# US 2014/0194300 A1 · Method and device for genetic map construction, method and device for haplotype analysis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0194300 A1 - Document ID: US20140194300A1 - Status: Published application - Kind: A1 - Application number: 14/240,955 - Filed: 2012-08-24 - Published: 2014-07-10 - Earliest claimed: 2011-08-26 - Assignees: Bgi Tech Solutions Co., Ltd. - Inventors: Luting Song; Di Shao; Zequn Zheng; Zhijun Zheng; Kui Wu; Shuheng Liang; Ye Tao; Yong Hou - Web page: https://patentgrep.com/patent/US20140194300A1 ## CPC - C12Q 1/6869: Methods for sequencing - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes - G16B 30/10: Sequence alignment; Homology search - C12Q 2600/172: Haplotypes - G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression - G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY ## Abstract Provided are the method and device for genetic map construction and the method and device for haplotype determination of a single cell. Wherein the method for genetic map construction includes: whole genome sequencing for at least a single cell from a same species, aligning the sequencing data to reference sequences respectively to determine genotypes of SNP sites, determining male parent a/female parent b typing results of SNP genotypes of a single cell based on the genotypes of SNP sites, dividing the chromosome of the species into linkage regions based on the male parent a/female parent b typing results of SNP genotypes, determining the variation ratio of a/b between two linkage regions to obtain recombination rate between every two continuous linkage regions, determining recombination map of a single cell according to the recombination rate, wherein the boundary site of a and b is the recombination site, determining the recombination rate of each recombination rate based on the recombination map to construct a genetic map of the species. ## Claims ### Claim 1 A method of constructing a genetic map, comprising following steps: whole genome sequencing for at least one single cell derived from a same species, to obtain every whole genome sequencing data of the at least one single cell; aligning the every whole genome sequencing data of the at least one single cell to a reference sequence respectively, to determine every SNP site genotypes of the at least one single cell; based on the every SNP site genotypes of the at least one single cell, deducing genotypes of two chromosomes from a male parent and a female parent respectively by means of a maximum parsimony of recombination, to determine male parent a/female parent b typing results of every SNP genotype of the at least one single cell; dividing a chromosome of the species into linkage regions based on the male parent a/female parent b typing results of every SNP genotype of the at least one single cell, wherein a standard of dividing the chromosome of the species into linkage regions is that: (1) the SNP number comprising continuous same a or b is at least 5; (2) a physical length of a selected linkage region is greater than 250 kb; for every one of the at least one single cell, determining a variation ratio of a/b between two continuous linkage regions, to obtain a recombination rate between every two continuous linkage regions; based on the recombination rate between every two continuous linkage regions, determining every recombination map of the at least one single cell, wherein a boundary site between a and b is a recombination site; and based on recombination maps of all single cells, determining a recombination rate of each recombination site, to construct the genetic map of the species. ### Claim 2 (canceled) ### Claim 3 (depends on 1) The method of claim 1, wherein the species is human. ### Claim 4 (depends on 1) The method of claim 1, wherein the single cell is a sperm cell. ### Claim 5 (canceled) ### Claim 6 (depends on 1) The method of claim 1, wherein the whole genome sequencing is performed using a Next-Generation or a Third-Generation sequencing platform. ### Claim 7 (depends on 1) The method of claim 1, wherein the step of whole genome sequencing for at least one single cell derived from a same species further comprises: lysing the single cell to release a whole genome DNA using an alkaline lysis method; amplifying the whole genome DNA, to obtain an amplified whole genome DNA; for the amplified whole genome DNA, constructing a sequencing-library, to obtain a sequencing-library of the single cell; and sequencing the sequencing-library of the single cell, to obtain the every whole genome sequencing data of the at least one single cell, wherein the single cell is a sperm cell, and the released whole genome DNA is subjected to a guanidine hydrochloride treatment after the single cell is lysed to release a whole genome DNA, and the guanidine hydrochloride