Solid-phase replication of long template libraries for NGS applications

The rapid development of next generation sequencingNGStechnologies has allowed researchers to make unprecedented progress in the analysis of genomesthe results of which have profound implications for human healthThe ability to fully characterize genomes remains unrealizedhoweverdue in large part to our inability to reconcile sequence information over long distancesCurrent long readlong templateand synthetic library technologies are underpoweredinconvenientand expensiveThey do not provide a high throughputsingleplatform solution for the de novo assembly of complex genomesRedVault Biosciences is developing an innovative long template NGS technology that could fundamentally advance our current genome analysis strategies and would have broad applications in the clinic and laboratoryOur previous Phase I studyRCAdemonstrated feasibility and validated the early components of the technology by creating long template dumbbellLTDlibraries from genomic DNA and analyzing highly processstrand displacing DNA polymerasesSolid phase rolling circle replicationRCRwas performed fromkb andkb genomic LTD libraries and bound products were thoroughly characterizedTo demonstrate application using current sequencing technologywe propose a second Phase I grant to show feasibility of our LTD approach with a commercial NGS platformIf successfulour overall goal is to fully develop and translate RedVaultandapos s LTD sequencing technology into a robust integrated NGS pipeline capable of resolving complex structural variation and improving de novo genome assemblySpecific Aimwill focus on standardizing LTD construction protocols with a commercialshortread NGS platformSpecific Aimwill demonstrate compatibility of RedVaultandapos s LTD RCR products with short read NGS sequencing chemistry using arrayed flow cellsshort read NGS components and reduced complexity single base extension experimentsSpecific Aimwill optimize compatibility and performance by iteratively sequencing and assembling the Celegans genome from multiple LTD libraries The study and sequencing of genomes has led to amazing discoveries in forensicshistoryand medicinePrevious methods associated with preparing a DNA sample for sequencing depend on computationally reconstructing small pieces of data to understand genomic structure and variationswhich is time intensiveerror proneand not accurate enough for large scale genomic informationThe research proposed here is oriented toward significantly improving the methods of sample preparationwhich will lead to improved efficiencyaccuracyand reduced costs to sequence DNAthereby making the technology more accessible to more people