Simultaneous determination of aneuploidy and fetal fraction

Abstract

The invention provides compositions and methods for simultaneously determining the presence or absence of fetal aneuploidy and the relative amount of fetal nucleic acids in a sample obtained form a pregnant female. The method encompasses the use of sequencing technologies and exploits the occurrence of polymorphisms to provide a streamlined noninvasive process applicable to the practice of prenatal diagnostics.

1 . A method for simultaneously quantifying at least one chromosome of interest and determining fetal fraction in a maternal sample comprising a mixture of fetal and maternal nucleic acid molecules, said method comprising:
 (a) sequencing at least a portion of said mixture of fetal and maternal nucleic acids to obtain (i) a plurality of sequence tags for said at least one chromosome of interest and for at least one normalizing chromosome, and (ii) a plurality of polymorphic sequences each comprising one or more polymorphic sites, wherein said polymorphic sequences and said plurality of sequences are obtained in the same sequencing run;   (b) using said plurality of sequence tags obtained in step (a) to obtain a first number of sequence tags for said at least one chromosome of interest and a second number of sequence tags for said at least one normalizing chromosome:   (c) quantifying relative amounts of said at least one chromosome of interest from said first and second number of sequence tags; and   (d) analyzing the polymorphic sequences from step (a) to obtain the percentage of fetal fraction of the,mixture of fetal and maternal nucleic acids.   
     
     
         2 . The method of  claim 1 , further comprising comparing said relative amounts of said at least one chromosome of interest to a threshold value to determine the presence or absence of a fetal aneuploidy, and wherein said maternal sample is a maternal plasma or serum sample. 
     
     
         3 . The method of  claim 1 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA (cfDNA) molecules. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein said polymorphic sites are located on the same or on different chromosomes. 
     
     
         8 . The method of  claim 1 , wherein each of said plurality of polymorphic sites comprises at feast one single nucleotide polymorphism (SNP). 
     
     
         9 . The method of  claim 8 , wherein said at least one SNP is a single SNP selected from each of said plurality of polymorphic target nucleic acids comprises a SNP selected from rs560681, rs1109037, rs9866013, rs13182883, rs13218440, rs7041158, rs740598, rs10773760, rs4530059, rs7205345, rs8078417, rs576261, rs2567608, rs430046, rs9951171, rs338882, rs10776839, rs9905977, rs1277284, rs258684, rs1347696, rs508485, rs9788670, rs8137254, rs3143, rs2182957, rs3739005, and rs530022. 
     
     
         10 . The method of  claim 8 , wherein said at least one SNP is a tandem SNP. 
     
     
         11 . The method of  claim 10 , wherein said tandem SNP is selected from sets of tandem SNPs rs7277033-rs2110153; rs2822654-rs1882882; rs368657-rs376635; rs2822731-rs2822732; rs1475881-rs7275487; rs1735976-rs2827016; rs447340-rs2824097; rs418989-rs13047336; rs987980-rs987981; rs4143392-rs4143391; rs1691324-rs13050434; rs11909758-rs9980111; rs2826842-rs232414; rs1980969-rs1980970; rs9978999-rs9979175; rs1034346-rs12481852; rs7509629-rs2828358; rs4817013-rs7277036; rs9981121-rs2829696; rs455921-rs2898102; rs2898102-rs458848; rs961301-rs2830208; rs2174536-rs458076; rs11088023-rs11088024; rs1011734-rs1011733; rs2831244-rs9789838; rs8132769-rs2831440; rs8134080-rs2831524; rs4817219-rs4817220; rs2250911-rs2250997; rs2831899-rs2831900; rs2831902-rs2831903; rs11088086-rs2251447; rs2832040-rs11088088; rs2832141-rs2246777; rs2832959-rs9980934; rs2833734-rs2833735; rs933121-rs933122; rs2834140-rs12626953; rs2834485-rs3453; rs9974986-rs2834703; rs2776266-rs2835001; rs1984014-rs1984015; rs7281674-rs2835316; rs13047304-rs13047322; rs2835545-rs4816551; rs2835735-rs2835736; rs13047608-rs2835826; rs2836550-rs2212596; rs2836660-rs2836661; rs465612-rs8131220; rs9980072-rs8130031; rs418359-rs2836926; rs7278447-rs7278858; rs385787-rs367001; rs367001-rs386095; rs2837296-rs2837297; and rs2837381-rs4816672. 
     
     
         12 . The method of  claim 1 , wherein each of said plurality of polymorphic sites comprises at least one short tandem repeat (STR). 
     
     
         13 . The method of  claim 1 , wherein each of said plurality of polymorphic Sites is an STR selected from CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D2S1338, Penta D, Penta E, D22S1045, D20S1082, D20S482, D18S853, D17S1301, D17S974, D14S1434, D12ATA63, D11S4463, D10S1438, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627 and D1GATA113. 
     
     
         14 . The method of  claim 12 , wherein said at least one STR is less than about 300 base pairs. 
     
     
         15 . The method of  claim 1 , wherein said sequencing is next generation sequencing (NGS). 
     
     
         16 . The method of  claim 1 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators. 
     
     
         17 . The method of  claim 1 , wherein said sequencing is sequencing-by-ligation. 
     
     
         18 . The method of  claim 1 , wherein said sequencing comprises an amplification. 
     
     
         19 . The method of  claim 1 , wherein said sequencing is single molecule sequencing. 
     
     
         20 . The method of  claim 2 , wherein said aneuploidy is a chromosomal aneuploidy. 
     
     
         21 . The method of  claim 20 , wherein said chromosomal aneuploidy is a trisomy or a monosomy. 
     
     
         22 . The method of  claim 20 , wherein said chromosomal aneuploidy is chosen from trisomy 8, trisomy 13, trisomy 15, trisomy 16, trisomy 18, trisomy 21, trisomy 22, monosomy X, and XXX. 
     
     
         23 . The method of  claim 2 , wherein said aneuploidy is a partial aneuploidy. 
     
     
         24 . The method of  claim 2 , further comprising calculating a chromosome dose for said chromosome of interest based on the number of said sequence tags for said chromosome of interest and for said at least one normalizing chromosome. 
     
     
         25 . The method of  claim 1 , further comprising comparing the number of tags that map to the first allele to the number of tags that map to the second allele of said one or more polymorphic sites. 
     
     
         26 . The method of  claim 1 , further comprising amplifying said sequences comprising said plurality of polymorphic sites. 
     
     
         27 . The method of  claim 1 , wherein said polymorphic sites are located on autosomes. 
     
     
         28 . The method of  claim 1 , wherein said quantifying the relative amounts of said at least one chromosome of interest comprises determining a ratio of said first and second number of sequence tags.