Methods for the diagnosis of fetal abnormalities

• US 8,137,912 B2
• Filed: 06/14/2007
• Issued: 03/20/2012
• Est. Priority Date: 06/14/2006
• Status: Active Grant

• Current Assignee: Gpb Scientific, LLC, Verinata Health Incorporated (Illumina Incorporated), Mehmet Toner
• Original Assignee: Gpb Scientific, LLC, Verinata Health Incorporated (Illumina Incorporated), The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Kapur, Ravi, Toner, Mehmet, Shoemaker, Daniel, Davis, Ronald W., Stoughton, Roland
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US11/763,245
• Publication Number:
  US 20080138809A1
• Time in Patent Office: 1,741 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2800/385 Congenital anomalies
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

The present invention relates to methods for detecting, enriching, and analyzing rare cells that are present in the blood, e.g. fetal cells. The invention further features methods of analyzing rare cell(s) to determine the presence of an abnormality, disease or condition in a subject, e.g. a fetus by analyzing a cellular sample from the subject.

Claims

1. A method for diagnosing the presence or absence of a fetal aneuploidy in a maternal blood sample including fetal and maternal genomic DNA, the method comprising:

(a) enriching the maternal blood sample for fetal genomic DNA to produce an enriched sample comprising fetal and maternal genomic DNA;

(b) obtaining fetal and maternal genomic DNA from the enriched sample;

(c) conducting whole genome amplification of both the fetal and maternal genomic DNA to obtain amplified fetal and amplified maternal genomic DNA molecules;

(d) performing ultra-deep sequencing of the amplified fetal and amplified maternal genomic DNA molecules of step (c) to produce partial or complete genome sequences corresponding to the amplified fetal and amplified maternal genomic DNA molecules, wherein the ultra-deep sequencing comprises sequencing at least a million of said amplified fetal and amplified maternal genomic DNA molecules in parallel; and

(e) analyzing the genome sequences of the amplified fetal and amplified maternal genomic DNA molecules of step (d) to count chromosomes without performing a single nucleotide polymorphism (SNP) analysis or a short tandem repeat (STR) analysis and thereby determine the presence or absence of said fetal aneuploidy.

2. The method of claim 1, wherein said sequencing comprises sequencing-by-synthesis.

3. The method of claim 1, wherein said sequencing comprises single molecule sequencing-by-synthesis.

4. The method of claim 1, wherein said sequencing comprises pyrosequencing.

5. The method of claim 4, wherein said sequencing comprises using a fiber optic plate that transmits a chemiluminescent signal generated by the sequencing reaction.

6. The method of claim 1, wherein said aneuploidy comprises trisomy.

7. The method of claim 6, wherein said trisomy comprises trisomy 13, 18, or 21.

8. The method of claim 1, wherein said sequencing comprises detection of nucleotide incorporation in substantially real time or real time.

9. The method of claim 1, wherein said sequencing generates at least 100,000 sequence reads per hour.

10. The method of claim 1, wherein said sequencing comprises obtaining sequence reads, and wherein there are at least 50 bases per read.

11. The method of claim 1, wherein said sequencing comprises using bridge amplification to generate clonal single molecule arrays.

12. The method of claim 1, wherein said whole genome amplification comprises multiple displacement amplification.

13. The method of claim 1, wherein said whole genome amplification comprises polymerase chain reaction (PCR) amplification.

14. The method of claim 13, wherein said PCR amplification comprises amplification with outer primers.

26. The method of claim 2, wherein said aneuploidy comprises monosomy, trisomy, tetrasomy, or pentasomy of one or more chromosomes.

27. The method of claim 26, wherein step (d) produces partial genome sequences corresponding to the amplified fetal and amplified maternal genomic DNA molecules.

28. The method of claim 27, wherein said trisomy ccmprises trisomy 13, 18, or 21.

15. A method for diagnosing the presence or absence of a fetal aneuploidy in a maternal blood sample including fetal and maternal genomic DNA, the method comprising:

(a) enriching the maternal blood sample for fetal genomic DNA to produce an enriched sample comprising fetal and maternal genomic DNA;

(b) obtaining fetal and maternal genomic DNA from the enriched sample;

(c) conducting whole genome amplification of both the fetal and maternal genomic DNA to obtain amplified fetal and amplified maternal genomic DNA molecules;

(d) performing ultra-deep sequencing of the amplified fetal and amplified maternal genomic DNA molecules of step (c) to produce partial or complete genome sequences corresponding to the amplified fetal and amplified maternal genomic DNA molecules, wherein the ultra-deep sequencing comprises conducting amplification of the amplified fetal and amplified maternal genomic DNA molecules of step (c) to produce at least a million clusters of immobilized fetal and maternal genomic DNA molecules in a flow cell and sequencing the clusters of said DNA molecules simultaneously in a sequencing reaction; and

(e) analyzing the genome sequences of the amplified fetal and amplified maternal genomic DNA molecules of step (d) to count chromosomes without performing a single nucleotide polymorphism (SNP) analysis or a short tandem repeat (STR) analysis and thereby determine the presence or absence of said fetal aneuploidy.

