BACKGROUND
despite the wealth of knowledge that comparative genomics has provided about the evolution of past and current life forms, the process of adaptation is still poorly understood.
predictive insight into how adaptation will occur -- which adaptive mutations are likely to arise and fix during selection -- is a daunting challenge.
progress in this area will further efforts to deter cancer progression, the emergence of new pathogens, and antibiotic resistance.
as a simple model for analyzing adaptation, experimental evolution of microorganisms provides the unique opportunity to catalog, monitor the dynamics, and measure the reproducibility of adaptation in real time.
in the past decade, the characterization of laboratory evolution outcomes beyond phenotypic analysis has been advanced by the use of dna microarrays.
in bacteria and yeast, these approaches have been applied to expose adaptive point mutations and structural rearrangements in response to carbon source replacement as well as carbon source and nutrient limitation  <cit> .
furthermore, studies have begun to unveil the effect of population dynamics on the outcomes of evolution, providing direct evidence of parallel evolution and clonal interference and support for historical contingencies  <cit> .
however, these studies are limited in that the precise nature of the adaptive point mutations or large-scale sequence rearrangements in the evolved organisms has not been defined systematically.
in recent years, however, a series of high-throughput dna sequencing technologies capable of producing gigabases of dna sequence information in a single experiment have been developed.
these technologies are transforming biological research, allowing the rapid identification of genetic variation intrinsic to diseases, behavior and other traits.
indeed, these technologies have been applied to a wide host of biological research problems, from enhancing our understanding of laboratory strains of bacteria by pinpointing suppressor mutations  <cit>  and mutational biases  <cit> , to identifying the genetic variation within all known serotypes of a major viral pathogen  <cit> .
in yeasts, they have been applied to analyze the genetic variation within and between wild and domestic populations  <cit> , the spectrum of spontaneous mutations in saccharomyces cerevisiae  <cit>  and the adaptive mutations in a pichia stipilis strain efficient in xylose fermentation  <cit> .
in bacteria, these sequencing technologies have recently been applied to study the rate of evolutionary adaptation over  <dig>  generations  <cit> .
furthermore, they have launched a personal genomics era that is revealing the breadth of genetic variation in the small number of individuals fully sequenced thus far  <cit> , and across targeted subgenomic portions in many individuals  <cit> , pinpointing disease causative alleles in exomes of affected individuals  <cit> .
here, we use whole-genome sequencing to reveal the repertoire of point mutations and copy number polymorphisms in an evolved s. cerevisiae strain.
we sequenced an adapted strain isolated after ~ <dig> generations of a continuous haploid culture under sulfate limitation, and compared this analysis to mutational profiling data obtained using array-based technologies  <cit> .
we generated high-depth sequencing data for the evolved and parental genomes, applied a heuristic approach to uncover single nucleotide polymorphisms  and a recursive segmentation algorithm to discover breakpoints and refine copy-number estimates, and devised a general approach to extract precise breakpoint sequence information from single-end short reads.
these breakpoints allowed us to delineate the structural rearrangement underlying a 5× copy number amplification of a ~ <dig> kb genomic segment.
RESULTS
we constructed dna sequencing libraries from genomic dna of the yeast strain dby <dig>  evolved in a sulfate-limited chemostat, and its haploid ancestor strain dby <dig>  <cit> .
we collected  <dig> , <dig> and  <dig> , <dig> single-end,  <dig> bp reads from the evolved and parent genomes, respectively, on an illumina genome analyzer ii platform .
more than 75% of the reads  were aligned to the s. cerevisiae reference genome using maq  <cit> .
following quality-filtering, these reads yielded a coverage of ~ <dig> % of the mappable nuclear genome of both strains with an average read-depth of  <dig> × and  <dig> × in the evolved and ancestor genomes, respectively .
the majority of reads  had a mapping quality score ≥ <dig>
a heuristic approach to point mutation detection
in sequencing the adapted strain, our aim was to reveal the full complement of genetic adaptations that had occurred during its evolution under sulfate limitation.
