Massively parallel technologies have reduced the per-base cost of DNA sequencing by several orders of magnitude. However, limited read lengths and a lack of methods to establish contiguity over even modest distances have prevented these technologies from achieving the high-quality, low-cost de novo assembly of mammalian genomes. Even as revolutionary sequencing technologies further mature, it may continue to be the case that the best technologies in terms of cost-per-base yield reads that are of an insufficient length or quality for the effective de novo assembly of large genomes. To address this critical need, we are exploiting high density, random, in vitro transposition as a novel means of physically shattering genomic DNA in creative ways that facilitate the recovery of contiguity information at different scales. Our project is divided into four aims, the first three of which are respectively directed at the development of massively parallel methods for determining short-range, mid-range, and long-range contiguity. These are: 1) a method for shattering genomic DNA with symmetric tags that post hoc inform the ordering of adjacent fragmentation events in a way that is entirely independent of the primary sequence content;2) a method for massively parallel, in vitro barcoding of fosmid or BAC-sized subsequences of a genome, thereby facilitating hierarchical assembly;3) an in situ method for converting stretched DNA molecules into adaptor-flanked libraries, such that reads generated by massively parallel sequencing will remain linearly ordered in terms of the XY coordinates at which they originate. In the fourth aim, we will integrate these methods to demonstrate: 1) the highly cost-effective de novo assembly of the mouse genome with a quality that exceeds that of the original assembly;2) the highly cost-effective haplotype resolved resequencing of a human genome.

Public Health Relevance

As we enter an era of personalized medicine, a deep understanding of the human genome will be increasingly important to public health, contributing to the unraveling of the genetic basis of human disease, as well as serving an increasing role in clinical diagnostics. The technologies developed by this project will accelerate progress towards these goals by enabling the affordable sequencing of haplotype-resolved human genomes. These same technologies will also facilitate the high-quality, cost-effective assembly of the genomes of other mammalian species, which inform our understanding of the human genome through evolutionary analysis.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG006283-03
Application #
8511772
Study Section
Special Emphasis Panel (ZHG1-HGR-N (M2))
Program Officer
Schloss, Jeffery
Project Start
2011-08-15
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
3
Fiscal Year
2013
Total Cost
$560,244
Indirect Cost
$196,449
Name
University of Washington
Department
Genetics
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Kronenberg, Zev N; Fiddes, Ian T; Gordon, David et al. (2018) High-resolution comparative analysis of great ape genomes. Science 360:
Cusanovich, Darren A; Hill, Andrew J; Aghamirzaie, Delasa et al. (2018) A Single-Cell Atlas of In Vivo Mammalian Chromatin Accessibility. Cell 174:1309-1324.e18
Cusanovich, Darren A; Reddington, James P; Garfield, David A et al. (2018) The cis-regulatory dynamics of embryonic development at single-cell resolution. Nature 555:538-542
Smukowski Heil, Caiti; Burton, Joshua N; Liachko, Ivan et al. (2018) Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C. Yeast 35:71-84
Cao, Junyue; Packer, Jonathan S; Ramani, Vijay et al. (2017) Comprehensive single-cell transcriptional profiling of a multicellular organism. Science 357:661-667
Bickhart, Derek M; Rosen, Benjamin D; Koren, Sergey et al. (2017) Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome. Nat Genet 49:643-650
Gasperini, Molly; Findlay, Gregory M; McKenna, Aaron et al. (2017) CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions. Am J Hum Genet 101:192-205
Ramani, Vijay; Deng, Xinxian; Qiu, Ruolan et al. (2017) Massively multiplex single-cell Hi-C. Nat Methods 14:263-266
Gordon, David; Huddleston, John; Chaisson, Mark J P et al. (2016) Long-read sequence assembly of the gorilla genome. Science 352:aae0344
Salipante, Stephen J; Adey, Andrew; Thomas, Anju et al. (2016) Recurrent somatic loss of TNFRSF14 in classical Hodgkin lymphoma. Genes Chromosomes Cancer 55:278-87

Showing the most recent 10 out of 27 publications