treatment is performed using a solution of guanidine hydrochloride having a final concentration of 2×10−3 to 3×10−1 mol/L. ### Claim 8 (canceled) ### Claim 9 (canceled) ### Claim 10 (canceled) ### Claim 11 (depends on 1) The method of claim 1, wherein the reference sequence is a known genomic sequence of the species. ### Claim 12 A device of constructing a genetic map, comprising: a single cell whole genome sequencing unit, for whole genome sequencing for at least one single cell derived from a same species, to obtain every whole genome sequencing data of the at least one single cell; an SNP site genotype statistical unit, connected to the single cell whole genome sequencing unit, for aligning the every whole genome sequencing data of the at least one single cell to a reference sequence respectively, to determine every SNP site genotypes of the at least one single cell; a parent typing unit, connected to the SNP site genotype statistical unit, for deducing genotypes of two chromosomes from a male parent and a female parent respectively by means of a maximum parsimony of recombination based on the every SNP site genotypes of the at least one single cell, to determine male parent a/female parent b typing results of every SNP genotype of the at least one single cell; a linkage region dividing unit, connected to the parent typing unit, for dividing a chromosome of the species into linkage regions based on the male parent a/female parent b typing results of every SNP genotype of the at least one single cell, wherein a standard of dividing the chromosome of the species into linkage regions is that: (1) the SNP number comprising continuous same a or b is at least 5; (2) a physical length of a selected linkage region is greater than 250 kb; a recombination rate between contiguous linkage regions calculating unit, connected to the linkage region dividing unit, for every one of the at least one single cell, determining a variation ratio of a/b between two continuous linkage regions, to obtain a recombination rate between every two continuous linkage regions, and based on the recombination rate between every two continuous linkage regions, determining every recombination map of the at least one single cell, wherein a boundary site between a and b is a recombination site; a recombination rate of the recombination site calculating unit, connected to the recombination rate between contiguous linkage regions calculating unit, for determining a recombination rate of each recombination site, to construct the genetic map of the species based on recombination maps of all single cells. ### Claim 13 (depends on 12) The device of claim 12, wherein the single cell whole genome sequencing unit further comprises: a cellular whole genome DNA obtaining subunit, for lysing the single cell to release a whole genome DNA using an alkaline lysis method; a whole genome DNA amplifying subunit, connected to the cellular whole genome DNA obtaining subunit, for amplifying the whole genome DNA, to obtain an amplified whole genome DNA; and a whole genome DNA sequencing subunit, connected to the whole genome DNA amplifying subunit, for constructing a sequencing-library for the amplified whole genome DNA, to obtain a sequencing-library of the single cell, and sequencing the sequencing-library of the single cell, to obtain the every whole genome sequencing data of the at least one single cell. ### Claim 14 (depends on 12) The device of claim 12, wherein the cellular whole genome DNA obtaining subunit is further suitable for subjecting the released whole genome DNA to a guanidine hydrochloride treatment after the single cell is lysed to release a whole genome DNA, wherein the whole genome DNA sequencing subunit is suitable for using at least one of a Next-Generation and a Third-Generation sequencing platform. ### Claim 15 (canceled) ### Claim 16 A method of determining a haplotype of a single cell, comprising following steps: whole genome sequencing the single cell, to obtain a whole genome sequencing data of the single cell; aligning the obtained whole genome sequencing data to a reference sequence, to determine SNP site genotypes of the single cell; based on the SNP site genotypes of the single cell, deducing genotypes of two chromosomes from a male parent and a female parent respectively by means of a maximum parsimony of recombination, to determine male parent a/female parent b typing results of the SNP site genotypes of the single cell; dividing a chromosome of the species into linkage regions based on the male parent a/female parent b typing results of the SNP site genotypes of the single cell, wherein a standard of dividing the chromosome of the species into linkage regions is that: (1) the SNP number comprising continuous same a or b is at least 5; (2) a physical length of a selected linkage region is