Rapid whole-genome mutational profiling using next-generation sequencing technologies

被引：223

作者：

Smith, Douglas R. ^{[1
]}

Quinlan, Aaron R. ^{[2
]}

Peckham, Heather E. ^{[3
]}

Makowsky, Kathryn ^{[1
]}

Tao, Wei ^{[1
]}

Woolf, Betty ^{[1
]}

Shen, Lei ^{[1
]}

Donahue, William F. ^{[1
]}

Tusneem, Nadeem ^{[1
]}

Stromberg, Michael P. ^{[2
]}

Stewart, Donald A. ^{[2
]}

Zhang, Lu ^{[2
]}

Ranade, Swati S. ^{[3
]}

Warner, Jason B. ^{[3
]}

Lee, Clarence C. ^{[3
]}

Coleman, Brittney E. ^{[3
]}

Zhang, Zheng ^{[3
,4
]}

McLaughlin, Stephen F. ^{[3
]}

Malek, Joel A. ^{[3
]}

Sorenson, Jon M. ^{[3
]}

Blanchard, Alan P. ^{[3
]}

Chapman, Jarrod ^{[5
]}

Hillman, David ^{[5
]}

Chen, Feng ^{[5
]}

Rokhsar, Daniel S. ^{[5
]}

McKernan, Kevin J. ^{[3
]}

Jeffries, Thomas W. ^{[6
]}

Marth, Gabor T. ^{[2
]}

Richardson, Paul M. ^{[5
]}

机构：

[1] Agencourt Biosci Corp, Beverly, MA 01915 USA

[2] Boston Coll, Dept Biol, Chestnut Hill, MA 02467 USA

[3] Appl Biosyst Inc, Beverly, MA 01915 USA

[4] Appl Biosyst Inc, Foster City, CA 94404 USA

[5] US DOE, Joint Genome Inst, Walnut Creek, CA 94598 USA

[6] US Forest Serv, Inst Microbial & Biochem Technol, Madison, WI 53726 USA

来源：

GENOME RESEARCH | 2008年 / 18卷 / 10期

关键词：

D O I：

10.1101/gr.077776.108

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 [生物化学与分子生物学]; 081704 [应用化学];

摘要：

Forward genetic mutational studies, adaptive evolution, and phenotypic screening are powerful tools for creating new variant organisms with desirable traits. However, mutations generated in the process cannot be easily identified with traditional genetic tools. We show that new high-throughput, massively parallel sequencing technologies can completely and accurately characterize a mutant genome relative to a previously sequenced parental (reference) strain. We studied a mutant strain of Pichia stipitis, a yeast capable of converting xylose to ethanol. This unusually efficient mutant strain was developed through repeated rounds of chemical mutagenesis, strain selection, transformation, and genetic manipulation over a period of seven years. We resequenced this strain on three different sequencing platforms. Surprisingly, we found fewer than a dozen mutations in open reading frames. All three sequencing technologies were able to identify each single nucleotide mutation given at least 10-15-fold nominal sequence coverage. Our results show that detecting mutations in evolved and engineered organisms is rapid and cost-effective at the whole-genome level using new sequencing technologies. Identification of specific mutations in strains with altered phenotypes will add insight into specific gene functions and guide further metabolic engineering efforts.

引用

页码：1638 / 1642

页数：5

共 13 条

[1]

Burke D., 2000, Methods in Yeast Genetics Plainview, NY, V2000

[2]

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