Advances In Single Molecule Real Time Smrt Sequencing

Author : Matthew Pendleton
Publisher :
ISBN 13 :
Total Pages : 163 pages
Book Rating : 4.:/5 (984 download)

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Download or read book Resolving Genome Complexity: Computational and Technological Methods written by Matthew Pendleton and published by . This book was released on 2017 with total page 163 pages. Available in PDF, EPUB and Kindle. Book excerpt: In the post-human genome era we have amassed endless genome sequencing data, but our ability to address variation between genomes has been largely restricted to small indels and single nucleotide variations. More complex regions of the genome, such as highly repetitive regions or those containing structural variation, have been largely ignored. This proposal aims to help elucidate these regions via a combination of computational and experimental approaches.First, we seek to build computational tools for identifying and computing large genomic differences by taking advantage of “third-generation” DNA sequencing technologies. This is done, in part, using a novel structural variation tool which uses a systematic framework for interrogating sequence “dot plots” (or groups of sequence “dot plots”) which have been historically used for visual identification of genome rearrangements, especially for complex comparative genomics. We combine these approaches with established tools for identifying single nucleotide variations (SNVs), to calculate multi-event co-variance in two constrained cancer projects: HCV viral quasispecies analysis in liver cancer, and FLT3 tyrosine kinase variation in acute myelogenous leukemia (AML). We then apply and extend these methods (along with existing methods for assembly, scaffolding, and phasing) in the context of the first amplification-free, diploid human genome assembly. In addition to enumerating far more structural variation than has been previously seen, this work provides the first human assembly that combines single molecule real time sequencing on Pacific BioSciences’ SMRT RT II sequencing platform with optical mapping generated by BioNano Genomics Irys mapping system. This hybrid strategy results in a de novo assembled genome that approaches reference quality.Second, to target the most complicated regions of the genome, we propose a pair of new amplification-free enrichment technologies. One approach combines an old mainstay of human genomics, the pulsed field gel electrophoresis, with the power of RNA guided cas9 endonuclease to enable enrichment of high molecular weight DNA from genome intervals that are difficult to manipulate with BAC cloning methods. The other approach employs an inactivated Cas9 protein engineered to permit bead based pull-down of a targeted region. We believe these approaches fill-in a needed gap, where resolution of structural variants, unambiguous haplotypic resolution and complete assembly would be impossible with existing targeting approaches but prohibitively expensive using high depth, long read whole genome sequencing.