Saccharomyces cerevisiae has proved to be an invaluable model in classical and molecular genetics... more Saccharomyces cerevisiae has proved to be an invaluable model in classical and molecular genetics studies. Despite several hundreds of isolates already available, the scientific community relies on the use of only a handful of unrelated strains. The lack of sequence information, haploid derivatives and genetic markers has prevented novel strains from being used. Here, we release a set of 55 S. cerevisiae and Saccharomyces paradoxus genetically tractable strains, previously sequenced in the Saccharomyces Genome Resequencing Project. These strains are stable haploid derivatives and ura3 auxotrophs tagged with a 6-bp barcode, recognized by a restriction enzyme to allow easy identification. We show that the specific barcode can be used to accurately measure the prevalence of different strains during competition experiments. These strains are now amenable to a wide variety of genetic experiments and can be easily crossed with each other to create hybrids and segregants, providing a valuable resource for breeding programmes and quantitative genetic studies. Three versions of each strain (haploid Mat a and Matα and diploid Mat a/α all as ura3∷KanMX-Barcode) are available through the National Culture Yeast Collection.
De novo sequencing of genomes is followed by annotation analyses aiming at identifying functional... more De novo sequencing of genomes is followed by annotation analyses aiming at identifying functional genomic features such as genes, non-coding RNAs or regulatory sequences, taking advantage of diverse datasets. These steps sometimes fail at detecting non-coding functional sequences: for example, origins of replication, centromeres and rDNA positions have proven difficult to annotate with high confidence. Here, we demonstrate an unconventional application of Chromosome Conformation Capture (3C) technique, which typically aims at deciphering the average 3D organization of genomes, by showing how functional information about the sequence can be extracted solely from the chromosome contact map. Specifically, we describe a combined experimental and bioinformatic procedure that determines the genomic positions of centromeres and ribosomal DNA clusters in yeasts, including species where classical computational approaches fail. For instance, we determined the centromere positions in Naumovozyma castellii, where these coordinates could not be obtained previously. Although computed centromere positions were characterized by conserved synteny with neighboring species, no consensus sequences could be found, suggesting that centromeric binding proteins or mechanisms have significantly diverged. We also used our approach to refine centromere positions in Kuraishia capsulata and to identify rDNA positions in Debaryomyces hansenii. Our study demonstrates how 3C data can be used to complete the functional annotation of eukaryotic genomes. The source code is provided in the Supplementary Material. This includes a zipped file with the Python code and a contact matrix of Saccharomyces cerevisiae. romain.koszul@pasteur.fr Supplementary data are available at Bioinformatics online.
Closing gaps in draft genome assemblies can be costly and time-consuming, and published genomes a... more Closing gaps in draft genome assemblies can be costly and time-consuming, and published genomes are therefore often left 'unfinished.' Here we show that genome-wide chromosome conformation capture (3C) data can be used to overcome these limitations, and present a computational approach rooted in polymer physics that determines the most likely genome structure using chromosomal contact data. This algorithm--named GRAAL--generates high-quality assemblies of genomes in which repeated and duplicated regions are accurately represented and offers a direct probabilistic interpretation of the computed structures. We first validated GRAAL on the reference genome of Saccharomyces cerevisiae, as well as other yeast isolates, where GRAAL recovered both known and unknown complex chromosomal structural variations. We then applied GRAAL to the finishing of the assembly of Trichoderma reesei and obtained a number of contigs congruent with the know karyotype of this species. Finally, we showed that GRAAL can accurately reconstruct human chromosomes from either fragments generated in silico or contigs obtained from de novo assembly. In all these applications, GRAAL compared favourably to recently published programmes implementing related approaches.
During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms a... more During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs. The aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses. Our results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations.
In a search for genes involved in cell-type-dependent chromosome instability, we have found a rol... more In a search for genes involved in cell-type-dependent chromosome instability, we have found a role for NEJ1, a regulator of nonhomologous end joining (NHEJ), in cells that survive in the absence of telomerase. In yeast, NHEJ is regulated by mating-type status through NEJ1, which is repressed in a/alpha cells. For efficient NHEJ, NEJ1 is required as part of a complex with LIF1 and DNL4, which catalyzes DNA ligation. In haploid cells without telomerase, we find that the absence of NEJ1 results in high frequencies of circular chromosomes in type II survivors (i.e., those typified by lengthened telomere repeat tracts). These telomere fusion events are DNL4 dependent. NEJ1 therefore has a role in protecting telomeres from end fusions by NHEJ in the absence of telomerase that contrasts with its role in promoting repair at sites of DNA double-strand breaks.
