Targeted resequencing is one of the fastest growing applications for next-generation sequencing technology. The ultimate goal is to look for causative mutations within discrete genomic loci in populations in order to enhance diagnostics and treatments. The availability of next-generation sequencing tools has dramatically increased sequencing throughput.
Some of the challenges and highlights in the field will be presented at CHI’s “Next-Generation Sequencing Conference” next month. Scientists will be reporting on cutting-edge technologies that include massively parallel analysis of DNA fragments linked to beads, microfluidics for droplet-based PCR, and microarray-focused enrichment of targeted sequences.
“Recent data suggests that disease may not be associated with a limited number of common variants, but more likely caused by a large number of rare variants,” suggests Jeff Olson, nucleic acid applications manager, RainDance Technologies (RDT). Targeted sequencing on next-generation sequencers is one of the technologies that can be applied to identify these rare variants in patient samples.
“We have created a whole new platform class using the simplicity and speed of droplet-based microfluidics to increase the efficiency of targeted sequencing,” Olson says. “Basically, we perform PCR in picoliter volume droplets, which avoid the challenges of alternative methods such as multiplexed PCR amplifications and hybridization.”
The technology provides a means for high-resolution analysis of genetic variation among individuals within populations. “The use of droplet-based microfluidics greatly improves uniformity and specificity of the targeted sequencing process, which increases the efficiency of next-generation sequencers.”
The key to accomplishing this process is, according to RainDance, its Sequence Enrichment solution, which begins with the design and generation of a PCR primer pair library in droplets capable of amplifying hundreds to thousands of genomic loci.
The RDT 1000 instrument merges these droplets containing the PCR primer pairs with droplets containing the genomic DNA sample and the PCR reaction mix. This forms a fully functional PCR reaction in the merged droplet. The RDT 1000 instrument can generate these PCR droplets at a rate of 10 million discrete droplets per hour, Olson says. The droplets are then thermocycled with an end result of thousands of PCR-amplified genomic loci in a single tube that can then be characterized by any of the next-generation sequencing platforms.
Identifying Sequence Variants
Ewen Kirkness, Ph.D., investigator in the genomic medicine group at the J. Craig Venter Institute, is utilizing the RainDance Technologies’ droplet-based platform.
“We are all on a learning curve with the new technologies for target preparation and sequencing. Until recently, Sanger sequencing of PCR products has been the gold standard. With high levels of sequence coverage, however, the newer sequencing technologies can reveal a more comprehensive collection of sequence variants in targeted regions of the genome. The challenge now is to optimize sample preparation for the most cost-effective use of the new sequencing technologies.”
Dr. Kirkness’ group is collaborating with Scripps Research Institute to identify genomic variants associated with healthy aging. “We are interested in the healthy elderly, and the specific genes that contribute to this condition. We’ve spent a few months considering the related literature, and have come up with an initial list of about 100 candidate genes. After comparing to matched controls, we hope to identify key genes that are involved in healthy longevity.”