Non-invasive determination of fetal inheritance of parental haplotypes at the genome-wide scale

Abstract

The present invention provides a method, device and a computer program for haplotyping single cells, such that a sample taken from a pregnant female, without directly sampling the fetus, provides the ability to non-invasively determine the fetal genome. The method can be performed by determining the parental and inherited haplotypes, or can be performed merely on the basis of the mother's genetic information, obtained preferably in a blood or serum sample. The novel device allows for sequence analysis of single chromosomes from a single cell, preferably by partitioning single chromosomes from a metaphase cell into long, thin channels where a sequence analysis can be performed.

1 - 18 . (canceled) 
     
     
         19 . A method of determining an appropriate set of markers that define a maternal haplotype, comprising determining alleles that are present at polymorphic loci in a first maternal haplotype but not at corresponding loci on a second maternal haplotype, wherein said determining is performed by the steps of:
 a. simultaneously isolating all of the chromosomes from a single metaphase cell in a sample from a first mother by partitioning said chromosomes into a plurality of channels of a microfluidic device and analyzing each of said chromosomes to determine a first set of maternal single nucleotide polymorphisms (SNPs);   b. simultaneously isolating all of the chromosomes from a single metaphase cell in a sample from a second mother by partitioning said chromosomes into a plurality of channels of a microfluidic device and analyzing each of said chromosomes to determine a second set of maternal single nucleotide polymorphisms (SNPs);   c. identifying SNPs which are present at loci in either of the first or second mother but absent at corresponding loci in the other mother; and   d. including in said appropriate set of markers defining a maternal haplotype those alleles at which SNPs exist in only one of the first or second mother.   
     
     
         20 . The method of  claim 19 , wherein the alleles that are present at polymorphic loci in the first maternal haplotype but not at corresponding loci on the second maternal haplotype are also not at corresponding loci on either paternal haplotype. 
     
     
         21 . A method of determining an appropriate set of markers that define a paternal haplotype, comprising determining alleles that are present at polymorphic loci in a first paternal haplotype but not at corresponding loci on a second paternal haplotype. 
     
     
         22 . The method of  claim 21 , wherein the alleles that are present at polymorphic loci in the first paternal haplotype but not at corresponding loci on the second paternal haplotype are also not at corresponding loci on either maternal haplotype. 
     
     
         23 . The method of  claim 21 , wherein the number of markers in the set can be increased by haplotype imputation. 
     
     
         24 . The methods of  claim 23 , wherein the haplotype imputation comprises statistically inferring allelic identities at any unmeasured loci by comparing observed alleles on the haplotype to be imputed with a database of previously documented haplotypes of which allelic identities are known at both measured and unmeasured loci. 
     
     
         25 . The method of  claim 23 , wherein the database is from a normal population. 
     
     
         26 . The method of  claim 23 , wherein the database is from a population of carriers with a particular disease that is genetically transmissible. 
     
     
         27 . A method of determining a minimum amount of digital sampling to achieve a desired confidence level as to which parental haplotypes are over-represented, comprising:
 a. estimating a fraction of fetal DNA present in the sample; and   b. estimating density of available markers.   
     
     
         28 . A method of estimating fetal DNA fraction comprising measuring relative representation of parental haplotypes, by
 a. obtaining a maternal sample from a female pregnant with at least one fetus, wherein said sample contains DNA from both the pregnant female and the fetus;   b. detecting the presence of a paternally inherited haplotype by the steps of:
 i. simultaneously isolating all of the chromosomes from a single metaphase cell of the fetus's father by partitioning said chromosomes into a plurality of channels of a microfluidic device and analyzing each of said chromosomes to determine a set of paternal single nucleotide polymorphisms (SNPs); 
 ii. simultaneously isolating all of the chromosomes from a single metaphase cell of the fetus's mother by partitioning said chromosomes into a plurality of channels of a microfluidic device and analyzing each of said chromosomes to determine a set of maternal single nucleotide polymorphisms (SNPs); 
 iii. determining all SNPs that are heterozygous in the father and homozygous in the mother to identify at various loci alleles present in the father and absent in the mother, thereby defining each of the father's haplotypes; and 
 iv. counting a number of representative alleles on each paternal haplotype to determine a representation of the two paternal haplotypes in the maternal sample containing DNA from the pregnant female and the fetus; 
 v. comparing the representation of the two paternal haplotypes to obtain a relative representation in the maternal sample containing DNA from the pregnant female and the fetus; 
 vi. determining an over-representation ε of one of the two paternal haplotypes in the maternal sample containing DNA from the pregnant female and the fetus; and 
 vii. correlating said over-representation ε with a paternally inherited haplotype; or 
   c. determining an over-representation of a maternal haplotype by:
 i. determining all SNPs that are heterozygous in the fetus's mother; and 
 ii. identifying alleles present in the mother but absent in the paternally inherited haplotype at each SNP locus to define the mother's haplotypes; 
 iii. counting a number of representative alleles on each maternal haplotype to determine a representation of the two maternal haplotypes; 
 iv. comparing the representation of the two maternal haplotypes to obtain a relative representation; 
 v. determining an over-representation ε of one of the two maternal haplotypes in the maternal sample containing DNA from the pregnant female and the fetus; and 
   d. correlating the over-representation ε with the fetal DNA fraction.   
     
     
         29 . The method of  claim 19 , wherein determining a set of markers that define a haplotype of an individual can be obtained by:
 a. comparing alleles at polymorphic loci across related family members; or   b. analyzing alleles at polymorphic loci on single DNA molecules or single chromosome molecules.   
     
     
         30 - 35 . (canceled) 
     
     
         36 . The method of  claim 21 , wherein determining a set of markers that define a haplotype of an individual can be obtained by:
 c. comparing alleles at polymorphic loci across related family members; or   d. analyzing alleles at polymorphic loci on single DNA molecules or single chromosome molecules.