16. The method of claim 15, wherein said sequencing comprises sequencing-by-synthesis.

17. The method of claim 15, wherein said sequencing reaction of step (d) is a sequencing-by-synthesis reaction using four fluorescently labeled modified nucleotides possessing termination properties, wherein each cycle of the sequencing-by-synthesis reaction occurs in the presence of all four nucleotides, and wherein laser excitation captures an image of emitted fluorescence from each cluster in the flow cell for each cycle of the reaction.

18. The method of claim 15, wherein said aneuploidy comprises trisomy.

19. The method of claim 15, wherein said sequencing comprises detection of nucleotide incorporation in substantially real time or real time.

20. The method of claim 15, wherein said sequencing generates at least 100,000 sequence reads per hour.

21. The method of claim 15, wherein said sequencing comprises obtaining sequence reads, and wherein there are at least 50 bases per read.

22. The method of claim 15, wherein the whole genome amplification comprises multiple displacement amplification.

23. The method of claim 15, wherein said whole genome amplification comprises polymerase chain reaction (PCR) amplification.

29. The method of claim 17, wherein said aneuploidy comprises monosomy, trisomy, tetrasomy, or pentasomy of one or more chromosomes.

30. The method of claim 29, wherein step (d) produces partial genome sequences corresponding to the amplified fetal and amplified maternal genomic DNA molecules.

31. The method of claim 30, wherein said trisomy comprises trisomy 13, 18, or 21.

24. A method for determining the presence or absence of a fetal aneuploidy in a maternal blood sample including fetal and maternal genomic DNA, the method comprising:

(a) enriching the maternal blood sample for fetal genomic DNA to produce an enriched sample comprising fetal and maternal genomic DNA;

(b) obtaining fetal and maternal genomic DNA from the enriched sample;

(c) conducting whole genome amplification of both the fetal and maternal genomic DNA to obtain amplified fetal and amplified maternal genomic DNA molecules;

(d) performing ultra-deep sequencing of the amplified fetal and amplified maternal genomic DNA molecules of step (c) to produce partial or complete genome sequences corresponding to the amplified fetal and amplified maternal genomic DNA molecules, wherein the ultra-deep sequencing comprises sequencing at least a million of said amplified fetal and amplified maternal genomic DNA molecules in parallel; and

(e) analyzing the genome sequences of the amplified fetal and amplified maternal genomic DNA molecules of step (d) to count chromosomes without determining a ratio of the amounts of maternal to paternal alleles and thereby determine the presence or absence of said fetal aneuploidy.

32. The method of claim 24, wherein said sequencing comprises sequencing-by-synthesis and said aneuploidy comprises monosomy, trisomy, tetrasomy, or pentasomy of one or more chromosomes.

33. The method of claim 32, wherein step (d) produces partial genome sequences corresponding to the amplified fetal and amplified maternal genomic DNA molecules.

34. The method of claim 33, wherein said trisomy comprises trisomy 13, 18, or 21.

25. A method for determining the presence or absence of a fetal aneuploidy in a maternal blood sample including fetal and maternal genomic DNA, the method comprising:

(a) enriching the maternal blood sample for fetal genomic DNA to produce an enriched sample comprising fetal and maternal genomic DNA;

(b) obtaining fetal and maternal genomic DNA from the enriched sample;

(c) conducting whole genome amplification of both the fetal and maternal genomic DNA to obtain amplified fetal and amplified maternal genomic DNA molecules;

(d) performing ultra-deep sequencing of the amplified fetal and amplified maternal genomic DNA molecules of step (c) to produce partial or complete genome sequences corresponding to the amplified fetal and amplified maternal genomic DNA molecules, wherein the ultra-deep sequencing comprises conducting amplification of the amplified fetal and amplified maternal genomic DNA molecules of step (c) to produce at least a million clusters of immobilized fetal and maternal genomic DNA molecules in a flow cell and sequencing the clusters of said DNA molecules simultaneously in a sequencing reaction; and

(e) analyzing the genome sequences of the amplified fetal and amplified maternal genomic DNA molecules of step (d) to count chrommomes without determining a ratio of the amounts of maternal to paternal alleles and thereby determine the presence or absence of said fetal aneuploidy.

35. The method of claim 25, wherein said sequencing comprises sequencing-by-synthesis and said aneuploidy comprises monosomy, trisomy, tetrasomy, or pentasomy of one or more chromosomes.

36. The method of claim 35, wherein step (d) produces partial genome sequences corresponding to the amplified fetal and amplified maternal genomic DNA molecules.

37. The method of claim 36, wherein said trisomy comprises trisomy 13, 18, or 21.