Dr. Han was one of a number of scientists who made presentations regarding target enrichment at the “Sequencing, Finishing, and Analysis in the Future” (SFAF) conference in Santa Fe, which was co-sponsored by the Los Alamos National Laboratory and DOE Joint Genome Institute.
According to Dr. Han, for the repetitive regions, PCR primers are based on conserved sections of the genes for 16S and 23S ribosomal RNA, which appear in many locations of bacterial genomes.
“We enrich the genome, put the enriched fragments onto the Pacific Biosciences sequencer, and sequence the repeats,” continued Dr. Han. “In many parts of the sequence there will be a unique sequence anchored at one or both ends of it, and that will help us to link these scaffolds together.”
This work, while promising, will remain unpublished for now, as the Joint Genome Institute has shifted its resources to other projects.
As target-enrichment strategies go, PCR-based methods have both advantages and disadvantages relative to hybridization-based methods, in which “bait” sequences are used to capture the targets of interest from genomic libraries.
“Hybridization methods are flexible and have multiple stop-start sites, and you can capture very large sizes, but they require library prep,” said Jennifer Carter Jones, Ph.D., a genomics field applications scientist at Agilent. “With PCR-based methods, you have to design PCR primers and you’re doing multiplexed PCR, so it’s limited in the size that you can target. But the workflow is quick because there’s no library preparation; you’re just doing PCR.”
Given these considerations, the choice of method depends on a given project’s specific questions and equipment.
“If you’re just thinking about them roughly, the high-throughput sequencers like the Illumina HiSeq system or Life Technologies SOLiD 5500 system, have tremendous capacity and can target very large capture sizes, so hybridization can be a good fit,” said Dr. Jones.
“And then there’s your desktop sequencers, which are limited in capacity, but you can get results very quickly, and so a PCR-based method may be advantageous.”
At the SFAF conference Dr. Jones focused on going beyond basic target enrichment and described new tools for more efficient NGS research. She discussed Agilent’s recently acquired HaloPlex technology, a hybrid system that includes both a hybridization step and a PCR step. Because no library preparation is required, sequencing results can be obtained in about six hours, making it suitable for clinical uses.
However, the hybridization step allows capture of targets of up to 5 megabases—longer than purely PCR-based methods can deliver.
The Agilent talk also provided details on the applications of SureSelect, the company’s hybridization technology, to Methyl-Seq and RNA-Seq research. With this technology, 120-mer baits hybridize to targets, then are pulled down with streptavidin-coated magnetic beads.
“The reason why a long bait is important,” said Dr. Jones, “is that it’s more tolerant of mismatches. So if you have large indels, we’ll still be able to capture and pull down your targets.”
Agilent’s SureSelectXT Human MethylSeq includes baits for capturing the 84 megabases of the genome, including 3.7 million CpGs, that are thought to be most important in determining a cell’s methylation state.