research community. "Our paper describes how this process can be carried out, but we think that our experience in using the technique could be leveraged by other research groups and save them the trial and error of instituting a new process," Dr. Krzyzanowski noted.