Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses

Abstract

Provided are compositions and processes that utilize genomic regions that are differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample. The compositions and processes described herein are particularly useful for non-invasive prenatal diagnostics, including the detection of chromosomal aneuploidies.

What is claimed is: 
     
         1 . A method for determining fetal fraction in a sample comprising:
 (a) enriching a sample nucleic acid for a plurality of polymorphic nucleic acid targets, which sample nucleic acid comprises fetal nucleic acid and maternal nucleic acid;   (b) obtaining nucleotide sequences for some or all of the nucleic acid targets by a sequencing process;   (c) analyzing the nucleotide sequences of (b); and   (d) determining fetal fraction based on the analysis of (c), wherein the polymorphic nucleic acid targets and number thereof result in at least five polymorphic nucleic acid targets being informative for determining the fetal fraction for at least 90% of samples.   
     
     
         2 . The method of  claim 1 , wherein the enriching comprises amplifying the plurality of polymorphic nucleic acid targets. 
     
     
         3 . The method of  claim 1 , wherein the maternal genotype and the paternal genotype at each of the polymorphic nucleic acid targets are not known prior to (a). 
     
     
         4 . The method of  claim 1 , wherein polymorphic nucleic acid targets having a minor allele population frequency of about 40% or more are selected. 
     
     
         5 . The method of  claim 1 , comprising determining an allele frequency in the sample for each of the polymorphic nucleic acid targets. 
     
     
         6 . The method of  claim 5 , wherein determining which polymorphic nucleic acid targets are informative comprises identifying informative genotypes by comparing each allele frequency to one or more fixed cutoff frequencies. 
     
     
         7 . The method of  claim 6 , wherein the fixed cutoff for identifying informative genotypes from non-informative homozygotes is about a 1% or greater shift in allele frequency. 
     
     
         8 . The method of  claim 6 , wherein the fixed cutoff for identifying informative genotypes from non-informative heterozygotes is about a 25% or greater shift in allele frequency. 
     
     
         9 . The method of  claim 5 , wherein determining which polymorphic nucleic acid targets are informative comprises identifying informative genotypes by comparing each allele frequency to one or more target-specific cutoff frequencies. 
     
     
         10 . The method of  claim 9 , wherein each target-specific cutoff frequency is determined based on the allele frequency variance for the corresponding polymorphic nucleic acid target. 
     
     
         11 . The method of  claim 5  further comprising determining an allele frequency mean. 
     
     
         12 . The method of  claim 11 , wherein fetal fraction is determined based, in part, on the allele frequency mean. 
     
     
         13 . The method of  claim 1 , wherein the fetal genotype at one or more informative polymorphic nucleic acid targets is heterozygous or the fetal genotype at one or more informative polymorphic nucleic acid targets is homozygous. 
     
     
         14 . The method of  claim 1 , wherein fetal fraction is determined with a coefficient of variance (CV) of 0.20 or less. 
     
     
         15 . The method of  claim 1 , wherein the polymorphic nucleic acid targets each comprise at least one single nucleotide polymorphism (SNP). 
     
     
         16 . The method of  claim 15 , wherein the SNPs are selected from:
 rs10413687, rs10949838, rs1115649, rs11207002, rs11632601, rs11971741, rs12660563, rs13155942, rs1444647, rs1572801, rs17773922, rs1797700, rs1921681, rs1958312, rs196008, rs2001778, rs2323659, rs2427099, rs243992, rs251344, rs254264, rs2827530, rs290387, rs321949, rs348971, rs390316, rs3944117, rs425002, rs432586, rs444016, rs4453265, rs447247, rs4745577, rs484312, rs499946, rs500090, rs500399, rs505349, rs505662, rs516084, rs517316, rs517914, rs522810, rs531423, rs537330, rs539344, rs551372, rs567681, rs585487, rs600933, rs619208, rs622994, rs639298, rs642449, rs6700732, rs677866, rs683922, rs686851, rs6941942, rs7045684, rs7176924, rs7525374, rs870429, rs949312, rs9563831, rs970022, rs985462, rs1005241, rs1006101, rs10745725, rs10776856, rs10790342, rs11076499, rs11103233, rs11133637, rs11974817, rs12102203, rs12261, rs12460763, rs12543040, rs12695642, rs13137088, rs13139573, rs1327501, rs13438255, rs1360258, rs1421062, rs1432515, rs1452396, rs1518040, rs16853186, rs1712497, rs1792205, rs1863452, rs1991899, rs2022958, rs2099875, rs2108825, rs2132237, rs2195979, rs2248173, rs2250246, rs2268697, rs2270893, rs244887, rs2736966, rs2851428, rs2906237, rs2929724, rs3742257, rs3764584, rs3814332, rs4131376, rs4363444, rs4461567, rs4467511, rs4559013, rs4714802, rs4775899, rs4817609, rs488446, rs4950877, rs530913, rs6020434, rs6442703, rs6487229, rs6537064, rs654065, rs6576533, rs6661105, rs669161, rs6703320, rs675828, rs6814242, rs6989344, rs7120590, rs7131676, rs7214164, rs747583, rs768255, rs768708, rs7828904, rs7899772, rs7900911, rs7925270, rs7975781, rs8111589, rs849084, rs873870, rs9386151, rs9504197, rs9690525, and rs9909561.   
     
