SEQUENCING METHODS AND COMPOSITIONS FOR PRENATAL DIAGNOSES

• US 20110201507A1
• Filed: 12/01/2010
• Published: 08/18/2011
• Est. Priority Date: 01/19/2010
• Status: Active Grant

• Current Assignee: Verinata Health Incorporated (Illumina Incorporated)
• Original Assignee: Verinata Health Incorporated (Illumina Incorporated)
• Inventors: Rhees, Brian Kent, Rava, Richard P., Heilek, Gabrielle, Chinnappa, Manjula, Comstock, David A.
• Granted Patent: US 9,657,342 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16H 10/40 for data related to laboratory analysis, e.g. patient specimen analysis

Abstract

The invention provides methods for determining aneuploidy and/or fetal fraction in maternal samples comprising fetal and maternal cfDNA by massively parallel sequencing. The method comprises a novel protocol for preparing sequencing libraries that unexpectedly improves the quality of library DNA while expediting the process of analysis of samples for prenatal diagnoses.

Claims

1. A method for determining a fetal chromosomal aneuploidy in a maternal sample comprising a mixture of fetal and maternal nucleic acids molecules, said method comprising:

(a) preparing a sequencing library from said mixture of fetal and maternal nucleic acid molecules;

wherein preparing said library comprises the consecutive steps of end-repairing, dA-tailing and adaptor ligating said nucleic acids;

(b) sequencing at least a portion of said nucleic acid molecules, thereby obtaining sequence information for a plurality of fetal and maternal nucleic acid molecules of a maternal sample;

(c) using the sequence information to obtain a chromosome dose for an aneuploid chromosome; and

(d) comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal aneuploidy.

2. The method of claim 1, further comprising:

(a) using the sequence information to identify a number of mapped sequence tags for at least one normalizing chromosome and for an aneuploid chromosome; and

(b) using the number of mapped sequence tags identified for said aneuploid chromosome and the number of mapped sequence tags identified for the at least one normalizing chromosome in step (a) to calculate a chromosome dose for said aneuploid chromosome as a ratio of the number of mapped sequence tags identified for said aneuploid chromosome and the number of mapped sequence tags identified for the at least one normalizing chromosome.

3. The method of claim 2, wherein step (b) comprises:

(i) calculating a sequence tag density ratio for said aneuploid chromosome, by relating the number of mapped sequence tags identified for said aneuploid chromosome in step (a) to the length of said aneuploid chromosome;

(ii) calculating a sequence tag density ratio for said at least one normalizing chromosome, by relating the number of mapped sequence tags identified for said at least one normalizing chromosome in step (a) to the length of said at least one normalizing chromosome; and

(iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a chromosome dose for said aneuploid chromosome, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for said aneuploid chromosome and the sequence tag density ratio for said at least one normalizing chromosome.

4. The method of claim 2, wherein said at least one normalizing chromosome is a chromosome having the smallest variability and/or the greatest differentiability.

5. The method of claim 2, wherein said aneuploid chromosome is chromosome 21, and said at least one normalizing chromosome is selected from chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14.

6. The method of claim 2, wherein said aneuploid chromosome is chromosome 21, and said at least one normalizing chromosome is a group of chromosomes selected from chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14.

7. The method of claim 2, wherein said aneuploid chromosome is chromosome 18, and said at least one normalizing chromosome is selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14.

8. The method of claim 2, wherein said aneuploid chromosome is chromosome 18, and said at least one normalizing chromosome is a group of chromosomes selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14.

9. The method of claim 2, wherein said aneuploid chromosome is chromosome 13, and said at least one normalizing chromosome is selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.

10. The method of claim 2, wherein said aneuploid chromosome is chromosome 13, and said at least one normalizing chromosome is a group of chromosomes selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.

11. The method of claim 2, wherein said aneuploid chromosome is chromosome X, and said at least one normalizing chromosome is selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.

12. The method of claim 2, wherein said aneuploid chromosome is chromosome X, and said at least one normalizing chromosome is a group of chromosomes selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.

25. The method of claim 1, 13 or 17, wherein said consecutive steps are performed in the absence of polyethylene glycol.

26. The method of claim 1, 13 or 17, wherein said consecutive steps are performed in less than 1 hour.

27. The method of claim 1, 13 or 17, wherein said consecutive steps exclude purification.

28. The method of claim 1, 13 or 17, wherein said maternal sample is a biological fluid selected from blood, plasma, serum, urine and saliva.

29. The method of claim 1, 13 or 17, wherein said fetal and maternal nucleic acid molecules are cell-free DNA (cfDNA) molecules.

30. The method of claim 1, 13 or 17, wherein said sequencing is next generation sequencing (NGS).

31. The method of claim 1, 13 or 17, wherein said sequencing is massively parallel sequencing using sequencing-by synthesis with reversible dye terminators.

32. The method of claim 1, 13 or 17, wherein said sequencing is massively parallel sequencing using sequencing-by-ligation.

33. The method of claim 1, 13 or 17, wherein said sequencing comprises an amplification.

34. The method of claim 1, 13 or 17, wherein said sequencing is single molecule sequencing.

35. A computer-readable medium having stored thereon computer-readable instructions for carrying out the method of claim 1, or 13.

