METHODS FOR DETECTING FETAL NUCLEIC ACIDS AND DIAGNOSING FETAL ABNORMALITIES

• US 20100216153A1
• Filed: 03/19/2010
• Published: 08/26/2010
• Est. Priority Date: 02/27/2004
• Status: Abandoned Application

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Helicos Biosciences Corporation
• Inventors: LAPIDUS, STANLEY, LIPSON, DORON, EFCAVITCH, J. WILLIAM, LETOVSKY, STANLEY, THOMPSON, JOHN F., MILOS, PATRICE
• Application Number:
  US12/727,824
• Publication Number:
  US 20100216153A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2535/122 Massive parallel sequencing
• 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 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

The invention generally relates to methods for detecting fetal nucleic acids and methods for diagnosing fetal abnormalities. In certain embodiments, the invention provides methods for determining whether fetal nucleic acid is present in a maternal sample including obtaining a maternal sample suspected to include fetal nucleic acids, and performing a sequencing reaction on the sample to determine presence of at least a portion of a Y chromosome in the sample, thereby determining that fetal nucleic acid is present in the sample. In other embodiments, the invention provides methods for quantitative or qualitative analysis to detect fetal nucleic acid in a maternal sample, regardless of the ability to detect the Y chromosome, particularly for samples including normal nucleic acids from a female fetus.

Claims

1. A method for determining a fetal abnormality, the method comprising:

obtaining a maternal sample;

sequencing at least a portion of nucleic acids in the sample;

comparing obtained sequence information to a reference sequence;

identifying fetal nucleic acid, if present, in the sample;

optionally, if fetal nucleic acid is present, determining whether the fetus has an abnormality.

2. The method of claim 1, wherein said reference sequence is selected from a maternal reference sequence, a fetal reference sequence, or a consensus human genomic sequence.

3. The method of claim 2, wherein said maternal reference sequence is selected from a sequence obtained from a buccal sample, a saliva sample, a urine sample, a breast nipple aspirate sample, a sputum sample, a tear sample, and an amniotic fluid sample.

4. The method of claim 1, wherein the sequencing reaction is a single molecule sequencing reaction.

5. The method of claim 4, wherein the single molecule sequencing reaction comprises sequencing by synthesis and/or sequencing by nanopore detection.

6. The method according to claim 1, wherein the maternal sample is a tissue or body fluid.

7. The method according to claim 6, wherein the body fluid is maternal blood, blood plasma, or serum.

8. The method according to claim 1, wherein the fetal nucleic acid is cell free circulating fetal nucleic acid.

9. The method according to claim 1, wherein prior to the sequencing step, the method further comprises enriching for fetal nucleic acid in the sample.

10. The method of claim 1, wherein said identifying step comprises a technique selected from sparse allele calling, targeted gene sequencing, identification of Y chromosomal material, enumeration, copy number analysis, and breakpoint analysis.

11. A method for determining whether a fetus has an abnormality, the method comprising:

obtaining a maternal sample comprising both maternal and fetal nucleic acids;

attaching a plurality of unique tags to nucleic acids in the sample, wherein each type of tag is associated with a different genomic region;

performing a sequencing reaction on the tagged nucleic acids to obtain tagged sequences; and

determining whether the fetus has an abnormality by quantifying the tagged sequences.

12. The method according to claim 11, wherein the different genomic region is at least a portion of a chromosome.

13. A method determining the presence of fetal nucleic acid, the method comprising:

obtaining a maternal sample;

sequencing nucleic acids in the sample, wherein said sequencing has an associated error rate;

determining whether fetal nucleic acid is present in the sample based at least in part on a quantitative measure of nucleic acid identified as fetal by said sequencing wherein the quantitative measure has a confidence level determined at least in part by the error rate.

