SIMULTANEOUS DETERMINATION OF ANEUPLOIDY AND FETAL FRACTION
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 determining aneuploidy and fetal fraction in a maternal sample comprising a mixture of fetal and maternal nucleic acid molecules, said method comprising:
(a) enriching said mixture for a plurality of polymorphic target nucleic;
(b) sequencing at least a portion of the enriched mixture obtained in step (a), wherein said sequencing comprises providing a plurality of sequence tags; and
(c) based on said sequencing, simultaneously determining said fetal fraction and said aneuploidy.
2 . The method of claim 1 , wherein said maternal sample is a biological fluid chosen from blood, plasma, serum, urine and saliva.
3 . The method of claim 1 , wherein said fetal and maternal nucleic acid molecules are cell-free DNA (cfDNA) molecules.
4 . The method of claim 1 , wherein said enriching comprises amplifying a plurality of polymorphic target nucleic acids in a portion of said mixture.
5 . The method of claim 1 , wherein said enriching comprises amplifying a plurality of polymorphic target nucleic acids in a portion of a purified mixture of fetal and maternal nucleic acids.
6 . The method of claim 1 , wherein said enriching comprises combining at least a portion of a first sequencing library of said mixture of fetal and maternal nucleic acid molecules with at least a portion of a second sequencing library of amplified polymorphic target nucleic acids.
7 . The method of claim 1 , wherein said polymorphic target nucleic acids are located on the same or on different chromosomes.
8 . The method of claim 1 , wherein each of said plurality of polymorphic target nucleic acids comprises at least 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 (SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16), rs 4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 & 56).
10 . The method of claim 8 , wherein said at least one SNP is a tandem SNP selected from sets of tandem SNPs rs7277033-rs2110153 (SEQ ID NOS 312 & 313); rs2822654-rs1882882 (SEQ ID NOS 314 & 315); rs368657-rs376635 (SEQ ID NOS 316 & 317); rs2822731-rs2822732 (SEQ ID NOS 318 & 319); rs1475881-rs7275487 (SEQ ID NOS 320 & 321); rs1735976-rs2827016 (SEQ ID NOS 322 & 323); rs447340-rs2824097 (SEQ ID NOS 324 & 325); rs418989-rs13047336 (SEQ ID NOS 326 & 327); rs987980-rs987981 (SEQ ID NOS 328 & 329); rs4143392-rs4143391 (SEQ ID NOS 330 & 331); rs1691324-rs13050434 (SEQ ID NOS 332 & 333); rs11909758-rs9980111 (SEQ ID NOS 334 & 335); rs2826842-rs232414 (SEQ ID NOS 336 & 337); rs1980969-rs1980970 (SEQ ID NOS 338 & 339); rs9978999-rs9979175 (SEQ ID NOS 340 & 341); rs1034346-rs12481852 (SEQ ID NOS 342 & 343); rs7509629-rs2828358 (SEQ ID NOS 344 & 345); rs4817013-rs7277036 (SEQ ID NOS 346 & 347); rs9981121-rs2829696 (SEQ ID NOS 348 & 349); rs455921-rs2898102 (SEQ ID NOS 350 & 351); rs2898102-rs458848 (SEQ ID NOS 352 & 353); rs961301-rs2830208 (SEQ ID NOS 354 & 355); rs2174536-rs458076 (SEQ ID NOS 356 & 357); rs11088023-rs11088024 (SEQ ID NOS 358 & 359); rs1011734-rs1011733 (SEQ ID NOS 360 & 361); rs2831244-rs9789838 (SEQ ID NOS 362 & 363); rs8132769-rs2831440 (SEQ ID NOS 364 & 365); rs8134080-rs2831524 (SEQ ID NOS 366 & 367); rs4817219-rs4817220 (SEQ ID NOS 368 & 369); rs2250911-rs2250997 (SEQ ID NOS 370 & 371); rs2831899-rs2831900 (SEQ ID NOS 372 & 373); rs2831902-rs2831903 (SEQ ID NOS 374 & 375); rs11088086-rs2251447 (SEQ ID NOS 376 & 377); rs2832040-rs11088088 (SEQ ID NOS 378 & 379); rs2832141-rs2246777 (SEQ ID NOS 380 & 381); rs2832959-rs9980934 (SEQ ID NOS 382 & 383); rs2833734-rs2833735 (SEQ ID NOS 384 & 385); rs933121-rs933122 (SEQ ID NOS 386 & 387); rs2834140-rs12626953 (SEQ ID NOS 388 & 389); rs2834485-rs3453 (SEQ ID NOS 390 & 391); rs9974986-rs2834703 (SEQ ID NOS 392 & 393); rs2776266-rs2835001 (SEQ ID NOS 394 & 395); rs1984014-rs1984015 (SEQ ID NOS 396 & 397); rs7281674-rs2835316 (SEQ ID NOS 398 & 399); rs13047304-rs13047322 (SEQ ID NOS 400 & 401); rs2835545-rs4816551 (SEQ ID NOS 402 & 403); rs2835735-rs2835736 (SEQ ID NOS 404 & 405); rs13047608-rs2835826 (SEQ ID NOS 406 & 407); rs2836550-rs2212596 (SEQ ID NOS 408 & 409); rs2836660-rs2836661 (SEQ ID NOS 410 & 411); rs465612-rs8131220 (SEQ ID NOS 412 & 413); rs9980072-rs8130031 (SEQ ID NOS 414 & 415); rs418359-rs2836926 (SEQ ID NOS 416 & 417); rs7278447-rs7278858 (SEQ ID NOS 418 & 419); rs385787-rs367001 (SEQ ID NOS 420 & 421); rs367001-rs386095 (SEQ ID NOS 422 & 423); rs2837296-rs2837297 (SEQ ID NOS 424 & 425); and rs2837381-rs4816672 (SEQ ID NOS 426 & 427).
