WO2014019275A1

WO2014019275A1 - Noninvasive detection of fetal health status

Info

Publication number: WO2014019275A1
Application number: PCT/CN2012/081435
Authority: WO
Inventors: Wei Wang; Yingrui Li; Yu Xu; Ping Liu; Guangqing SUN; Xi Zeng
Original assignee: Bgi Shenzhen Co., Limited; Bgi Shenzhen
Priority date: 2012-07-31
Filing date: 2012-09-14
Publication date: 2014-02-06
Also published as: HK1208890A1; CN104619858A; CN104619858B

Abstract

The present invention discloses a method to identify biomarkers of fetal health status from foreign organism such as a virus, a bacterium, a fungus and a parasite, and uses thereof for noninvasive determination of fetal health status. And the present invention also provides a system and a kit which are used in the method.

Description

NONINVASIVE DETECTION OF FETAL HEALTH STATUS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and benefits of Chinese Patent Application Serial No. 201210271283.4, entitled "Method for determining fetal health status" filed July 31, 2012, the entire contents of which are herein incorporated by reference for all purposes.

Field

[0002] The disclosure relates to noninvasive methods for the determination of the health status of a fetus. More particularly, this disclosure relates to methods for the detection of fetal health status by assessing biomarkers related to a foreign organism in a biological sample from a pregnant female.

Background

[0003] Conventional prenatal diagnostic methods with invasive procedures, such as chorionic villus sampling and amniocentesis, carry potential risks for both fetuses and mothers.

Noninvasive screening of fetal aneuploidy using maternal serum markers and ultrasound are available but have limited sensitivity and specificity. Kagan, et al., Human Reproduction (2008) 23:1968-1975; Malone, et al., N Engl J Med (2005) 353:2001-2011.

[0004] With the development of DNA sequencing technology, noninvasive detection technology has been widely applied in clinical diagnosis of some diseases, to provide a new and effective method. Cell free fetal DNA can be detected in maternal peripheral blood. Lo et al., Lancet 350:485 487 (1997); Lo et al., Am. J. hum. Genet. 62:768-775 (1998). Though the quantity of fetal DNA in maternal peripheral blood is low, a new generation of sequencing techniques has been successfully applied to detect genetic abnormalities for fetal disease diagnosis.

[0005] Viruses, bacteria, parasites, fungi and other pathogens after infection with pregnant women can have adverse consequences on the fetus, such as abortion, stillbirth, and birth defects. Feng & Shen, Obstetrics and Gynecology, (People's Medical Publishing House, pp. 150-154). Some of the common clinical infection pathogens during pregnancy are: HBV, HCV, HIV, TORCH {Toxoplasma gondii, rubella virus, cytomegalovirus, and Herpes simplex virus), syphilis. [0006] At present, detection methods for HBV, HCV, HIV, TORCH, and syphilis commonly used in hospitals are mainly through assessment of the peripheral blood of pregnant women with ELISA based methods for the detection of antibodies against the pathogens, which indirectly reflect the pregnant women's infection status. These methods are generally not effective for detecting fetal infection. Some of the limitations of the methods are: 1) that only a few known pathogens can be detected; 2) that each pathogen requires a corresponding antibody detection reagent; 3) that the amount of blood needed increases with the number of pathogens to be detected; 4) that the methods are time consuming; and 5) that the methods do not favor batch testing.

[0007] The most fatal drawback of the ELISA based methods is that they are not suitable for a judgment of fetal infection. For example, TORCH detection is mainly determined by ELISA based methods for assessment of IgM and IgG in maternal peripheral blood, followed by determining the infection state of the pregnant women and inferring the possible effects on the fetus. Determining fetal infection sometimes requires further examinations, such as

amniocentesis for the detection of IgM or PCR of fetal nucleic acid to determine whether the fetus is infected by the pathogen. However, amniocentesis has a 1% miscarriage risk, which makes it difficult to be accepted by pregnant women and their families. Because IgM and IgG indirectly reflect the infection status of the body, and the antibodies are only produced from the pathogen at a certain period of time, there is a false negative probability. Additionally, because the hospitals use different detection kits, and there lacks a uniform standard of quality control, test results from different hospitals may not be consistent. Inconsistence in test results reduces patients' trust and acceptance to the test, which makes hospitals hesitant in using ELISA based TORCH screening methods of pregnant women.

