Research Project
9847444
Summary ?Third-generation? long-read sequencing technologies are increasingly sought after for the analysis of difficult genomic regions, such as the major histocompatibility complex (MHC), the human leukocyte antigen (HLA) loci and genomes of cancers, pathogens and viral insertions, as well as plant and animal genomes. Such methods provide researchers with more contextual information, enabling analysis of highly complex genomic regions. The ability to utilize large, contiguous DNA segments as sequencing templates greatly assists in detecting and characterizing structural variants, obtaining accurate sequence information across complex and GC-rich genomic loci, conducting de novo sequence assembly, phasing long-range haplotype information, and finding new or unexpected sequence elements, such as viral integration sites, inversions, duplications and other types of chromosomal rearrangements. Many targeted capture technologies can only pull down shorter (<1 kb) regions or require denaturation steps that can limit the size of recovered DNA, and thus cannot take advantage of these long-read technologies. Here we propose to develop a method that will significantly improve genome sequencing by providing a simple and rapid method for targeted capture of long (>50 kb) genomic DNA segments without DNA denaturation. This will provide researchers with a simple to use kit that can reduce sequencing costs and improve the quality of their results, particularly in highly complex genomic regions. The proposed method, called CAPTR, is based on the use of the CRISPR Cas9 system. By employing this system in a novel way, we can achieve targeted long-range capture from non-denatured DNA in a fast, single tube assay that is directly compatible with all common NGS library prep workflows. In this Phase I feasibility study, we will develop this capture method and test its ability to enable capture and sequencing of a complex and highly relevant region of the human genome: the human leukocyte antigen (HLA) region. Milestones for success include demonstrating at least 100x target enrichment, sequencing of a >50 kb segment of non-denatured genomic DNA, and obtaining methylation status information by direct sequencing of DNA captured by this method. The method leverages Generation Biotech?s 20 years of experience in targeted long-range genomic DNA capture by magnetic beads. If successful, this technology will greatly improve the ability of researchers to obtain accurate information from very difficult genomic regions that generate persistent problems with current methods. This can lead to extension of this technique to the clinical realm, where it can speed the advent of personalized medicine? providing faster, more accurate, and less expensive diagnostics and immunotherapies for cancers, better matches for organ transplants, and more effective treatment for immune-related diseases and infections.