     
         17 . The method of  claim 16 , wherein the SNPs are selected from:
 rs10413687, rs10949838, rs1115649, rs11207002, rs11632601, rs11971741, rs12660563, rs13155942, rs1444647, rs1572801, rs17773922, rs1797700, rs1921681, rs1958312, rs196008, rs2001778, rs2323659, rs2427099, rs243992, rs251344, rs254264, rs2827530, rs290387, rs321949, rs348971, rs390316, rs3944117, rs425002, rs432586, rs444016, rs4453265, rs447247, rs4745577, rs484312, rs499946, rs500090, rs500399, rs505349, rs505662, rs516084, rs517316, rs517914, rs522810, rs531423, rs537330, rs539344, rs551372, rs567681, rs585487, rs600933, rs619208, rs622994, rs639298, rs642449, rs6700732, rs677866, rs683922, rs686851, rs6941942, rs7045684, rs7176924, rs7525374, rs870429, rs949312, rs9563831, rs970022, and rs985462.   
     
     
         18 . The method of  claim 16 , wherein the SNPs are selected from:
 rs1005241, rs1006101, rs10745725, rs10776856, rs10790342, rs11076499, rs11103233, rs11133637, rs11974817, rs12102203, rs12261, rs12460763, rs12543040, rs12695642, rs13137088, rs13139573, rs1327501, rs13438255, rs1360258, rs1421062, rs1432515, rs1452396, rs1518040, rs16853186, rs1712497, rs1792205, rs1863452, rs1991899, rs2022958, rs2099875, rs2108825, rs2132237, rs2195979, rs2248173, rs2250246, rs2268697, rs2270893, rs244887, rs2736966, rs2851428, rs2906237, rs2929724, rs3742257, rs3764584, rs3814332, rs4131376, rs4363444, rs4461567, rs4467511, rs4559013, rs4714802, rs4775899, rs4817609, rs488446, rs4950877, rs530913, rs6020434, rs6442703, rs6487229, rs6537064, rs654065, rs6576533, rs6661105, rs669161, rs6703320, rs675828, rs6814242, rs6989344, rs7120590, rs7131676, rs7214164, rs747583, rs768255, rs768708, rs7828904, rs7899772, rs7900911, rs7925270, rs7975781, rs8111589, rs849084, rs873870, rs9386151, rs9504197, rs9690525, and rs9909561.   
     
     
         19 . The method of  claim 1 , wherein the polymorphic nucleic acid targets and number thereof result in at least five polymorphic nucleic acid targets being informative for determining the fetal fraction for at least 95% of samples. 
     
     
         20 . The method of  claim 1 , wherein the polymorphic nucleic acid targets and number thereof result in at least ten polymorphic nucleic acid targets being informative for determining the fetal fraction for at least 90% of samples. 
     
     
         21 . The method of  claim 1 , wherein 10 or more polymorphic nucleic acid targets are enriched. 
     
     
         22 . The method of  claim 21 , wherein about 40 to about 100 polymorphic nucleic acid targets are enriched. 
     
     
         23 . The method of  claim 1 , wherein the sequencing process comprises a sequencing by synthesis method. 
     
     
         24 . The method of  claim 23 , wherein the sequencing by synthesis method comprises about 36 cycles or about 27 cycles. 
     
     
         25 . The method of  claim 1 , wherein the sample nucleic acid is cell-free DNA. 
     
     
         26 . The method of  claim 1 , wherein the sample nucleic acid is obtained from a pregnant human female subject. 
     
     
         27 . The method of  claim 1 , wherein the sample nucleic acid is from plasma or serum.