copy number variation and point mutation analysis of this strain assessed by use of orf array and genomic tiling array approaches, respectively, provided a data set against which to compare the efficacy of whole-genome sequencing  <cit> .
to identify point mutations, we examined base calls using criteria that allowed comparisons between nucleotide positions in the evolved and parental short read data sets over the great majority of the genome.
we performed snp calling following two heuristic approaches, one that monitors commonly applied filters for read-depth and various quality scores, and one that is a simplified approach that monitors read-depth and the frequency of base-calls at each position.
for the simplified approach, we required at least  <dig> reads per position with ≥80% concordant snp calls in the evolved genome, and at least  <dig> reads per position with ≥70% concordant calls for a different base in the parental genome.
this approach recapitulated known features in the evolved genome while maximizing breadth, yielding a high-confidence set of snps and that allowed us to examine up to 91% of the mappable bases in the yeast genome.
we obtained modest gains in coverage following the simplified snp calling approach .
applying these heuristics, we detected four single-point differences between the laboratory evolved strain and that of its parent: the previously-described point mutations at pbp <dig> , sgf <dig> , and upf <dig>   <cit> , and an additional point mutation intergenic to rrn <dig> and ypk <dig>  which we confirmed by sanger sequencing .
the function of the rna polymerase i-specific transcription initiation factor rrn <dig> is conserved between yeast and humans  <cit> .
in humans, tif-ia  modulates ribosomal gene expression in response to nutrient limitation via the mtor pathway  <cit> .
the expression of rrn <dig> is less than half the level in the evolved strain compared to its ancestor, suggesting that this snp may be regulatory and part of the adaptive response in this lineage.
the finding of an additional snp in the sequencing data but missed by array-based comparative genome sequencing is consistent with the estimated 15% false negative rate for the snpscanner algorithm  <cit> .
in addition, we searched for small insertions and deletions via gapped alignment of reads unmapped in the maq alignment, requiring that candidate indel coordinates were sequenced as wildtype in the comparison strain.
however, no small insertion or deletion differences between the evolved and parental genomes could be detected .
segmentation-based detection of copy number polymorphisms
previous analysis of the evolved and parental strains using comparative genome hybridizations on microarrays  detected a genomic amplification spanning the high-affinity sulfate transporter sul <dig> locus in the genome of the laboratory-evolved strain, providing a 50% fitness advantage relative to the ancestral genome  <cit> .
sul <dig> gene amplification was a common adaptation in continuous cultures under sulfate limitation, occurring in  <dig> of  <dig> adapted clones from various experimental evolutions  <cit> .
however, the precise breakpoints and nature of the structural rearrangement has not been defined for these amplifications.
we compiled the read-depth data from evolved and parental genome sequencing data sets and calculated the ratio in read-depth for each base across the genome.
we used these data to perform circular binary segmentation  with dnacopy, a software package designed to partition the genome into segments of equal copy number from high-density array cgh data  <cit> .
dnacopy formulates the problem of copy-number variation  analysis as a change-point detection problem, whereby change-points correspond to the genomic locations of copy number transitions.
the algorithm searches for change-points by recursively partitioning the genome and performing a maximal t-statistic test between segment ratios, joining segments with comparable ratios.
we reasoned that this approach should detect transitions in ratios of sequencing read-depth as well, and applied it to search for such transitions throughout the yeast genome .
indeed, segmentation of sequencing depth ratios detected an ~ <dig> kb segment harboring sul <dig>  with an estimated 5× amplification .
these breakpoint coordinates are in close proximity to estimated coordinates obtained from tiling array data   <cit> , but have narrower uncertainty windows .
the read depth-based copy number analysis yielded a value close to an integer, suggesting it may be a reliable approximation of genomic copy number.
breakpoint sequence determination and analysis of rearrangement structure
we sought to pinpoint with single-base resolution the structural rearrangements underlying the sul <dig> amplification.