14. A method for determining whether a fetus has an abnormality, the method comprising:

obtaining a maternal sample suspected to contain maternal and fetal nucleic acids;

sequencing nucleic acids in the sample;

comparing obtained sequence information to a reference sequence;

identifying regions of sequence match or mismatch between the obtained sequence information and the reference sequence;

confirming the presence of fetal nucleic acid in the sample; and

optionally determining whether the fetus has an abnormality based upon the results of the identifying step.

15. A method for determining whether fetal nucleic acid is present in a maternal sample, the method comprising:

obtaining a maternal sample suspected to include fetal nucleic acids; and

performing a sequencing reaction on the sample that is capable of detecting presence of at least a portion of a Y chromosome in the sample if such portion is present; and

determining that fetal nucleic acid is present in the sample.

16. The method according to claim 15, wherein the sequencing reaction is a single molecule sequencing reaction.

17. The method according to claim 15, wherein the maternal sample is a tissue or body fluid.

18. The method according to claim 17, wherein the body fluid is maternal blood, blood plasma, or serum.

19. The method according to claim 15, further comprising:

performing a quantitative analysis on the obtained sequences to detect presence of fetal nucleic acid if the Y chromosome is not detected in the sample.

20. A method for determining proper function of an assay used for detection of an abnormality in a fetus, the method comprising:

obtaining a maternal sample suspected to include fetal nucleic acids;

determining whether at least a portion of a Y chromosome is present in the sample; and

optionally performing a quantitative analysis on the obtained sequences to detect presence of nucleic acid from a normal female fetus if the Y chromosome is not detected in the sample, thereby determining that that the assay is functioning properly.

21. A method for determining whether a fetus has an abnormality, the method comprising:

obtaining a maternal sample comprising both maternal and fetal nucleic acids;

performing a sequencing reaction on the sample to obtain sequence information on nucleic acids in the sample;

comparing the obtained sequence information to sequence information from a reference genome, thereby determining whether the fetus has an abnormality;

detecting presence of at least a portion of a Y chromosome in the sample; and

distinguishing false negatives from true negatives if the Y chromosome is not detected in the sample.

22. The method according to claim 21, wherein distinguishing comprises:

performing a quantitative analysis selected from the group consisting of copy number analysis;

sparse allele calling;

targeted resequencing; and

inversion analysis.

23. A method for determining whether a fetus has an abnormality, the method comprising:

obtaining a maternal sample comprising both maternal and fetal nucleic acids;

performing a sequencing reaction on the sample to obtain sequence information on nucleic acids in the sample;

comparing the obtained sequence information to sequence information from a reference genome, thereby determining whether the fetus has an abnormality; and

distinguishing false negatives from true negatives.

24. The method according to claim 23, wherein distinguishing comprises:

assaying the sample for presence of at least a portion of a Y chromosome; and

optionally performing a quantitative analysis on the obtained sequences to detect presence of nucleic acid from a normal female fetus if the Y chromosome is not detected in the sample.

25. The method according to claim 24, wherein the quantitative analysis is accomplished by a technique selected from the group consisting of:

copy number analysis;

sparse allele calling;

targeted resequencing; and

breakpoint analysis.

26. The method according to claim 23, wherein the distinguishing comprises performing a quantitative analysis on the obtained sequences to detect presence of nucleic acid from a normal fetus.

27. The method according to claim 26, wherein the quantitative analysis is accomplished by a technique selected from the group consisting of copy number analysis;

sparse allele calling;

targeted resequencing; and

breakpoint analysis.

28. The method according to claim 23, wherein prior to the performing step, the method further comprises enriching for the fetal nucleic acids in the sample.

29. The method according to claim 23, wherein the sequencing reaction is a single molecule sequencing by synthesis reaction.

30. The method according to claim 23, wherein the maternal sample is a tissue or body fluid.

31. The method according to claim 30, wherein the body fluid is maternal blood, blood plasma, or serum.

32. The method according to claim 23, wherein the abnormality results from a chromosomal aberration.

33. The method according to claim 23, wherein the abnormality results from fetal aneuploidy.

34. The method according to claim 23, wherein the abnormality is selected from the group consisting of Down syndrome (trisomy of chromosome 21), Edward syndrome (trisomy of chromosome 18), and Patau syndrome (trisomy of chromosome 13).