Recent sequencing efforts and experiments have advanced our understanding of genome evolution in ... more Recent sequencing efforts and experiments have advanced our understanding of genome evolution in yeasts, particularly the Saccharomyces yeasts. The ancestral genome of the Saccharomyces sensu stricto complex has been subject to both whole-genome duplication, followed by massive sequence loss and divergence, and segmental duplication. In addition the subtelomeric regions are subject to further duplications and rearrangements via ectopic exchanges. Translocations and other gross chromosomal rearrangements that break down syntenic relationships occur; however, they do not appear to be a driving force of speciation. Analysis of single genomes has been fruitful for hypothesis generation such as the whole-genome duplication, but comparative genomics between close and more distant species has proven to be a powerful tool in testing these hypotheses as well as elucidating evolutionary processes acting on the genome. Future work on population genomics and experimental evolution will keep yeast at the forefront of studies in genome evolution.
Genome analysis of over 70 Saccharomyces strains revealed the existence of five groups of genetic... more Genome analysis of over 70 Saccharomyces strains revealed the existence of five groups of genetically diverged S. cerevisiae wild-type isolates, which feature distinct genetic backgrounds and reflect the natural diversity existing among the species. The strains originated from different geographical and ecological niches (Malaysian, West African, North American, Wine/European and Sake) and represent clean, non-mosaic lineages of S. cerevisiae, meaning that their genomes differ essentially by monomorphic and private SNPs. In this study, one representative strain for each of the five S. cerevisiae clean lineages was selected and mutated for several auxotroph genes by clean markerless deletions, so that all dominant markers remained available for further genetic manipulations. A set of 50 strains was assembled, including eight haploid and two diploid strains for each lineage. These strains carry different combinations of leu2∆0, lys2∆0, met15∆0, ura3∆0 and/or ura3∆::KanMX-barcoded deletions with marker configurations resembling that of the BY series, which will allow large-scale crossing with existing deletion collections. This new set of genetically tractable strains provides a powerful tool kit to explore the impact of natural variation on complex biological processes.
Saccharomyces cerevisiae has proved to be an invaluable model in classical and molecular genetics... more Saccharomyces cerevisiae has proved to be an invaluable model in classical and molecular genetics studies. Despite several hundreds of isolates already available, the scientific community relies on the use of only a handful of unrelated strains. The lack of sequence information, haploid derivatives and genetic markers has prevented novel strains from being used. Here, we release a set of 55 S. cerevisiae and Saccharomyces paradoxus genetically tractable strains, previously sequenced in the Saccharomyces Genome Resequencing Project. These strains are stable haploid derivatives and ura3 auxotrophs tagged with a 6-bp barcode, recognized by a restriction enzyme to allow easy identification. We show that the specific barcode can be used to accurately measure the prevalence of different strains during competition experiments. These strains are now amenable to a wide variety of genetic experiments and can be easily crossed with each other to create hybrids and segregants, providing a valuable resource for breeding programmes and quantitative genetic studies. Three versions of each strain (haploid Mat a and Matα and diploid Mat a/α all as ura3∷KanMX-Barcode) are available through the National Culture Yeast Collection.
De novo sequencing of genomes is followed by annotation analyses aiming at identifying functional... more De novo sequencing of genomes is followed by annotation analyses aiming at identifying functional genomic features such as genes, non-coding RNAs or regulatory sequences, taking advantage of diverse datasets. These steps sometimes fail at detecting non-coding functional sequences: for example, origins of replication, centromeres and rDNA positions have proven difficult to annotate with high confidence. Here, we demonstrate an unconventional application of Chromosome Conformation Capture (3C) technique, which typically aims at deciphering the average 3D organization of genomes, by showing how functional information about the sequence can be extracted solely from the chromosome contact map. Specifically, we describe a combined experimental and bioinformatic procedure that determines the genomic positions of centromeres and ribosomal DNA clusters in yeasts, including species where classical computational approaches fail. For instance, we determined the centromere positions in Naumovozyma castellii, where these coordinates could not be obtained previously. Although computed centromere positions were characterized by conserved synteny with neighboring species, no consensus sequences could be found, suggesting that centromeric binding proteins or mechanisms have significantly diverged. We also used our approach to refine centromere positions in Kuraishia capsulata and to identify rDNA positions in Debaryomyces hansenii. Our study demonstrates how 3C data can be used to complete the functional annotation of eukaryotic genomes. The source code is provided in the Supplementary Material. This includes a zipped file with the Python code and a contact matrix of Saccharomyces cerevisiae. romain.koszul@pasteur.fr Supplementary data are available at Bioinformatics online.