to do so, we established a strategy to detect breakpoints in shotgun, single-end, short-read sequencing data.
we unearthed breakpoint-spanning reads in the evolved genome sequencing data set by assembling unmapped reads into contigs using velvet, a short-read de novo assembly algorithm  <cit> .
these contigs were blat-aligned to the reference genome sequence in an attempt to detect signatures of novel structures in the genome.
this approach yielded three contigs composed of subsequences with alignments to the mappable nuclear genome.
of these contigs, two aligned to sequences within the predicted amplification boundaries .
performing this analysis on the ancestor genome sequencing data yielded two contigs at distinct coordinates , from which we estimate the likelihood of the signatures detected in the evolved genome arising independently of the amplification and found within the predicted boundaries to be very low .
the breakpoint-matching contigs are small  and contain inversions of nearby genomic sequences overlapped by ≤ <dig> bp .
these breakpoints occur within the ctp <dig> and pca <dig> coding sequences, and would result in  <dig> and  <dig> amino acid truncations, respectively.
in addition, we found reads spanning the wild-type sequence at these coordinates, indicating that full-length copies of these genes are retained in the genome of the adapted strain.
in combination with the inversions in breakpoint-spanning contigs, the observation of reads that conform to the wild-type sequences across the predicted rearrangement boundaries suggests that the 5× amplification spanning chr2:  <dig> - <dig>  is structured as tandem inversions along the chromosome .
this arrangement was validated by southern blot analysis of the region with three different restriction enzymes and two probes .
the fact that at both breakpoints we found short homologous sequences 7- <dig> bp long overlapping the segmental inversions suggests that short homologous nucleotide tracts may be involved in driving the large structural rearrangements.
among the contigs with subsequence alignments to the mappable reference genome, the preponderance were composed of mitochondrial sequences .
this abundance of contigs of unmapped reads composed of mitochondrial subsequences was recapitulated with the ancestor genome data, but we found little overlap between structures observed in the two mitochondrial genomes .
we did not observe copy number gains or losses at these coordinates, which are also covered at high-depth with wild-type sequences.
these observations may reflect mitochondrial heteroplasmy in the form of structural rearrangements.
discussion
evolutionary adaptation is a fundamental biological phenomenon with far-reaching implications in cancer, antibiotic resistance, infectious disease management, and ecology.
cataloging the precise genetic changes that underlie an organism's responses to defined selective environments can yield insights not only into the pathways that define fitness in these environments, but also into the breadth and reproducibility of adaptive mutations.
the generation of such data requires approaches that can rapidly assay entire genomes with single-base resolution to expose point mutations and at megabase-scale to define structural rearrangements.
in this study, we applied whole-genome sequencing to identify the genetic changes in a laboratory-evolved yeast strain, following on similar work in bacteria and phage  <cit> .
this analysis revealed changes in the genome corresponding to single nucleotide polymorphisms and copy number amplifications, with specific gains over array-based methodologies.
we produced high-depth genome sequences for a yeast strain evolved under nutrient limitation and its parental strain.
the genome sequence of the parent was critical in allowing us to pinpoint mutations acquired during experimental evolution, and serves as a template against which to compare the genomes of other descendent strains.
we foresee the practice of maintaining high-quality genome sequences for stock strains in laboratories becoming pervasive as it facilitates rapid mutational profiling in laboratory evolution and selection experiments, and is readily applicable to a wide host of organisms and conditions.
we attempted a variety of filtering schemes for snp-calling, incorporating thresholds for consensus base quality, mapping quality of supporting reads, and consensus quality of adjacent bases.
however, these approaches generally reduced the coverage of the genome that could be analyzed or introduced false positives .
we therefore adopted heuristics that allowed us to derive a set of high-confidence snp calls while examining the majority of the haploid genome.
this approach detected a novel and potentially regulatory snp in the evolved genome near the rrn <dig> locus, encoding a nutrient-responsive transcriptional regulator, which is present at high frequency in the evolving population.
read-depth information in high-throughput sequencing data yielded clear signals of copy number variation between strains.