greater than 250 kb; based on the determined linkage region, determining the haplotype of the single cell, wherein a set of a or b in the same linkage regions of the whole genome constituents the haplotype of the single cell. ### Claim 17 (depends on 16) The method of claim 16, wherein the single cell derives from a mammal. ### Claim 18 (canceled) ### Claim 19 (depends on 16) The method of claim 16, wherein the single cell is a germ cell, and the germ cell is a sperm cell. ### Claim 20 (depends on 16) The method of claim 16, wherein the whole genome sequencing is performed using a Next-Generation or a Third-Generation sequencing platform. ### Claim 21 (depends on 16) The method of claim 16, wherein the step of whole genome sequencing the single cell, to obtain a whole genome sequencing data of the single cell further comprises: lysing the single cell to release a whole genome DNA using an alkaline lysis method; amplifying the whole genome DNA, to obtain an amplified whole genome DNA; for the amplified whole genome DNA, constructing a sequencing-library, to obtain a sequencing-library of the single cell; and sequencing the sequencing-library of the single cell, to obtain the every whole genome sequencing data of the at least one single cell, wherein the single cell is a sperm cell, the released whole genome DNA is subjected to a guanidine hydrochloride treatment after the single cell is lysed to release a whole genome DNA, and the guanidine hydrochloride treatment is performed using a solution of guanidine hydrochloride having a concentration of 2×10−3 to 3×10−1 mol/L. ### Claim 22 (canceled) ### Claim 23 (canceled) ### Claim 24 (depends on 21) The method of claim 21, wherein the single cell derives from human, and the solution of guanidine hydrochloride having a concentration of 2×10−3 mol/L. ### Claim 25 (depends on 16) The method of claim 16, wherein the reference sequence is a known genomic sequence of the species. ### Claim 26 A device of determining a haplotype of a single cell, comprising: a single cell whole genome sequencing unit, for whole genome sequencing the single cell, to obtain a whole genome sequencing data of the single cell; an SNP site genotype statistical unit, connected to the single cell whole genome sequencing unit, for aligning the obtained whole genome sequencing data to a reference sequence, to determine SNP site genotypes of the single cell; a parent typing unit, connected to the SNP site genotype statistical unit, for deducing genotypes of two chromosomes from a male parent and a female parent respectively by means of a maximum parsimony of recombination based on the SNP site genotypes of the single cell, to determine male parent a/female parent b typing results of the SNP site genotypes of the single cell; a linkage region (bin) dividing unit, connected to the parent typing unit, connected to the parent typing unit, for dividing a chromosome of the species into linkage regions (bin) based on the male parent a/female parent b typing results of the SNP site genotypes of the single cell, wherein a standard of dividing the chromosome of the species into linkage regions is that: (1) the SNP number comprising continuous same a or b is at least 5; (2) a physical length of a selected linkage region is greater than 250 kb; a haplotype determining unit, connected to the linkage region dividing unit, for determining the haplotype of the single cell based on the determined linkage region, wherein a set of a or b in the same linkage regions of the whole genome constituents the haplotype of the single cell. ### Claim 27 (depends on 26) The device of claim 26, wherein the single cell whole genome sequencing unit further comprises: a cellular whole genome DNA obtaining subunit, for lysing the single cell to release a whole genome DNA using an alkaline lysis method; a whole genome DNA amplifying subunit, connected to the cellular whole genome DNA obtaining subunit, for amplifying the whole genome DNA, to obtain an amplified whole genome DNA; and a whole genome DNA sequencing subunit, connected to the whole genome DNA amplifying subunit, for constructing a sequencing-library for the amplified whole genome DNA, to obtain a sequencing-library of the single cell, and sequencing the sequencing-library of the single cell, to obtain the every whole genome sequencing data of the at least one single cell. ### Claim 28 (depends on 27) The device of claim 27, wherein the cellular whole genome DNA obtaining subunit is further suitable for subjecting the released whole genome DNA to a guanidine hydrochloride treatment after the single cell is lysed to release a whole genome DNA. ### Claim 29 (depends on 27) The device of claim 27, wherein the whole genome DNA sequencing subunit is suitable for using at least one of a Next-Generation and a Third-Generation sequencing platform.