11 . The method of claim 1 , wherein each of said plurality of polymorphic target nucleic acids comprises at least one short tandem repeat (STR).
12 . The method of claim 1 , wherein each of said plurality of polymorphic target nucleic acids 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, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627 and D1GATA113.
13 . The method of claim 11 , wherein said at least one STR is less than about 300 base pairs.
14 . The method of claim 1 , wherein said sequencing is next generation sequencing (NGS).
15 . The method of claim 1 , wherein said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
16 . The method of claim 1 , wherein said sequencing is sequencing-by-ligation.
17 . The method of claim 1 , wherein said sequencing comprises an amplification.
18 . The method of claim 1 , wherein said sequencing is single molecule sequencing.
19 . The method of claim 1 , wherein said aneuploidy is a chromosomal or a partial aneuploidy.
20 . The method of claim 1 , wherein said aneuploidy is a chromosomal aneuploidy chosen from trisomy 8, trisomy 13, trisomy 15, trisomy 16, trisomy 18, trisomy 21, trisomy 22, monosomy X, and XXX.
21 . The method of claim 1 , where determining said aneuploidy comprises calculating a chromosome dose based on the number of said sequence tags for a chromosome of interest and for a normalizing chromosome, and comparing said dose to a threshold value.
22 . The method of claim 1 , wherein determining said fetal fraction comprises identifying at least one informative polymorphic site in said enriched mixture, and calculating the fetal fraction from the amount of fetal and maternal polymorphic sites in said enriched sample.
23 . A composition comprising at least one set of primers for amplifying at least one SNP in a sample comprising a mixture of nucleic acid molecules, wherein said at least one SNP is chosen from rs560681 (SEQ ID NOS 1 & 2), rs1109037 (SEQ ID NOS 3 & 4), rs9866013 (SEQ ID NOS 5 & 6), rs13182883 (SEQ ID NOS 7 & 8), rs13218440 (SEQ ID NOS 9 & 10), rs7041158 (SEQ ID NOS 11 & 12), rs740598 (SEQ ID NOS 13 & 14), rs10773760 (SEQ ID NOS 15 & 16), rs 4530059 (SEQ ID NOS 17 & 18), rs7205345 (SEQ ID NOS 19 & 20), rs8078417 (SEQ ID NOS 21 & 22), rs576261 (SEQ ID NOS 23 & 24), rs2567608 (SEQ ID NOS 25 & 26), rs430046 (SEQ ID NOS 27 & 28), rs9951171 (SEQ ID NOS 29 & 30), rs338882 (SEQ ID NOS 31 & 32), rs10776839 (SEQ ID NOS 33 & 34), rs9905977 (SEQ ID NOS 35 & 36), rs1277284 (SEQ ID NOS 37 & 38), rs258684 (SEQ ID NOS 39 & 40), rs1347696 (SEQ ID NOS 41 & 42), rs508485 (SEQ ID NOS 43 & 44), rs9788670 (SEQ ID NOS 45 & 46), rs8137254 (SEQ ID NOS 47 & 48), rs3143 (SEQ ID NOS 49 & 50), rs2182957 (SEQ ID NOS 51 & 52), rs3739005 (SEQ ID NOS 53 & 54), and rs530022 (SEQ ID NOS 55 & 56).
24 . The composition of claim 23 , wherein said at least one set of primers is chosen from primer sets of SEQ ID NOs: 57-112.
25 . A composition comprising at least one set of primers for amplifying at least one STR in a sample comprising a mixture of nucleic acid molecules, wherein said at least one STR is chosen 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, D10S1435, D10S1248, D9S2157, D9S1122, D8S1115, D6S1017, D6S474, D5S2500, D4S2408, D4S2364, D3S4529, D3S3053, D2S1776, D2S441, D1S1677, D1S1627 and D1GATA113.
26 . The composition of claim 25 , wherein said at least one set of primers is selected from primer sets of SEQ ID NOs:113-196.
27 . The composition of claim 23 or claim 25 , wherein said mixture comprises fetal and maternal nucleic acid molecules
28 . The composition of claim 23 or claim 25 , wherein said sample is a maternal plasma sample.
29 . The composition of claim 23 or claim 25 , wherein said nucleic acids are cfDNA molecules.
30 . A kit for preparing a sequencing library for massively parallel sequencing of fetal and maternal nucleic acid molecules, said kit comprising a composition comprising at least one set of primers for amplifying at least one polymorphic nucleic acid in said fetal and maternal nucleic acid molecules.
31 . The kit of claim 30 , wherein said polymorphic nucleic acid comprises a SNP or an STR.
32 . The kit of claim 30 , wherein said sample is a maternal plasma sample.
33 . The kit of claim 30 , wherein said nucleic acids are cfDNA molecules.
34 . The kit of claim 30 , wherein said sequencing is single molecule sequencing.
35 . The kit of claim 30 , wherein said massively parallel sequencing is sequencing-by-synthesis with reversible dye terminators.
36 . The kit of claim 30 , wherein said massively parallel sequencing is sequencing-by-ligation.