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  • errataqwerty errataqwerty Jan 31, 2013 10:18 AM Flag

    New Sequenom patent

    Title:METHODS FOR DETECTING FETAL NUCLEIC ACIDS AND DIAGNOSING FETAL ABNORMALITIES
    Document Type and Number:United States Patent Application 20130022977 Kind Code:A1

    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.

    Inventors:Lapidus, Stanley (Bedford, NH, US)
    Thompson, John F. (Warwick, RI, US)
    Lipson, Doron (Chestnut Hill, MA, US)
    Milos, Patrice (Cranston, RI, US)
    Efcavitch, William J. (San Carlos, CA, US)
    Letovsky, Stanley (Milton, MA, US)
    Application Number:13/619039
    Publication Date:01/24/2013
    Filing Date:09/14/2012
    View Patent Images:Download PDF 20130022977
    PDF help Export Citation:Click for automatic bibliography generation

    Assignee:SEQUENOM, INC (San Diego, CA, US)
    Primary Class:435/6.11
    Other Classes:977/924
    International Classes:C12Q1/68; B82Y15/00

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    • I just searched the USPTO's website using their quick search functions for issued patents. i searched using the application number, inventor, title, and assignee....and no such patent was found.

      Sentiment: Strong Sell

      • 2 Replies to n_s_sherlock
      • You my friend are no sherlock -

        What is claimed is:

        1. 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, wherein the determining comprises: partitioning the genome into bins; and measuring a correlation between a number of counts in each bin and its GC content, wherein a statistically significant negative or positive correlation indicates existence of GC bias; correcting the sequence information to account for the GC bias; comparing corrected sequence information to a reference sequence; identifying fetal nucleic acid in the sample; determining whether the fetus has an abnormality from the fetal nucleic acid sequence.

        2. The method according to claim 1, wherein the reference sequence is selected from the group consisting of a maternal reference sequence, a fetal reference sequence, and a consensus human genomic sequence.

        3. The method according to claim 1, 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 according to claim 1, wherein sequencing is single molecule sequencing.

        5. The method according to claim 4, wherein single molecule sequencing 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 according to claim 1, 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.

        11. The method according to claim 1, wherein correcting comprises: selecting a subset of bins within a given range such that average GC content per chromosome is equalized.

        12. The method according to claim 1, 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.

        13. The method according to claim 1, 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.

        14. The method according to claim 1, wherein the determining comprises: comparing measured depth of coverage in chromosome regions to a normal control that was processed with the sample.

        15. The method according to claim 1, wherein the sample is suspected to contain fetal nucleic acid.

        Description:RELATED APPLICATION
        This application is a divisional of U.S. patent application Ser. No. 12/727,824, filed Mar. 19, 2010, which is a continuation-in-part of U.S. patent application Ser. No. 12/709,057, filed Feb. 19, 2010, which is a continuation-in-part of U.S. patent application Ser. No. 11/067,102, filed Feb. 25, 2005, which claims priority to and the benefit of U.S. patent application No. 60/548,704, filed Feb. 27, 2004, the contents of each of which are incorporated by reference herein in their entireties.

        FIELD OF THE INVENTION
        The invention generally relates to methods for detecting fetal nucleic acids and methods for diagnosing fetal abnormalities.

        BACKGROUND
        Fetal aneuploidy (e.g., Down syndrome, Edward syndrome, and Patau syndrome) and other chromosomal aberrations affect 9 of 1,000 live births (Cunningham et al. in Williams Obstetrics, McGraw-Hill, New York, p. 942, 2002). Chromosomal abnormalities are generally diagnosed by karyotyping of fetal cells obtained by invasive procedures such as chorionic villus sampling or amniocentesis. Those procedures are associated with potentially significant risks to both the fetus and the mother. Noninvasive screening using maternal serum markers or ultrasound are available but have limited reliability (Fan et al., PNAS, 105(42):16266-16271, 2008).

        Since the discovery of intact fetal cells in maternal blood, there has been intense interest in trying to use those cells as a diagnostic window into fetal genetics (Fan et al., PNAS, 105(42):16266-16271, 2008). The discovery that certain amounts (between about 3% and about 6%) of cell-free fetal nucleic acids exist in maternal circulation has led to the development of noninvasive PCR based prenatal genetic tests for a variety of traits. A problem with those tests is that PCR based assays trade off sensitivity for specificity, making it difficult to identify particular mutations. Further, due to the stochastic nature of PCR, a population of molecules that is present in a small amount in the sample often is overlooked, such as fetal nucleic acid in a sample from a maternal tissue or body fluid. In fact, if rare nucleic acid is not amplified in the first few rounds of amplification, it becomes increasingly unlikely that the rare event will ever be detected.

        Additionally, there is also the potential that fetal nucleic acid in a maternal sample is degraded and not amendable to PCR amplification due to the small size of the nucleic acid.