we applied a segmentation algorithm to refine the copy number estimate for the sul <dig> amplification on chromosome  <dig>  following detection of copy number polymorphisms, we developed a general approach for detecting breakpoint sequences from single-end, short read sequencing data.
applied to the data of the genome of the evolved strain, this approach yielded upstream and downstream breakpoint predictions with single-base resolution for the sul <dig> amplification.
using the derived breakpoint sequences, we predicted a ~ <dig> kb genomic rearrangement supporting the copy number amplification of the sul <dig> locus, and we validated this rearrangement experimentally .
the breakpoint sequences observed share the structural features of palindrome formation triggered by double-strand breaks near short inverted segments or by incorrect cleavage by a holliday junction resolvase at an inverted repeat-mediated cruciform structure, and subsequent ligation of hairpins  <cit> .
narayanan et al.
<cit>  have previously established this later mechanism in the development of inverted head-to-tail clusters of cup <dig> and sfa <dig> between inserted alu repeats in yeast centromeres.
thus, the observed breakpoint sequences in reads that match to two genomic locations provide insights into the structure of the segmental duplications and further evidence for the involvement of regions of micro-homology  in driving large-scale genomic rearrangements  <cit> .
CONCLUSIONS
in this study, we demonstrate the ability of high-throughput sequencing to catalog the genetic changes underlying adaptation in a yeast strain evolved in a sulfate-limited environment.
we generated high-depth genome sequences for evolved and parental yeast strains.
we describe approaches effective at identifying single-nucleotide polymorphisms, as well as detecting the location of and quantifying copy number changes in the evolved genome with respect to that of its parental strain.
furthermore, we developed a general approach for detecting breakpoint sequences in single-end, short read sequencing data.
this approach yielded breakpoint predictions with single base resolution for a ~ <dig> kb amplification harboring the sul <dig> gene, providing insights into the mechanisms that may have facilitated the expansion underlying this adaptive rearrangement.
as such, this study suggests that the combination of whole-genome sequencing and experimental evolution is a powerful approach to study the features that restrain and promote genomic plasticity, defining possible routes of adaptation and outcomes of evolution.
in addition, such studies should yield valuable functional information on the relationship between fitness and adaptations at both the single-gene and genomic levels  <cit> .
scaling these studies to population-level analysis will allow us to frame this knowledge within the context of population forces, which may yield new insights into evolutionary dynamics.

 despite the wealth of knowledge that comparative genomics has provided about the evolution of past and current life forms, the process of adaptation is still poorly understood. predictive insight into how adaptation will occur -- which adaptive mutations are likely to arise and fix during selection -- is a daunting challenge. progress in this area will further efforts to deter cancer progression, the emergence of new pathogens, and antibiotic resistance. as a simple model for analyzing adaptation, experimental evolution of microorganisms provides the unique opportunity to catalog, monitor the dynamics, and measure the reproducibility of adaptation in real time. in the past decade, the characterization of laboratory evolution outcomes beyond phenotypic analysis has been advanced by the use of dna microarrays. in bacteria and yeast, these approaches have been applied to expose adaptive point mutations and structural rearrangements in response to carbon source replacement as well as carbon source and nutrient limitation. furthermore, studies have begun to unveil the effect of population dynamics on the outcomes of evolution, providing direct evidence of parallel evolution and clonal interference and support for historical contingencies. however, these studies are limited in that the precise nature of the adaptive point mutations or large-scale sequence rearrangements in the evolved organisms has not been defined systematically. in recent years, however, a series of high-throughput dna sequencing technologies capable of producing gigabases of dna sequence information in a single experiment have been developed. these technologies are transforming biological research, allowing the rapid identification of genetic variation intrinsic to diseases, behavior and other traits. indeed, these technologies have been applied to a wide host of biological research problems, from enhancing our understanding of laboratory strains of bacteria by pinpointing suppressor mutations and mutational biases, to identifying the genetic variation within all known serotypes of a major viral pathogen. in yeasts, they have been applied to analyze the genetic variation within and between wild and domestic populations, the spectrum of spontaneous mutations in saccharomyces cerevisiae and the adaptive