[0008] While the most sensitive method to directly detect the presence of pathogens is pathogen culture, it is time consuming and has a higher failure rate. Therefore, pathogen culture has not seen large-scale clinical application.

Summary of the Invention

[0009] The current disclosure is directed to methods for noninvasive detection of fetal health status by assessing one or more polynucleotide, polypeptide and/or small molecule from a foreign organism in a biological sample from a pregnant female.

[0010] Therefore, in one aspect, provided herein is a method for determining the status of health of a fetus, which method comprises: a) assessing the identity and/or level of one or more polynucleotide, polypeptide and/or small molecule from a foreign organism in a biological sample from a pregnant female; b) analyzing said identity and/or level of said foreign

polynucleotide, polypeptide and/or small molecule; and c) determining the status of health of said fetus, wherein the status of health of said fetus is not HBV infection. In some embodiments, the status of health of the fetus is not an infection by the foreign organism. In some embodiments, the small molecule may be related to host metabolism.

[0011] In one embodiment the fetus is a human fetus. In another embodiment the analysis is conducted using one or more normal controls. In yet another embodiment the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material. In still another embodiment the foreign organism is selected from the group consisting of: a virus, a bacterium, a fungus and a parasite. In a further embodiment the status of health is selected from the group consisting of: genetic abnormality, and developmental abnormality. In some embodiments, the biological sample is obtained from at least 2, 5, 10, 20, 50, 100, 200, 500 or 1000 pregnant females.

[0012] In one embodiment, the genetic abnormality is a fetal aneuploidy. In another embodiment the fetal aneuploidy is a disorder for an autosome selected from the group consisting of trisomy 13, 18 and 21. In some embodiments, the fetal aneuploidy is a disorder for a sex chromosome selected from the group consisting of XO, XXX, XXY and XYY.

[0013] In a second aspect, provided herein is a method for identifying a bio marker for the status of health of a fetus, which method comprises: a) assessing the identity and/or level of a polynucleotide, polypeptide and/or small molecule from a foreign organism in a biological sample from one or more pregnant female; b) conducting statistical analysis of said identity and/or level of said foreign polynucleotide, polypeptide and/or small molecule; and c) identifying said non-human polynucleotide, polypeptide or small molecule as a biomarker for said status of health if statistically significant association is established, wherein the status of health of said fetus is not HBV infection, or the biological sample is obtained from at least 100 pregnant females. In some embodiments, the status of health of the fetus is not an infection by the foreign organism. In some embodiments, the small molecule may be related to host metabolism.

[0014] In some embodiments, the fetus is a human fetus. In some embodiments, the statistical analysis is conducted using one or more normal controls. In yet another embodiment the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material. In still another embodiment the foreign organism is selected from the group consisting of: a virus, a bacterium, a fungus and a parasite. In a further embodiment the status of health is selected from the group consisting of: genetic abnormality, and developmental abnormality.

[0015] In one embodiment, the genetic abnormality is a fetal aneuploidy. In another embodiment the fetal aneuploidy is a disorder for an autosome selected from the group consisting of trisomy 13, 18 and 21. In some embodiments, the fetal aneuploidy is a disorder for a sex chromosome selected from the group consisting of XO, XXX, XXY and XYY.

[0016] In some embodiments, the bio marker is selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedro virus, Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalomyocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37,

Choristoneura occidentalis granulo virus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6.

[0017] In some embodiments, the biological sample is obtained from at least 2, 5, 10, 20, 50, 100, 200, 500 or 1000 pregnant females.

