title: Rapid whole-genome mutational profiling using next-generation sequencing technologies.
abstract: 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.
authors.affiliation: Agencourt Bioscience Corporation, Beverly, Massachusetts 01915, USA. douglas.smith@agencourt.com
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Smith
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Quinlan
authors.forename: Heather E
authors.initials: HE
authors.lastname: Peckham
authors.forename: Kathryn
authors.initials: K
authors.lastname: Makowsky
authors.forename: Wei
authors.initials: W
authors.lastname: Tao
authors.forename: Betty
authors.initials: B
authors.lastname: Woolf
authors.forename: Lei
authors.initials: L
authors.lastname: Shen
authors.forename: William F
authors.initials: WF
authors.lastname: Donahue
authors.forename: Nadeem
authors.initials: N
authors.lastname: Tusneem
authors.forename: Michael P
authors.initials: MP
authors.lastname: Stromberg
authors.forename: Donald A
authors.initials: DA
authors.lastname: Stewart
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.forename: Swati S
authors.initials: SS
authors.lastname: Ranade
authors.forename: Jason B
authors.initials: JB
authors.lastname: Warner
authors.forename: Clarence C
authors.initials: CC
authors.lastname: Lee
authors.forename: Brittney E
authors.initials: BE
authors.lastname: Coleman
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhang
authors.forename: Stephen F
authors.initials: SF
authors.lastname: McLaughlin
authors.forename: Joel A
authors.initials: JA
authors.lastname: Malek
authors.forename: Jon M
authors.initials: JM
authors.lastname: Sorenson
authors.forename: Alan P
authors.initials: AP
authors.lastname: Blanchard
authors.forename: Jarrod
authors.initials: J
authors.lastname: Chapman
authors.forename: David
authors.initials: D
authors.lastname: Hillman
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Rokhsar
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: McKernan
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Jeffries
authors.forename: Gabor T
authors.initials: GT
authors.lastname: Marth
authors.forename: Paul M
authors.initials: PM
authors.lastname: Richardson
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D016681:Genome, Fungal; D009154:Mutation; D010843:Pichia / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
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