mutations in a pichia stipilis strain efficient in xylose fermentation. in bacteria, these sequencing technologies have recently been applied to study the rate of evolutionary adaptation over generations. furthermore, they have launched a personal genomics era that is revealing the breadth of genetic variation in the small number of individuals fully sequenced thus far, and across targeted subgenomic portions in many individuals, pinpointing disease causative alleles in exomes of affected individuals. here, we use whole-genome sequencing to reveal the repertoire of point mutations and copy number polymorphisms in an evolved s. cerevisiae strain. we sequenced an adapted strain isolated after ~ generations of a continuous haploid culture under sulfate limitation, and compared this analysis to mutational profiling data obtained using array-based technologies. we generated high-depth sequencing data for the evolved and parental genomes, applied a heuristic approach to uncover single nucleotide polymorphisms and a recursive segmentation algorithm to discover breakpoints and refine copy-number estimates, and devised a general approach to extract precise breakpoint sequence information from single-end short reads. these breakpoints allowed us to delineate the structural rearrangement underlying a 5× copy number amplification of a ~ kb genomic segment.  we constructed dna sequencing libraries from genomic dna of the yeast strain dby evolved in a sulfate-limited chemostat, and its haploid ancestor strain dby. we collected, and, single-end, bp reads from the evolved and parent genomes, respectively, on an illumina genome analyzer ii platform. more than 75% of the reads were aligned to the s. cerevisiae reference genome using maq. following quality-filtering, these reads yielded a coverage of ~ % of the mappable nuclear genome of both strains with an average read-depth of × and × in the evolved and ancestor genomes, respectively. the majority of reads had a mapping quality score ≥ a heuristic approach to point mutation detection in sequencing the adapted strain, our aim was to reveal the full complement of genetic adaptations that had occurred during its evolution under sulfate limitation. copy number variation and point mutation analysis of this strain assessed by use of orf array and genomic tiling array approaches, respectively, provided a data set against which to compare the efficacy of whole-genome sequencing. to identify point mutations, we examined base calls using criteria that allowed comparisons between nucleotide positions in the evolved and parental short read data sets over the great majority of the genome. we performed snp calling following two heuristic approaches, one that monitors commonly applied filters for read-depth and various quality scores, and one that is a simplified approach that monitors read-depth and the frequency of base-calls at each position. for the simplified approach, we required at least reads per position with ≥80% concordant snp calls in the evolved genome, and at least reads per position with ≥70% concordant calls for a different base in the parental genome. this approach recapitulated known features in the evolved genome while maximizing breadth, yielding a high-confidence set of snps and that allowed us to examine up to 91% of the mappable bases in the yeast genome. we obtained modest gains in coverage following the simplified snp calling approach. applying these heuristics, we detected four single-point differences between the laboratory evolved strain and that of its parent: the previously-described point mutations at pbp, sgf, and upf, and an additional point mutation intergenic to rrn and ypk which we confirmed by sanger sequencing. the function of the rna polymerase i-specific transcription initiation factor rrn is conserved between yeast and humans. in humans, tif-ia modulates ribosomal gene expression in response to nutrient limitation via the mtor pathway. the expression of rrn is less than half the level in the evolved strain compared to its ancestor, suggesting that this snp may be regulatory and part of the adaptive response in this lineage. the finding of an additional snp in the sequencing data but missed by array-based comparative genome sequencing is consistent with the estimated 15% false negative rate for the snpscanner algorithm. in addition, we searched for small insertions and deletions via gapped alignment of reads unmapped in the maq alignment, requiring that candidate indel coordinates were sequenced as wildtype in the comparison strain. however, no small insertion or deletion differences between the evolved and parental genomes could be detected. segmentation-based detection of copy number polymorphisms previous analysis of the evolved and parental strains using comparative genome hybridizations on microarrays detected a genomic amplification spanning the high-affinity sulfate transporter sul locus in the genome of the laboratory-evolved strain, providing a 50% fitness advantage relative to the ancestral genome. sul gene amplification was a common adaptation in continuous cultures under sulfate limitation, occurring in of adapted clones from various experimental evolutions. however, the precise breakpoints and