[0018] In a third aspect, provided herein is a method for determining the status of health of a fetus, which method comprises: a) assessing the identity and/or level of one or more biomarkers identified using the methods disclosed herein in a biological sample from a pregnant female; b) analyzing said identity and/or level of said biomarkers; and c) determining the status of health of said fetus.

[0019] In some embodiments, the fetus is a human fetus. In some embodiments, the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material. In some embodiments, the biomarker is a polynucleotide. In some embodiments, step a) comprises purifying cell- free DNA from the biological sample. In some embodiments, the DNA is amplified. In some embodiments, the DNA is constructed into a library. In some embodiments, the DNA is sequenced. In some embodiments, the DNA is sequenced by high-throughput sequencing. In some embodiments, step b) comprises comparing the sequences obtained from step a) to the sequences of the biomarkers. In some embodiments, the sequences obtained from step a) that match human genomic sequences are removed before step b).

[0020] In a fourth aspect, provided in this disclosure is a panel of bio markers for determining trisomy 21 of a human fetus, which panel comprises two or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedro virus, Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalo myocarditis virus, Hepatitis C virus genotype 1,

Enterobacteria phage Ml 3, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6.

[0021] In a fifth aspect, provided herein is a method for determining the status of health of a fetus, which method comprises: a) assessing the identity and/or level of one or more biomarkers from a foreign organism by sequencing of a polynucleotide in a biological sample from a pregnant female; b) analyzing said identity and/or level of said one or more biomarkers; and c) determining the status of health of said fetus. In some embodiments, high-throughput sequencing is used by the method.

[0022] In some embodiments, the fetus is a human fetus. In some embodiments, the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material. In some embodiments, step a) comprises purifying cell-free DNA from the biological sample. In some embodiments, the DNA is amplified. In some embodiments, the DNA is constructed into a library. In some embodiments, step b) comprises comparing the sequences obtained from step a) to the sequences of the biomarkers. In some embodiments, the sequences obtained from step a) that match human genomic sequences are removed before step b). In some embodiments, the foreign organism is selected from the group consisting of: HBV, HCV, HIV, TORCH {Toxoplasma gondii, rubella virus, cytomegalovirus, and Herpes simplex virus), and syphilis.

[0023] In a sixth aspect, provided herein is a system for determining the status of health of a fetus, which system comprises: a) means for assessing the identity and/or level of one or more polynucleotide, polypeptide and/or small molecule from a foreign organism in a biological sample from a pregnant female; and b) means for analyzing said identity and/or level of said foreign polynucleotide, polypeptide and/or small molecule. In some embodiments, the status of health of said fetus is not HBV infection. In some embodiments, the small molecule may be related to host metabolism. In some embodiments, the means for assessing the identity and/or level of one or more polynucleotide, polypeptide and/or small molecule may be a sequencing device. In some embodiments, the means for analyzing said identity and/or level of said foreign polynucleotide, polypeptide and/or small molecule may be a computer.

[0024] In a sixth aspect, provided herein is a kit for determining the status of health of a fetus comprising: a) a biomarker disclosed herein; and b) an instruction on how to use said biomarker.

Brief Description of the Drawings

[0025] Figure 1 shows an exemplary process for identifying biomarkers for health status of a fetus.

Detailed Description of the Invention I. Definitions

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[0027] As used herein, the singular forms "a", "an", and "the" include plural references unless indicated otherwise. For example, "a" dimer includes one or more dimers.

[0028] The term "biomarker" or "marker" as used herein refers generally to a molecule, including a gene, protein, carbohydrate structure, glycolipid, or small molecule, the expression of which in or on a mammalian tissue or cell or secreted can be detected by known methods (or methods disclosed herein) and is predictive or can be used to predict (or aid prediction) for a health status of a fetus.

