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1. WO2011157846 - METHODS FOR HAPLOTYPING SINGLE CELLS

Publication Number WO/2011/157846
Publication Date 22.12.2011
International Application No. PCT/EP2011/060211
International Filing Date 20.06.2011
IPC
C12Q 1/68 2006.1
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
68involving nucleic acids
CPC
C12Q 1/6874
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
68involving nucleic acids
6869Methods for sequencing
6874involving nucleic acid arrays, e.g. sequencing by hybridisation
C12Q 1/6886
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
68involving nucleic acids
6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
6883for diseases caused by alterations of genetic material
6886for cancer
C12Q 1/701
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms
70involving virus or bacteriophage
701Specific hybridization probes
C12Q 2600/172
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
2600Oligonucleotides characterized by their use
172Haplotypes
G16B 20/00
GPHYSICS
16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
20ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
G16B 20/10
GPHYSICS
16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
20ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
10Ploidy or copy number detection
Applicants
  • KATHOLIEKE UNIVERSITEIT LEUVEN [BE]/[BE] (AllExceptUS)
  • VERMEESCH, Joris [BE]/[BE] (UsOnly)
  • VOET, Thierry [BE]/[BE] (UsOnly)
  • ZAMANI ESTEKI, Masoud [IR]/[BE] (UsOnly)
Inventors
  • VERMEESCH, Joris
  • VOET, Thierry
  • ZAMANI ESTEKI, Masoud
Agents
  • LAENEN, Bart, Roger, Albert
Priority Data
1010232.518.06.2010GB
Publication Language English (en)
Filing Language English (EN)
Designated States
Title
(EN) METHODS FOR HAPLOTYPING SINGLE CELLS
(FR) PROCÉDÉS D'HAPLOTYPAGE DE CELLULES ISOLÉES
Abstract
(EN) We developed a generic approach to type genome-wide single nucleotide polymorphisms in single human cells and to reconstruct for the first time genome-wide haplotypes of single- or dual-cell derived genotypes. Proof-of-principle is delivered for EBV-transformed lymphoblastoid cells as well as human blastomeres. To this end, multiple displacement amplified DNA samples of single cells were hybridized to Affymetrix 250K SNP-arrays. Different algorithmic designs were subsequently developed to assess from the single-cell derived SNP-probe intensities the sequence of syntenic alleles and to pinpoint accurately the majority of parental homologous recombination sites across the entire genome using a linkage-based approach. This included the development of algorithms that rectify a large part of the discrepant allelic assignments in raw single or dual-cell derived haplotypes. This method to infer genome-wide haplotypes from the analysis of only one or two cells has tremendous applicative value. It has the capacity to revolutionize not only genetic diagnosis of preimplantation in vitro fertilized human embryos in the clinic, but also animal breeding programs by enabling genome-wide quantitative trait loci selection at the embryonic level. In addition, it allows to further scrutinize drivers of haplotype diversity, mainly meiotic homologous recombination as well as somatic (homologous) recombination processes that occur often during (human) tumorigenesis.
(FR) Nous avons mis au point une démarche générique de typage de polymorphismes mononucléotidiques pangénomiques dans des cellules humaines isolées, ainsi que, pour la première fois, de reconstruction d'haplotypes pangénomiques de génotypes issus de cellules isolées ou doubles. Le principe a été validé pour des cellules lymphoblastoïdes transformées par l'EVB ainsi que pour des blastomères humains. A cette fin, des échantillons d'ADN amplifiés à multiples déplacements issus de cellules isolées ont été hybridés à des puces SNP Affymetrix 250K. Différents modèles algorithmiques ont été développés par la suite pour évaluer à partir de l'intensité de complexes SNP-sonde dérivés de cellules isolées la séquence d'allèles synténiques et pour localiser précisément la majorité des sites de recombinaison homologue parentaux dans l'intégralité du génome en utilisant une démarche fondée sur les liaisons. Pour cela, il a fallu mettre au point des algorithmes capables de rectifier une grande partie des affectations alléliques divergentes dans les haplotypes bruts obtenus à partir de cellules isolées ou doubles. Ce procédé consistant à inférer des haplotypes pangénomiques à partir de l'analyse d'une ou deux cellules seulement présente un intérêt remarquable pour ce qui est de ses applications. Ce procédé a la capacité de révolutionner non seulement le diagnostic génétique préimplantatoire d'embryons humain fécondés in vitro dans la pratique clinique, mais également les programmes de sélection d'animaux en permettant la sélection de loci à caractère quantitatif pangénomiques au niveau embryonnaire. En outre, cela permet d'étudier de plus près les éléments catalysant la diversité des haplotypes, principalement la recombinaison homologue méiotique, ainsi que les processus de recombinaison somatique (homologue) se produisant souvent lors de la genèse de tumeurs (humaines).
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