        There is a need for methods that can noninvasively detect fetal nucleic acids and diagnose fetal abnormalities.

        SUMMARY
        The invention generally relates to methods for detecting fetal nucleic acids and for diagnosing fetal abnormalities. Methods of the invention take advantage of sequencing technologies, particularly single molecule sequencing-by-synthesis technologies, to detect fetal nucleic acid in maternal tissues or body fluids. Methods of the invention are highly sensitive and allow for the detection of the small population of fetal nucleic acids in a maternal sample, generally without the need for amplification of the nucleic acid in the sample.

        Methods of the invention involve sequencing nucleic acid obtained from a maternal sample and distinguishing between maternal and fetal nucleic acid. Distinguishing between maternal and fetal nucleic acid identifies fetal nucleic acid, thus allowing the determination of abnormalities based upon sequence variation. Such abnormalities may be determined as single nucleotide polymorphisms, variant motifs, inversions, deletions, additions, or any other nucleic acid rearrangement or abnormality.

        Methods of the invention are also used to determine the presence of fetal nucleic acid in a maternal sample by identifying nucleic acid that is unique to the fetus. For example, one can look for differences between obtained sequence and maternal reference sequence; or can involve the identification of Y chromosomal material in the sample. The maternal sample may be a tissue or body fluid. In particular embodiments, the body fluid is maternal blood, maternal blood plasma, or maternal serum.

        The invention also provides a way to confirm the presence of fetal nucleic acid in a maternal sample by, for example, looking for unique sequences or variants.

        The sequencing reaction may be any sequencing reaction. In particular embodiments, the sequencing reaction is a single molecule sequencing reaction. Single-molecule sequencing is shown for example in Lapidus et al. (U.S. Pat. No. 7,169,560), Lapidus et al. (U.S. patent application number 2009/0191565), Quake et al. (U.S. Pat. No. 6,818,395), Harris (U.S. Pat. No. 7,282,337), Quake et al. (U.S. patent application number 2002/0164629), and Braslaysky, et al. PNAS (USA), 100: 3960-3964 (2003), the contents of each of these references is incorporated by reference herein in its entirety.

        Briefly, in some implementations, a single-stranded nucleic acid (e.g., DNA or cDNA) is hybridized to oligonucleotides attached to a surface of a flow cell. The oligonucleotides may be covalently attached to the surface or various attachments other than covalent linking as known to those of ordinary skill in the art may be employed. Moreover, the attachment may be indirect, e.g., via the polymerases of the invention directly or indirectly attached to the surface. The surface may be planar or otherwise, and/or may be porous or non-porous, or any other type of surface known to those of ordinary skill to be suitable for attachment. The nucleic acid is then sequenced by imaging or otherwise detecting the polymerase-mediated addition of fluorescently-labeled nucleotides incorporated into the growing strand surface oligonucleotide, at single molecule resolution. In certain embodiments, the nucleotides used in the sequencing reaction are not chain terminating nucleotides.

        Because the Y chromosome will only be present if the fetal nucleic acid is from a male, methods of the invention may further include performing a quantitative assay on the obtained sequences to detect presence of fetal nucleic acid if the Y chromosome is not detected in the sample. Such quantitative assays include copy number analysis, sparse allele calling, targeted resequencing, and breakpoint analysis.

        The ability to detect fetal nucleic acid in a maternal sample allows for development of a noninvasive diagnostic assay to #$%$ whether a fetus has an abnormality. Thus, another aspect of the invention provides noninvasive methods for determining whether a fetus has an abnormality. Methods of the invention may involve obtaining a sample including 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.

        An important aspect of a diagnostic assay is the ability of the assay to distinguish between false negatives (no detection of fetal nucleic acid when in fact it is present) and true negatives (detection of nucleic acid from a healthy fetus). Methods of the invention provide this capability. If the Y chromosome is detected in the maternal sample, methods of the invention assure that the assay is functioning properly, because the Y chromosome is associated only with males and will be present in a maternal sample only if male fetal nucleic acid is present in the sample. Some methods of the invention provide for further quantitative or qualitative analysis to distinguish between false negatives and true negatives, regardless of the ability to detect the Y chromosome, particularly for samples including normal nucleic acids from a female fetus. Such additional quantitative analysis may include copy number analysis, sparse allele calling, targeted resequencing, and breakpoint analysis.

        Another aspect of the invention provides methods for determining whether a fetus has an abnormality, including obtaining a maternal sample comprising both maternal and fetal nucleic acids; attaching unique tags to nucleic acids in the sample, in which each tag is associated with a different chromosome; 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. In certain embodiments, the tags include unique nucleic acid sequences.

        Sentiment: Strong Buy

      • Go to freepatentsonline

    • Do you have a link for this mnew patent? Thanks

 
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