High throughput sequencing enables detailed analysis of S. cerevisiae DNA replication
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Conrad Nieduszynski
PROVIDER: E-GEOD-42243 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:For rapid characterisation of replication dynamics in wild-type cells from an unperturbed cell cycle we have obtained replicating (S phase) and non-replicating (G2 phase) cells by fluorescence activated cell sorting (FACS) and subjected the DNA to copy number analysis.
2013-06-20 | GSE42243 | GEO
Project description:We have used three complementary deep sequencing approaches to characterise the temporal order of genome replication in S. cerevisiae. Each approach measures the increase in DNA copy number as a genomic region is replicated (in a large population of cells). We find that the use of deep sequencing provided high spatial resolution. For maximum temporal resolution we have measured replication dynamics at multiple time points during a synchronous S phase. To pinpoint replication origins we have synchronously released a checkpoint mutant (rad53-delta) into S phase under conditions of depleted dNTPs (200 mM hydroxyurea; HU) and measured the increase in DNA copy number after 60 minutes. Finally for rapid characterisation of replication dynamics in wild-type cells from an unperturbed cell cycle we have obtained replicating (S phase) and non-replicating (G2 phase) cells by fluorescence activated cell sorting (FACS) and subjected the DNA to copy number analysis. We have compared and contrasted these approaches and developed the tools required for interpreting the deep sequencing data. Measurement of genome replication time for various S. cerevisiae strains. For each strain two samples were analysed: a replicating sample (from S phase) and a non-replicating sample (from G2 phase).
2013-10-02 | E-GEOD-48212 | biostudies-arrayexpress
Project description:We have used three complementary deep sequencing approaches to characterise the temporal order of genome replication in S. cerevisiae. Each approach measures the increase in DNA copy number as a genomic region is replicated (in a large population of cells). We find that the use of deep sequencing provided high spatial resolution. For maximum temporal resolution we have measured replication dynamics at multiple time points during a synchronous S phase. To pinpoint replication origins we have synchronously released a checkpoint mutant (rad53-delta) into S phase under conditions of depleted dNTPs (200 mM hydroxyurea; HU) and measured the increase in DNA copy number after 60 minutes. Finally for rapid characterisation of replication dynamics in wild-type cells from an unperturbed cell cycle we have obtained replicating (S phase) and non-replicating (G2 phase) cells by fluorescence activated cell sorting (FACS) and subjected the DNA to copy number analysis. We have compared and contrasted these approaches and developed the tools required for interpreting the deep sequencing data.
2013-10-02 | GSE48212 | GEO
Project description:Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3-Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore DDK at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites. Measurement of genome replication time for various S. cerevisiae strains. For each strain two samples were analysed: a replicating sample (from S phase) and a non-replicating sample (from G2 phase).
2013-06-06 | E-GEOD-41982 | biostudies-arrayexpress
Project description:DNA replication initiates from defined locations called replication origins; some origins are highly active whereas others are dormant and rarely used. Origins also differ in their activation time resulting in particular genomic regions replicating at characteristic times and in a defined temporal order. Here we report the comparison of genome replication in four budding yeast species: Saccharomyces cerevisiae, S. paradoxus, S. arboricolus and S. bayanus. First, we find that the locations of active origins are predominantly conserved between species, whereas dormant origins are poorly conserved. Second, we generated genome-wide replication profiles for each of these species and discovered that the temporal order of genome replication is highly conserved. Therefore, active origins are not only conserved in location, but also activation time. Only a minority of these conserved origins show differences in activation time between these species. To gain insight as to the mechanisms by which origin activation time is regulated we generated replication profiles for a S. cerevisiae / S. bayanus hybrid strain and find that there are both local and global regulators of origin function. Measurement of genome replication time from 4 budding yeast species (Saccharomyces cerevisiae, S. paradoxus, S. arboricolus and S. bayanus) and a hybrid (between Saccharomyces cerevisiae and S. bayanus). For each strain two samples were analysed: a replicating sample (from S phase) and a non-replicating sample (from G2 phase) The reference genome sequences for each yeast species are available as Series supplementary file [sac*.fa]
2012-06-19 | E-GEOD-36045 | biostudies-arrayexpress
Project description:DNA-replication is a key process in life and can lead to disease when disturbed. Cell-type specific early and late replication domains have been discovered throughout genomes by analysis of DNA from populations of cells. However, cell to cell differences and the association of these differences with other cellular processes remain largely elusive. Here we demonstrate for the first time that consecutive domains of early and late DNA-replication can be detected in single S-phase cells using array comparative genomic hybridization, providing proof-of-concept for a novel tool to investigate DNA-replication genome wide at the single-cell level. Furthermore, methods to profile the genome of a single cell for DNA-copy number aberrations are revolutionizing both basic genome research and clinical genetic diagnosis. It is thus important to apprehend not only technical but also biological reasons for false positive copy number detection. None of the current single-cell copy number calling methods distinguishes between a cell in G1-, S- or G2/M-phase of the cell cycle and mostly use cells isolated randomly from populations. We demonstrate that the oscillating pattern between early and late replicating loci instigates significantly more false-positive DNA copy-number calls in a diploid cell in S-phase cells than in G1- or G2/M-phase, depending on the specific aCGH-signal normalization method used. We propose a work-flow to detect single cells in S-phase and to correct for DNA-replication bias before copy number profiling. DNA was extracted from many cells from 1 S-phase, 1 G1-phase and 1 G2/Mphase enriched cell population from an EBV-transformed lymphoblastoid cell line from a male carrying a 750kb amplification on the short arm of chromosome 4. These test samples were hybridized comparatively to commercial male reference DNA.
