Rapid whole-genome mutational profiling using next-generation sequencing technologies.
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.
Smith
Douglas R
DR
Agencourt Bioscience Corporation, Beverly, Massachusetts 01915, USA. douglas.smith@agencourt.com
Quinlan
Aaron R
AR
Peckham
Heather E
HE
Makowsky
Kathryn
K
Tao
Wei
W
Woolf
Betty
B
Shen
Lei
L
Donahue
William F
WF
Tusneem
Nadeem
N
Stromberg
Michael P
MP
Stewart
Donald A
DA
Zhang
Lu
L
Ranade
Swati S
SS
Warner
Jason B
JB
Lee
Clarence C
CC
Coleman
Brittney E
BE
Zhang
Zheng
Z
McLaughlin
Stephen F
SF
Malek
Joel A
JA
Sorenson
Jon M
JM
Blanchard
Alan P
AP
Chapman
Jarrod
J
Hillman
David
D
Chen
Feng
F
Rokhsar
Daniel S
DS
McKernan
Kevin J
KJ
Jeffries
Thomas W
TW
Marth
Gabor T
GT
Richardson
Paul M
PM
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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