Sequence enrichment of long DNA to enable new modes of genomic analysis

Sequence enrichment of long DNA to enable new modes of genomic analysis SBIR grantProject Summary Abstract The assembly of phased maternal and paternal haplotypes from human genomic DNA is compromised when short DNA fragments are used for sequencingThe analysis of DNA methylation modifications is also severely compromised when short DNA is sequencedA technology recently developed by PetaOmicsIncenables DNA sequence enrichment that yields long DNA fragments with sizes in the range ofbpWith future improvementsthe capture of fragments ofbp should be possibleThe technology is novel and disruptiveand has strong potential for successful commercialization via sales of cost effective DNA target enrichment kitsAny region in the genome can be targeted for sequence enrichmentincluding regions with very high sequence variabilityThe enriched DNA is double strandedand both strands retain all the biologically relevant DNA methylation informationThe widespread utilization of the technology will advance the field of genomicsespecially when deployed in population studies of complex human diseaseswhere it can help to unravel the functional impact of alternative diplotypesspecific combinations of maternal and paternal genetic informationThis Phase I research project aims to refine this novel technology by optimizing probe chemistry as well as DNA capture protocolsThe utility of the technology will be demonstrated by targeting for sequence enrichment a segment ofmegabases located within the MHC locus in human chromosomecharacterized by complex structural variation that includes variable numbers of sequence repeatsThe sequence enriched material will consist of longoverlappingdouble stranded DNA fragments derived from the MHC region of interestThe Pacific BiosciencesPacBiolong read DNA sequencing platform will be used to sequence the enriched DNAand to perform a de novo sequence assembly of the targeted region of the human genomeThe sequence enriched material will additionally be used for perform DNA methylation sequencing using the PacBio platformLong sequencing reads will be informative with regards to a subset ofhydroxy methylcytosine modificationsand will be used to assemble very long DNA methylation contigsCompletion of these aims will set the stage for a Phase II project directed to demonstrating genomewide applicabilityand potential for rapid commercialization of sequence enrichment kitsas well as to deployment of novel DNA methylation sequencing applications of the technology that will have a dramatic impact in human epigeneticspopulation studiesand medical research in general!Sequence enrichment of long DNA to enable new modes of genomic analysis SBIR grant! Project Narrative There is a need for DNA sequence enrichment approaches that are enabling for powerfulyet low cost modes of DNA sequencing analysis based on targeted capture of longdouble stranded DNA moleculesThis Phase I research project aims to refine this novel technology and to demonstrate its utilization for sequence enrichment of a large and structurally complex segment of the MHC locus in human chromosomeincluding the de novo assembly of long phased haplotypesWe will demonstrate significant improvements and substantive advantages over existing methodsincluding enhanced capabilities for DNA methylation analysis!