35. A method for determining whether a fetus has an abnormality, the method comprising:

obtaining a maternal sample comprising both maternal and fetal nucleic acids;

performing a sequencing reaction on the sample to obtain sequence information on nucleic acids in the sample;

comparing the obtained sequence information to sequence information from a reference genome, thereby determining whether the fetus has an abnormality;

detecting presence of at least a portion of a Y chromosome in the sample; and

distinguishing false negatives from true negatives if the Y chromosome is not detected in the sample.

36. The method according to claim 35, wherein prior to the performing step, the method further comprises enriching for the fetal nucleic acids in the sample.

37. The method according to claim 35, wherein distinguishing comprises:

performing a quantitative analysis selected from the group consisting of;

copy number analysis;

sparse allele calling;

targeted resequencing; and

breakpoint analysis.

38. The method according to claim 35, wherein the comparing step comprises comparison of measured depth of sequencing coverage in a region of interest to expected depth of coverage in a diploid sample with the same total coverage.

39. The method according to claim 38, wherein the region of interest is an entire chromosome.

40. The method according to claim 38, wherein the region of interest is a part of a chromosome.

41. The method according to claim 38, wherein where the expected depth of coverage is determined by average coverage from a control population.

42. The method according to claim 38, wherein the expected depth of coverage is determined by comparison to majority of regions of the same sample.

43. The method according to claim 38, wherein the expected depth of coverage is determined by a model of the observed distribution of coverage values in the sample which partitions the space of coverage depth into ranges corresponding to different copy numbers.

44. A method for determining whether a fetus has an abnormality, the method comprising:

obtaining a maternal sample comprising both maternal and fetal nucleic acids;

attaching unique tags to nucleic acids in the sample, wherein each tag is associated with a different chromosome;

performing a sequencing reaction on the tagged nucleic acids to obtain tagged sequences; and

optionally determining whether the fetus has an abnormality by quantifying the tagged sequences.

45. The method according to claim 44, wherein the tags comprise unique nucleic acid sequences.

46. A method for determining whether fetal nucleic acid is present in a maternal sample, the method comprising:

obtaining a maternal sample suspected to include fetal nucleic acids;

selecting at least two unique k-mers for detection in the sample; and

determining whether fetal nucleic acid is present in the maternal sample based on the ratio of the unique k-mers.

47. The method according to claim 46, further comprising diagnosing an abnormality in the fetus based on analysis of the detected sequence.

48. The method according to claim 46, wherein the unique sequences comprise one or more single nucleotide polymorphisms.

49. The method according to claim 46, further comprising the step of determining an amount of each of said unique k-mers.

50. The method according to claim 46, wherein said determining step comprises sequencing at least a portion of said nucleic acid.

51. The method according to claim 46, wherein the maternal sample is a tissue or body fluid.

52. The method according to claim 46, wherein the body fluid is maternal blood, blood plasma, or serum.

53. A method for identifying a fetal abnormality, the method comprising the steps of:

sequencing nucleic acid from a maternal sample;

distinguishing fetal nucleic acid from maternal nucleic acid;

confirming presence or absence of said fetal nucleic acid; and

optionally identifying an abnormality based upon a sequence variant in said fetal nucleic acid.

54. The method of claim 53, wherein said confirming step comprises identifying false negative results in said distinguishing step.

55. The method of claim 53, wherein said confirming step comprises identifying false positive results in said distinguishing step.

56. The method of claim 53, wherein said confirming step comprises identification of nucleic acid associated with a Y chromosome.

57. The method of claim 53, wherein said distinguishing step comprises comparing obtained sequence to one or more reference sequence.

58. The method of claim 57, wherein said reference sequence is selected from a human genome consensus sequence, a maternal reference sequence, a paternal reference sequence, and a fetal reference sequence.

