Denaturation-Free DNA Target Enrichment and Direct Sequencing of Large Chromosomal Segments

SummaryThird generationlong read sequencing technologies are increasingly sought after for the analysis of difficult genomic regionssuch as the major histocompatibility complexMHCthe human leukocyte antigenHLAloci and genomes of cancerspathogens and viral insertionsas well as plant and animal genomesSuch methods provide researchers with more contextual informationenabling analysis of highly complex genomic regionsThe ability to utilize largecontiguous DNA segments as sequencing templates greatly assists in detecting and characterizing structural variantsobtaining accurate sequence information across complex and GC rich genomic lociconducting de novo sequence assemblyphasing long range haplotype informationand finding new or unexpected sequence elementssuch as viral integration sitesinversionsduplications and other types of chromosomal rearrangementsMany targeted capture technologies can only pull down shorterandltkbregions or require denaturation steps that can limit the size of recovered DNAand thus cannot take advantage of these long read technologiesHere we propose to develop a method that will significantly improve genome sequencing by providing a simple and rapid method for targeted capture of longandgtkbgenomic DNA segments without DNA denaturationThis will provide researchers with a simple to use kit that can reduce sequencing costs and improve the quality of their resultsparticularly in highly complex genomic regionsThe proposed methodcalled CAPTRis based on the use of the CRISPR CassystemBy employing this system in a novel waywe can achieve targeted long range capture from non denatured DNA in a fastsingle tube assay that is directly compatible with all common NGS library prep workflowsIn this Phase I feasibility studywe will develop this capture method and test its ability to enable capture and sequencing of a complex and highly relevant region of the human genomethe human leukocyte antigenHLAregionMilestones for success include demonstrating at leastx target enrichmentsequencing of a andgtkb segment of non denatured genomic DNAand obtaining methylation status information by direct sequencing of DNA captured by this methodThe method leverages Generation Biotech syears of experience in targeted long range genomic DNA capture by magnetic beadsIf successfulthis technology will greatly improve the ability of researchers to obtain accurate information from very difficult genomic regions that generate persistent problems with current methodsThis can lead to extension of this technique to the clinical realmwhere it can speed the advent of personalized medicineproviding fastermore accurateand less expensive diagnostics and immunotherapies for cancersbetter matches for organ transplantsand more effective treatment for immune related diseases and infections Narrative Many regions of the genome being studies by researchers are highly complex and require isolation of large contiguous segments of that region in order to give information about itscontextMany DNA capture methods are not able to provide these researchers with such large regionsThis project is aimed at developing a novel method of capturing long segments of specific genomic regions that will give researchers the contextual information they need to study these regions