[0029] The term "chromosomal abnormality" refers to a deviation between the structure of the subject chromosome and a normal homologous chromosome. The term "normal" refers to the predominate karyotype or banding pattern found in healthy individuals of a particular species. A chromosomal abnormality can be numerical or structural, and includes but is not limited to aneuploidy, polyploidy, inversion, a trisomy, a monosomy, duplication, deletion, deletion of a part of a chromosome, addition, addition of a part of chromosome, insertion, a fragment of a chromosome, a region of a chromosome, chromosomal rearrangement, and translocation. A chromosomal abnormality can be correlated with presence of a pathological condition or with a predisposition to develop a pathological condition. As defined herein, a single nucleotide polymorphism ("SNP") is not a chromosomal abnormality.

[0030] Monosomy X (XO, absence of an entire X chromosome) is the most common type of Turner syndrome, occurring in 1 in 2500 to 1 in 3000 live-born girls (Sybert and McCauley N Engl J Med (2004) 351:1227-1238). XXY syndrome is a condition in which human males have an extra X chromosome, existing in roughly 1 out of every 1000 males (Bock,

Understanding Klinefelter Syndrome: A Guide for XXY Males and Their Families. NIH Pub. No. 93-3202 (1993)). XYY syndrome is an aneuploidy of the sex chromosomes in which a human male receives an extra Y chromosome, giving a total of 47 chromosomes instead of the more usual 46, affecting 1 in 1000 male births while potentially leading to male infertility (Aksglaede, et al., / Clin Endocrinol Metab (2008) 93:169-176).

[0031] Turner syndrome encompasses several conditions, of which monosomy X (XO, absence of an entire sex chromosome, the Barr body) is most common. Typical females have two X chromosomes, but in Turner syndrome, one of those sex chromosomes is missing. Occurring in 1 in 2000 to 1 in 5000 phenotypic females, the syndrome manifests itself in a number of ways. Klinefelter syndrome is a condition in which human males have an extra X chromosome. In humans, Klinefelter syndrome is the most common sex chromosome disorder and the second most common condition caused by the presence of extra chromosomes. The condition exists in roughly 1 out of every 1,000 males. XYY syndrome is an aneuploidy of the sex chromosomes in which a human male receives an extra Y chromosome, giving a total of 47 chromosomes instead of the more usual 46. This produces a 47, XYY karyotype. This condition is usually

asymptomatic and affects 1 in 1000 male births while potentially leading to male infertility.

[0032] Trisomy 13 (Patau syndrome), trisomy 18 (Edward syndrome) and trisomy 21 (Down syndrome) are the most clinically important autosomal trisomies and how to detect them has always been the hot topic. Detection of above fetal chromosomal aberration has great

significance in prenatal diagnosis (Ostler, Diseases of the eye and skin: a color atlas. Lippincott Williams & Wilkins. pp. 72. ISBN 9780781749992 (2004); Driscoll and Gross, N Engl J Med (2009) 360: 2556-2562; Kagan, et al., Human Reproduction (2008) 23:1968-1975). [0033] The term "reference unique reads" refers to fragments of a chromosome that have a unique sequence. Therefore, such fragments can be unambiguously assigned to a single chromosomal location. Reference unique reads of a chromosome may be constructed based on a published reference genome sequence, such as hgl8 or hgl9 for human being.

[0034] The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single- stranded deoxyribonucleic acid ("DNA"), as well as triple-, double- and single- stranded ribonucleic acid ("RNA"). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides

(containing D-ribose), including tRNA, rRNA, hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g. , peptide nucleic acids ("PNAs")) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, OR., as NeuGene^®) polymers, and other synthetic sequence- specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. Thus, these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' to P5' phosphoramidates, 2'-0-alkyl- substituted RNA, hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, "caps," substitution of one or more of the nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g. , methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g. , phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g. , aminoalkylphosphoramidates,

aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including enzymes (e.g., nucleases), toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelates (of, e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the

polynucleotide or oligonucleotide. [0035] As used herein, "sequence identity" or "identity" or "homology" in the context of two protein sequences (or nucleotide sequences) includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. The portion of the amino acid sequence or nucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. When percentage of sequence identity is used in reference to proteins it is recognised that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acids are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percentage sequence identity may be adjusted upwards to correct for the conservative nature of the substitutions. Sequences, which differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making these adjustments are well known to persons skilled in the art. The percentage is calculated by determining the number of positions at which the identical amino acid or nucleic acid base residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is give a score of zero, a conservative substitution is given a score between 0 and 1. The scoring of conservative substitutions is calculated, e.g.

according to the algorithm of Meyers and Miller (Computer Applic. Biol. ScL, 1998, 4, 11-17).