59. A method for analyzing nucleic acids in a sample, the method comprising:

sequencing a sample to obtain nucleic acid sequence information;

determining an amount of GC bias in the sequence information;

correcting the sequence information to account for the GC bias; and

analyzing the corrected information.

60. The method according to claim 59, wherein determining comprises:

partitioning the sequence information into bins; and

measuring a correlation between a number of chromosome counts in each bin and its GC content, wherein a statistically significant negative or positive correlation indicates existence of GC bias.

61. The method according to claim 59, wherein correcting comprises:

selecting a subset of bins within a given range such that average GC content per chromosome is equalized.

62. The method according to claim 59, wherein correcting comprises:

modeling a correlation between GC content and chromosome counts across a set of bins; and

adjusting the effect of the GC bias by subtracting the GC-dependent component from the chromosome count in each bin based upon the modeling.

63. The method according to claim 59, wherein correcting comprises:

obtaining an average sequence coverage per bin over a number of controls; and

dividing the obtained coverage in the sample by the mean of the controls.

64. The method according to claim 59, wherein the sample is a tissue or body fluid.

65. The method according to claim 64, wherein the sample is suspected to contain fetal nucleic acid.

66. The method according to claim 65, wherein the body fluid is maternal blood, blood plasma, or serum.

67. The method according to claim 59, wherein sequencing is single molecule sequencing and/or sequencing by nanopore detection.

68. The method according to claim 67, wherein single molecule sequencing is sequencing by synthesis and/or sequencing by nanopore detection.

69. A method for identifying a fetal abnormality, the method comprising:

obtaining a maternal sample;

sequencing at least a portion of nucleic acids in the sample to obtain sequence information;

determining an amount of GC bias in the sequence information;

correcting the sequence information to account for the GC bias;

comparing corrected sequence information to a reference sequence;

identifying fetal nucleic acid, if present, in the sample;

optionally, if fetal nucleic acid is present, determining whether the fetus has an abnormality.

70. The method according to claim 69, wherein the reference sequence is selected from a maternal reference sequence, a fetal reference sequence, or a consensus human genomic sequence.

71. The method according to claim 69, wherein said maternal reference sequence is selected from a sequence obtained from a buccal sample, a saliva sample, a urine sample, a breast nipple aspirate sample, a sputum sample, a tear sample, and an amniotic fluid sample.

72. The method according to claim 69, wherein sequencing is single molecule sequencing.

73. The method according to claim 72, wherein single molecule sequencing comprises sequencing by synthesis and/or sequencing by nanopore detection.

74. The method according to claim 69, wherein the maternal sample is a tissue or body fluid.

75. The method according to claim 74, wherein the body fluid is maternal blood, blood plasma, or serum.

76. The method according to claim 69, wherein the fetal nucleic acid is cell free circulating fetal nucleic acid.

77. The method according to claim 69, wherein prior to the sequencing step, the method further comprises enriching for fetal nucleic acid in the sample.

78. The method according to claim 69, wherein the identifying step comprises a technique selected from sparse allele calling, targeted gene sequencing, identification of Y chromosomal material, enumeration, copy number analysis, and inversion analysis.

79. The method according to claim 69, wherein determining comprises:

partitioning the sequence information into bins; and

measuring a correlation between a number of chromosome counts in each bin and its GC content, wherein a statistically significant negative or positive correlation indicates existence of GC bias.

80. The method according to claim 69, wherein correcting comprises:

selecting a subset of bins within a given range such that average GC content per chromosome is equalized.

81. The method according to claim 69, wherein correcting comprises:

modeling a correlation between GC content and chromosome counts across a set of bins; and

adjusting the effect of the GC bias by subtracting the GC-dependent component from the chromosome count in each bin based upon the modeling.

82. The method according to claim 69, wherein correcting comprises:

obtaining an average sequence coverage per bin over a number of controls and

dividing the obtained coverage in the sample by the mean of the controls.

83. The method according to claim 69, wherein the determining comprises:

comparing measured depth of coverage in chromosome regions to a normal control that was processed with the sample.