Closing gaps in draft genome assemblies can be costly and time-consuming, and published genomes a... more Closing gaps in draft genome assemblies can be costly and time-consuming, and published genomes are therefore often left 'unfinished.' Here we show that genome-wide chromosome conformation capture (3C) data can be used to overcome these limitations, and present a computational approach rooted in polymer physics that determines the most likely genome structure using chromosomal contact data. This algorithm--named GRAAL--generates high-quality assemblies of genomes in which repeated and duplicated regions are accurately represented and offers a direct probabilistic interpretation of the computed structures. We first validated GRAAL on the reference genome of Saccharomyces cerevisiae, as well as other yeast isolates, where GRAAL recovered both known and unknown complex chromosomal structural variations. We then applied GRAAL to the finishing of the assembly of Trichoderma reesei and obtained a number of contigs congruent with the know karyotype of this species. Finally, we showed that GRAAL can accurately reconstruct human chromosomes from either fragments generated in silico or contigs obtained from de novo assembly. In all these applications, GRAAL compared favourably to recently published programmes implementing related approaches.
During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms a... more During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs. The aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses. Our results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations.
In a search for genes involved in cell-type-dependent chromosome instability, we have found a rol... more In a search for genes involved in cell-type-dependent chromosome instability, we have found a role for NEJ1, a regulator of nonhomologous end joining (NHEJ), in cells that survive in the absence of telomerase. In yeast, NHEJ is regulated by mating-type status through NEJ1, which is repressed in a/alpha cells. For efficient NHEJ, NEJ1 is required as part of a complex with LIF1 and DNL4, which catalyzes DNA ligation. In haploid cells without telomerase, we find that the absence of NEJ1 results in high frequencies of circular chromosomes in type II survivors (i.e., those typified by lengthened telomere repeat tracts). These telomere fusion events are DNL4 dependent. NEJ1 therefore has a role in protecting telomeres from end fusions by NHEJ in the absence of telomerase that contrasts with its role in promoting repair at sites of DNA double-strand breaks.
Recent sequencing efforts and experiments have advanced our understanding of genome evolution in ... more Recent sequencing efforts and experiments have advanced our understanding of genome evolution in yeasts, particularly the Saccharomyces yeasts. The ancestral genome of the Saccharomyces sensu stricto complex has been subject to both whole-genome duplication, followed by massive sequence loss and divergence, and segmental duplication. In addition the subtelomeric regions are subject to further duplications and rearrangements via ectopic exchanges. Translocations and other gross chromosomal rearrangements that break down syntenic relationships occur; however, they do not appear to be a driving force of speciation. Analysis of single genomes has been fruitful for hypothesis generation such as the whole-genome duplication, but comparative genomics between close and more distant species has proven to be a powerful tool in testing these hypotheses as well as elucidating evolutionary processes acting on the genome. Future work on population genomics and experimental evolution will keep yeast at the forefront of studies in genome evolution.
Genome analysis of over 70 Saccharomyces strains revealed the existence of five groups of genetic... more Genome analysis of over 70 Saccharomyces strains revealed the existence of five groups of genetically diverged S. cerevisiae wild-type isolates, which feature distinct genetic backgrounds and reflect the natural diversity existing among the species. The strains originated from different geographical and ecological niches (Malaysian, West African, North American, Wine/European and Sake) and represent clean, non-mosaic lineages of S. cerevisiae, meaning that their genomes differ essentially by monomorphic and private SNPs. In this study, one representative strain for each of the five S. cerevisiae clean lineages was selected and mutated for several auxotroph genes by clean markerless deletions, so that all dominant markers remained available for further genetic manipulations. A set of 50 strains was assembled, including eight haploid and two diploid strains for each lineage. These strains carry different combinations of leu2∆0, lys2∆0, met15∆0, ura3∆0 and/or ura3∆::KanMX-barcoded deletions with marker configurations resembling that of the BY series, which will allow large-scale crossing with existing deletion collections. This new set of genetically tractable strains provides a powerful tool kit to explore the impact of natural variation on complex biological processes.
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