nature of the structural rearrangement has not been defined for these amplifications. we compiled the read-depth data from evolved and parental genome sequencing data sets and calculated the ratio in read-depth for each base across the genome. we used these data to perform circular binary segmentation with dnacopy, a software package designed to partition the genome into segments of equal copy number from high-density array cgh data. dnacopy formulates the problem of copy-number variation analysis as a change-point detection problem, whereby change-points correspond to the genomic locations of copy number transitions. the algorithm searches for change-points by recursively partitioning the genome and performing a maximal t-statistic test between segment ratios, joining segments with comparable ratios. we reasoned that this approach should detect transitions in ratios of sequencing read-depth as well, and applied it to search for such transitions throughout the yeast genome. indeed, segmentation of sequencing depth ratios detected an ~ kb segment harboring sul with an estimated 5× amplification. these breakpoint coordinates are in close proximity to estimated coordinates obtained from tiling array data, but have narrower uncertainty windows. the read depth-based copy number analysis yielded a value close to an integer, suggesting it may be a reliable approximation of genomic copy number. breakpoint sequence determination and analysis of rearrangement structure we sought to pinpoint with single-base resolution the structural rearrangements underlying the sul amplification. to do so, we established a strategy to detect breakpoints in shotgun, single-end, short-read sequencing data. we unearthed breakpoint-spanning reads in the evolved genome sequencing data set by assembling unmapped reads into contigs using velvet, a short-read de novo assembly algorithm. these contigs were blat-aligned to the reference genome sequence in an attempt to detect signatures of novel structures in the genome. this approach yielded three contigs composed of subsequences with alignments to the mappable nuclear genome. of these contigs, two aligned to sequences within the predicted amplification boundaries. performing this analysis on the ancestor genome sequencing data yielded two contigs at distinct coordinates, from which we estimate the likelihood of the signatures detected in the evolved genome arising independently of the amplification and found within the predicted boundaries to be very low. the breakpoint-matching contigs are small and contain inversions of nearby genomic sequences overlapped by ≤ bp. these breakpoints occur within the ctp and pca coding sequences, and would result in and amino acid truncations, respectively. in addition, we found reads spanning the wild-type sequence at these coordinates, indicating that full-length copies of these genes are retained in the genome of the adapted strain. in combination with the inversions in breakpoint-spanning contigs, the observation of reads that conform to the wild-type sequences across the predicted rearrangement boundaries suggests that the 5× amplification spanning chr2: - is structured as tandem inversions along the chromosome. this arrangement was validated by southern blot analysis of the region with three different restriction enzymes and two probes. the fact that at both breakpoints we found short homologous sequences 7- bp long overlapping the segmental inversions suggests that short homologous nucleotide tracts may be involved in driving the large structural rearrangements. among the contigs with subsequence alignments to the mappable reference genome, the preponderance were composed of mitochondrial sequences. this abundance of contigs of unmapped reads composed of mitochondrial subsequences was recapitulated with the ancestor genome data, but we found little overlap between structures observed in the two mitochondrial genomes. we did not observe copy number gains or losses at these coordinates, which are also covered at high-depth with wild-type sequences. these observations may reflect mitochondrial heteroplasmy in the form of structural rearrangements. discussion evolutionary adaptation is a fundamental biological phenomenon with far-reaching implications in cancer, antibiotic resistance, infectious disease management, and ecology. cataloging the precise genetic changes that underlie an organism's responses to defined selective environments can yield insights not only into the pathways that define fitness in these environments, but also into the breadth and reproducibility of adaptive mutations. the generation of such data requires approaches that can rapidly assay entire genomes with single-base resolution to expose point mutations and at megabase-scale to define structural rearrangements. in this study, we applied whole-genome sequencing to identify the genetic changes in a laboratory-evolved yeast strain, following on similar work in bacteria and phage. this analysis revealed changes in the genome corresponding to single nucleotide polymorphisms and copy number amplifications, with specific gains over array-based methodologies. we produced high-depth genome sequences for a yeast strain evolved under nutrient limitation