[0036] As used herein, the term "homologue" is used to refer to a nucleic acid which differs from a naturally occurring nucleic acid (i.e., the "prototype" or "wild-type" nucleic acid) by minor modifications to the naturally occurring nucleic acid, but which maintains the basic nucleotide structure of the naturally occurring form. Such changes include, but are not limited to: changes in one or a few nucleotides, including deletions (e.g., a truncated version of the nucleic acid) insertions and/or substitutions. A homologue can have enhanced, decreased, or

substantially similar properties as compared to the naturally occurring nucleic acid. A

homologue can be complementary or matched to the naturally occurring nucleic acid.

Homologues can be produced using techniques known in the art for the production of nucleic acids including, but not limited to, recombinant DNA techniques, chemical synthesis, etc. [0037] As used herein, "complementary or matched" means that two nucleic acid sequences have at least 50% sequence identity. Preferably, the two nucleic acid sequences have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity.

"Complementary or matched" also means that two nucleic acid sequences can hybridize under low, middle and/or high stringency condition(s).

[0038] As used herein, "substantially complementary or substantially matched" means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively, "substantially complementary or substantially matched" means that two nucleic acid sequences can hybridize under high stringency condition(s).

[0039] In general, the stability of a hybrid is a function of the ion concentration and temperature. Typically, a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Moderately stringent hybridization refers to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule. The hybridized nucleic acid molecules generally have at least 60% identity, including for example at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS, at 42°C. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in O.lx SSPE, and 0.1% SDS at 65°C.

[0040] Low stringency hybridization refers to conditions equivalent to hybridization in 10% formamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS at 22°C, followed by washing in lx SSPE, 0.2% SDS, at 37°C. Denhardt's solution contains 1% FicoU, 1% polyvinylpyrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art.

[0041] As used herein, high-throughput screening (HTS) refers to processes that test a large number of samples, such as samples of diverse chemical structures against disease targets to identify "hits" (see, e.g., Broach, et ah, High throughput screening for drug discovery, Nature, 384:14-16 (1996); Janzen, et al., High throughput screening as a discovery tool in the

pharmaceutical industry, Lab Robotics Automation 8261-265 (1996); Fernandes, P.B., Letter from the society president, J. Biomol. Screening 2:1 (1997); Burbaum, et al., New technologies for high-throughput screening, Curr. Opin. Chem. Biol. 1:72-78 (1997)). HTS operations are highly automated and computerized to handle sample preparation, assay procedures and the subsequent processing of large volumes of data.

[0042] It will be apparent to those skilled in the art that a number of different sequencing methods and variations can be used. In one embodiment, the sequencing is done using massively parallel sequencing. "Massively parallel sequencing" means techniques for sequencing millions of fragments of nucleic acids, e.g., using attachment of randomly fragmented genomic DNA to a planar, optically transparent surface and solid phase amplification to create a high density sequencing flow cell with millions of clusters, each containing -1,000 copies of template per sq. cm. These templates are sequenced using four-color DNA sequencing-by- synthesis technology. Massively parallel sequencing, such as that achievable on the 454 platform (Roche) (Margulies, et al., Nature (2005) 437:376-380), Illumina Genome Analyzer (or Solexa™ platform) or SOLiD System (Applied Biosystems) or the Helicos True Single Molecule DNA sequencing technology (Harris, et al., Science (2008) 320:106-109), the single molecule, real-time (SMRT™ ) technology of Pacific Biosciences, and nanopore sequencing (Soni and Meller, Clin Chem (2007) 53:1996-2001), allow the sequencing of many nucleic acid molecules isolated from a specimen at high orders of multiplexing in a parallel fashion (Dear, Brief Funct Genomic Proteomic (2003) 1:397-416). Each of these platforms sequences clonally expanded or even non-amplified single molecules of nucleic acid fragments. Commercially available sequencing equipment may be used in obtaining the sequence information of the polynucleotide fragments. The presently used sequencing is preferably carried out without a preamplification or cloning step, but may be combined with amplification-based methods in a microfluidic chip having reaction chambers for both PCR and microscopic template-based sequencing. Only about 30 bp of random sequence information are needed to identify a sequence as belonging to a specific human chromosome. Longer sequences can uniquely identify more particular targets. In the present case, a large number of 35 bp reads were obtained. Further description of a massively parallel sequencing method is found in Rogers and Ventner, Nature (2005) 437:326-327.

