A new generation of technologies is poised to reduce the cost of DNA sequencing by over two orders of magnitude. However, the routine sequencing of full human genomes will continue to be prohibitively expensive in the context of studies that require even modest sample sizes. However, it is frequently the case that investigators are interested in identifying germline variation or somatic mutations in a particular subset of the genome. Examples of genomic subsets that are highly relevant in the context of specific studies include: (a) a locus to which a disease phenotype has been mapped (i.e. a contiguous genomic region);and (b) the exons of genes belonging to a specific disease-related pathway (i.e. a large set of short, discontiguous sequences). Such subsets total to megabases in length, raising the question of how they can be efficiently isolated without performing hundreds to thousands of PCR reactions per genome. Our ability to take advantage of the power of next-generation sequencing technologies is markedly impaired by the lack of a corresponding targeting method, analogous to PCR that is matched to the scale at which the new sequencing platforms will routinely operate. To address this critical need, we will explore several novel strategies for """"""""genome partitioning"""""""". Our goal is to develop these strategies into broadly available methods that enable the selective and uniform amplification of complex, arbitrary subsets of a mammalian genome in a single reaction.
Our specific aims are: (1) to develop an enzymatic method for the uniform amplification of large sets of exon sequences from a human genome;(2) to develop a hybridization-based method for the selective amplification of contiguous megabase-scale regions from a human genome;(3) to integrate these methods with next-generation sequencing technologies, validating their utility by performing targeted variation discovery in a small number of individuals.
As we enter an era of """"""""personalized medicine"""""""", DNA sequencing technology will be increasingly important to public health, contributing towards the unraveling of the genetic basis of human disease, as well as serving an increasing role in clinical diagnostics. Next-generation sequencing technologies have the potential to markedly accelerate genetics research, but are markedly hindered by the lack of equivalently powerful methods to target specific subsets of the human genome. We propose here to develop technologies that meet this critical need.
| Knowles, Michael R; Ostrowski, Lawrence E; Loges, Niki T et al. (2013) Mutations in SPAG1 cause primary ciliary dyskinesia associated with defective outer and inner dynein arms. Am J Hum Genet 93:711-20 |
| Knowles, Michael R; Leigh, Margaret W; Ostrowski, Lawrence E et al. (2013) Exome sequencing identifies mutations in CCDC114 as a cause of primary ciliary dyskinesia. Am J Hum Genet 92:99-106 |
| Bamshad, Michael J; Ng, Sarah B; Bigham, Abigail W et al. (2011) Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet 12:745-55 |
| Hannibal, Mark C; Buckingham, Kati J; Ng, Sarah B et al. (2011) Spectrum of MLL2 (ALR) mutations in 110 cases of Kabuki syndrome. Am J Med Genet A 155A:1511-6 |
| George, Renee D; McVicker, Graham; Diederich, Rachel et al. (2011) Trans genomic capture and sequencing of primate exomes reveals new targets of positive selection. Genome Res 21:1686-94 |
| Cooper, Gregory M; Goode, David L; Ng, Sarah B et al. (2010) Single-nucleotide evolutionary constraint scores highlight disease-causing mutations. Nat Methods 7:250-1 |
| Ng, Sarah B; Buckingham, Kati J; Lee, Choli et al. (2010) Exome sequencing identifies the cause of a mendelian disorder. Nat Genet 42:30-5 |
| Ng, Sarah B; Bigham, Abigail W; Buckingham, Kati J et al. (2010) Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat Genet 42:790-3 |
| Roach, Jared C; Glusman, Gustavo; Smit, Arian F A et al. (2010) Analysis of genetic inheritance in a family quartet by whole-genome sequencing. Science 328:636-9 |
| Mamanova, Lira; Coffey, Alison J; Scott, Carol E et al. (2010) Target-enrichment strategies for next-generation sequencing. Nat Methods 7:111-8 |
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