and its parental strain. the genome sequence of the parent was critical in allowing us to pinpoint mutations acquired during experimental evolution, and serves as a template against which to compare the genomes of other descendent strains. we foresee the practice of maintaining high-quality genome sequences for stock strains in laboratories becoming pervasive as it facilitates rapid mutational profiling in laboratory evolution and selection experiments, and is readily applicable to a wide host of organisms and conditions. we attempted a variety of filtering schemes for snp-calling, incorporating thresholds for consensus base quality, mapping quality of supporting reads, and consensus quality of adjacent bases. however, these approaches generally reduced the coverage of the genome that could be analyzed or introduced false positives. we therefore adopted heuristics that allowed us to derive a set of high-confidence snp calls while examining the majority of the haploid genome. this approach detected a novel and potentially regulatory snp in the evolved genome near the rrn locus, encoding a nutrient-responsive transcriptional regulator, which is present at high frequency in the evolving population. read-depth information in high-throughput sequencing data yielded clear signals of copy number variation between strains. we applied a segmentation algorithm to refine the copy number estimate for the sul amplification on chromosome following detection of copy number polymorphisms, we developed a general approach for detecting breakpoint sequences from single-end, short read sequencing data. applied to the data of the genome of the evolved strain, this approach yielded upstream and downstream breakpoint predictions with single-base resolution for the sul amplification. using the derived breakpoint sequences, we predicted a ~ kb genomic rearrangement supporting the copy number amplification of the sul locus, and we validated this rearrangement experimentally. the breakpoint sequences observed share the structural features of palindrome formation triggered by double-strand breaks near short inverted segments or by incorrect cleavage by a holliday junction resolvase at an inverted repeat-mediated cruciform structure, and subsequent ligation of hairpins. narayanan et al. have previously established this later mechanism in the development of inverted head-to-tail clusters of cup and sfa between inserted alu repeats in yeast centromeres. thus, the observed breakpoint sequences in reads that match to two genomic locations provide insights into the structure of the segmental duplications and further evidence for the involvement of regions of micro-homology in driving large-scale genomic rearrangements.  in this study, we demonstrate the ability of high-throughput sequencing to catalog the genetic changes underlying adaptation in a yeast strain evolved in a sulfate-limited environment. we generated high-depth genome sequences for evolved and parental yeast strains. we describe approaches effective at identifying single-nucleotide polymorphisms, as well as detecting the location of and quantifying copy number changes in the evolved genome with respect to that of its parental strain. furthermore, we developed a general approach for detecting breakpoint sequences in single-end, short read sequencing data. this approach yielded breakpoint predictions with single base resolution for a ~ kb amplification harboring the sul gene, providing insights into the mechanisms that may have facilitated the expansion underlying this adaptive rearrangement. as such, this study suggests that the combination of whole-genome sequencing and experimental evolution is a powerful approach to study the features that restrain and promote genomic plasticity, defining possible routes of adaptation and outcomes of evolution. in addition, such studies should yield valuable functional information on the relationship between fitness and adaptations at both the single-gene and genomic levels. scaling these studies to population-level analysis will allow us to frame this knowledge within the context of population forces, which may yield new insights into evolutionary dynamics. 
both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. experimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. this study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. in addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity. however, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. these boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement. applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5× gene amplification.
 experimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. however, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. new dna sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined.  we sequenced > % of the genome of a laboratory-evolved strain of the yeast saccharomyces cerevisiae and its ancestor at >28× depth. both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5× gene amplification. these boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement.  this study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. in addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity. 