36. The computer-readable medium of claim 35, having stored thereon computer-readable instructions for carrying out a method comprising the steps:

(a) using sequence information obtained from a plurality of fetal and maternal nucleic acid molecules in a maternal plasma sample to identify a number of mapped sequence tags for a chromosome of interest;

(b) using sequence information obtained from a plurality of fetal and maternal nucleic acid molecules in a maternal plasma sample to identify a number of mapped sequence tags for at least one normalizing chromosome;

(c) using the number of mapped sequence tags identified for a chromosome of interest in step (a) and the number of mapped sequence tags identified for the at least one normalizing chromosome in step (b) to calculate a chromosome dose for said a chromosome of interest; and

(d) comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal aneuploidy.

37. The computer-readable medium of claim 35, wherein said chromosome of interest is selected from chromosomes 21, 13, 18 and X.

38. A computer processing system which is adapted or configured to perform the method of any of claims 1 or 13.

39. The computer processing system of claim 38, which is adapted or configured to perform the following steps:

(a) using sequence information obtained from a plurality of fetal and maternal nucleic acid molecules in a maternal plasma sample to identify a number of mapped sequence tags for a chromosome of interest;

(b) using sequence information obtained from a plurality of fetal and maternal nucleic acid molecules in a maternal plasma sample to identify a number of mapped sequence tags for at least one normalizing chromosome;

(c) using the number of mapped sequence tags identified for a chromosome of interest in step (a) and the number of mapped sequence tags identified for the at least one normalizing chromosome in step (b) to calculate a chromosome dose for a chromosome of interest; and

(d) comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal aneuploidy.

40. The computer processing system of claim 38, wherein said chromosome of interest is selected from chromosomes 21, 13, 18 and X.

41. An apparatus adapted or configured to perform the method of any of claims 1 and 13.

42. The apparatus of claim 41, which is adapted or configured for identifying fetal aneuploidy in a maternal plasma sample comprising fetal and maternal nucleic acid molecules, and wherein said apparatus comprises:

(a) a sequencing device adapted or configured for sequencing at least a portion of the nucleic acid molecules in a maternal plasma sample comprising fetal and maternal nucleic acid molecules, thereby generating sequence information; and

(b) a computer processing system configured to perform the following steps;

(i) using sequence information generated by the sequencing device to identify a number of mapped sequence tags for a chromosome of interest;

(ii) using sequence information generated by the sequencing device to identify a number of mapped sequence tags for at least one normalizing chromosome;

(iii) using the number of mapped sequence tags identified for a chromosome of interest in step (i) and the number of mapped sequence tags identified for the at least one normalizing chromosome in step (ii) to calculate a chromosome dose for a chromosome of interest; and

(iv) comparing said chromosome dose to at least one threshold value, and thereby identifying the presence or absence of fetal aneuploidy.

43. The apparatus of claim 41, wherein said chromosome of interest is selected from chromosomes 21, 13, 18 and X.

13. A method for determining the presence or absence of an aneuploidy in a maternal sample comprising a mixture of fetal and maternal nucleic acid molecules, said method comprising:

(a) preparing a sequencing library from said mixture;

wherein preparing said library comprises the consecutive steps of end-repairing, dA-tailing and adaptor ligating said fetal and maternal nucleic acid molecules;

(b) sequencing at least a portion of said library, wherein said sequencing comprises providing a plurality of sequence tags; and

(c) based on said sequencing, determining the presence or absence of aneuploidy in said sample.

14. The method of claim 13, wherein said aneuploidy is a chromosomal or a partial aneuploidy.

15. The method of claim 13, 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.

16. The method of claim 13, wherein step (c) 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.

17. A method for determining the fraction of fetal nucleic acids in a maternal sample comprising a mixture of fetal and maternal nucleic acid molecules, said method comprising:

(a) amplifying a plurality of polymorphic target nucleic acids in a portion of said mixture;

(b) preparing a sequencing library of the amplified product obtained in step (a) wherein preparing said library comprises the consecutive steps of end-repairing, dA-tailing and adaptor ligating said fetal and maternal nucleic acid molecules;

(c) sequencing at least a portion of said sequencing library; and

(d) based on said sequencing, determining said fraction.

18. The method of claim 17, wherein each of said plurality of polymorphic target nucleic acids comprises at least one nucleotide polymorphism (SNP).

19. The method of claim 17, wherein each of said plurality of polymorphic target nucleic acid comprises at least one short tandem repeat (STR).

20. The method of claim 17, wherein determining said fraction comprises determining the number of fetal and maternal sequence tags mapped to a reference target genome comprising of said at least one polymorphic nucleic acid.

21. The method of claim 17, wherein said at least one SNP is a single 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), rs4530059 (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).

22. The method of claim 17, wherein said at least one SNP is a tandem SNP selected from tandem SNP pairs 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).

23. The method of claim 17, wherein said at least one STR is selected from CSF1PO, FGA, TH01, 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.

24. The method of claim 17, further comprising determining the presence or absence of aneuploidy in said maternal sample.