2013-01-08 | E-GEOD-38757 | biostudies-arrayexpress
Project description:DNA-replication is a key process in life and can lead to disease when disturbed. Cell-type specific early and late replication domains have been discovered throughout genomes by analysis of DNA from populations of cells. However, cell to cell differences and the association of these differences with other cellular processes remain largely elusive. Here we demonstrate for the first time that consecutive domains of early and late DNA-replication can be detected in single S-phase cells using array comparative genomic hybridization, providing proof-of-concept for a novel tool to investigate DNA-replication genome wide at the single-cell level. Furthermore, methods to profile the genome of a single cell for DNA-copy number aberrations are revolutionizing both basic genome research and clinical genetic diagnosis. It is thus important to apprehend not only technical but also biological reasons for false positive copy number detection. None of the current single-cell copy number calling methods distinguishes between a cell in G1-, S- or G2/M-phase of the cell cycle and mostly use cells isolated randomly from populations. We demonstrate that the oscillating pattern between early and late replicating loci instigates significantly more false-positive DNA copy-number calls in a diploid cell in S-phase cells than in G1- or G2/M-phase, depending on the specific aCGH-signal normalization method used. We propose a work-flow to detect single cells in S-phase and to correct for DNA-replication bias before copy number profiling. DNA was extracted from many cells from 1 S-phase, 1 G1-phase and 1 G2/Mphase enriched cell population for two female normal control EBV-transformed lymphoblastoid cell lines. These test samples were hybridized comparatively to commercial male reference DNA.
2013-01-08 | E-GEOD-38756 | biostudies-arrayexpress
Project description:DNA-replication is a key process in life and can lead to disease when disturbed. Cell-type specific early and late replication domains have been discovered throughout genomes by analysis of DNA from populations of cells. However, cell to cell differences and the association of these differences with other cellular processes remain largely elusive. Here we demonstrate for the first time that consecutive domains of early and late DNA-replication can be detected in single S-phase cells using array comparative genomic hybridization, providing proof-of-concept for a novel tool to investigate DNA-replication genome wide at the single-cell level. Furthermore, methods to profile the genome of a single cell for DNA-copy number aberrations are revolutionizing both basic genome research and clinical genetic diagnosis. It is thus important to apprehend not only technical but also biological reasons for false positive copy number detection. None of the current single-cell copy number calling methods distinguishes between a cell in G1-, S- or G2/M-phase of the cell cycle and mostly use cells isolated randomly from populations. We demonstrate that the oscillating pattern between early and late replicating loci instigates significantly more false-positive DNA copy-number calls in a diploid cell in S-phase cells than in G1- or G2/M-phase, depending on the specific aCGH-signal normalization method used. We propose a work-flow to detect single cells in S-phase and to correct for DNA-replication bias before copy number profiling. DNA was extracted from many cells from 1 S-phase, 1 G1-phase and 1 G2/Mphase enriched cell population from an EBV-transformed lymphoblastoid cell line from a male carrying a 750kb amplification on the short arm of chromosome 4. These test samples were hybridized comparatively to commercial male reference DNA.
2013-01-08 | E-GEOD-41777 | biostudies-arrayexpress
Project description:Measurment of the ToR by comapring the DNA content of the S phase and the DNA content of the G1 phase, the method is based on the method used in the paper - Replication timing of the human genome by Woodfine et al. that was published in Hum. Mol. Genet. 2004. Briefly, after staining with Propidium Iodide, cells in G1 and S phase of the cell cycle are sorted by FACS sorter (ARIA or Vantage), DNA is then extracted from the fractions and differentially labeled with dUTP-Cy3 or dUTP-Cy5 using direct labeling (Agilent labeling kit). The labeled DNA is cohybridized to the array. Early replicating sequences correspond to the spots with high S/G1 ratio and late replicating sequences correspond to the spots with low S/G1 ratio. two mouse tissues were measured (MEF and L1210), 2 replicates for each sample
2010-05-25 | E-GEOD-17229 | biostudies-arrayexpress
Project description:DNA-replication is a key process in life and can lead to disease when disturbed. Cell-type specific early and late replication domains have been discovered throughout genomes by analysis of DNA from populations of cells. However, cell to cell differences and the association of these differences with other cellular processes remain largely elusive. Here we demonstrate for the first time that consecutive domains of early and late DNA-replication can be detected in single S-phase cells using array comparative genomic hybridization, providing proof-of-concept for a novel tool to investigate DNA-replication genome wide at the single-cell level. Furthermore, methods to profile the genome of a single cell for DNA-copy number aberrations are revolutionizing both basic genome research and clinical genetic diagnosis. It is thus important to apprehend not only technical but also biological reasons for false positive copy number detection. None of the current single-cell copy number calling methods distinguishes between a cell in G1-, S- or G2/M-phase of the cell cycle and mostly use cells isolated randomly from populations. We demonstrate that the oscillating pattern between early and late replicating loci instigates significantly more false-positive DNA copy-number calls in a diploid cell in S-phase cells than in G1- or G2/M-phase, depending on the specific aCGH-signal normalization method used. We propose a work-flow to detect single cells in S-phase and to correct for DNA-replication bias before copy number profiling. The genome of 2 S-phase, 1 M-phase and 1 G-phase single cells were amplified using the Sureplex amplification system. These test samples were hybridized comparatively to commercial male reference DNA,
2013-01-08 | E-GEOD-38759 | biostudies-arrayexpress