[0043] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used

interchangeably herein to refer to polymers of amino acids of any length, e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more amino acids. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non- amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0044] An "antibody" is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule, and can be an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE. IgY, which is the major antibody type in avian species such as chicken, is also included within the definition. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.

[0045] As used herein, the term "specifically binds" refers to the binding specificity of a specific binding pair. Recognition by an antibody of a particular target in the presence of other potential targets is one characteristic of such binding. Specific binding involves two different molecules wherein one of the molecules specifically binds with the second molecule through chemical or physical means. The two molecules are related in the sense that their binding with each other is such that they are capable of distinguishing their binding partner from other assay constituents having similar characteristics. The members of the binding component pair are referred to as ligand and receptor (anti-ligand), specific binding pair (SBP) member and SBP partner, and the like. A molecule may also be an SBP member for an aggregation of molecules; for example an antibody raised against an immune complex of a second antibody and its corresponding antigen may be considered to be an SBP member for the immune complex.

[0046] As used herein, a "small molecule related to host metabolism" refers to a low molecular weight organic compound which may be produced by a foreign organism such as a virus, a bacterium, a fungus or a parasite. Typically, it is not a polymer and has effect on host metabolism. Some molecules can bind with high affinity to a biopolymer such as protein, nucleic acid, or polysaccharide, and in addition alter the activity or function of the biopolymer.

Examples may include vitamins, antibiotics, and so on. [0047] As used herein, "biological sample" refers to any sample obtained from a living or viral source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can be obtained. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. For example, isolated nucleic acids that are amplified constitute a biological sample. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom.

[0048] It is understood that aspects and embodiments of the invention described herein include "consisting" and/or "consisting essentially of aspects and embodiments.

[0049] Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

II. Determining the Status of Health of a Fetus

[0050] Provided herein is a method for determining the status of health of a fetus, which method comprises: a) assessing the identity and/or level of one or more polynucleotide, polypeptide and/or small molecule from a foreign organism in a biological sample from a pregnant female; b) analyzing said identity and/or level of said foreign polynucleotide,

polypeptide and/or small molecule; and c) determining the status of health of said fetus, wherein the status of health of said fetus is not HBV infection. In some embodiments, the status of health of the fetus is not an infection by the foreign organism. The steps of operation may be carried out in no specific order. In some embodiments, the fetus may be a human fetus. In some

embodiments, the small molecule may be related to host metabolism.

[0051] To assess the identity and/or level of one or more polynucleotide, sequence

information of polynucleotide fragments can be obtained by sequencing template DNA obtained from a biological sample. In some embodiments, the template DNA contains both maternal DNA and fetal DNA. In some embodiments, the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material. In some embodiments, template DNA is obtained from blood of a pregnant female. Blood may be collected using any standard technique for blood drawing including but not limited to

venipuncture. For example, blood can be drawn from a vein from the inside of the elbow or the back of the hand. Blood samples can be collected from a pregnant female at any time during fetal gestation. For example, blood samples can be collected from human females at 1-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, or 40-44 weeks of fetal gestation, and preferably between 8-28 weeks of fetal gestation.

[0052] The polynucleotide fragments are aligned to reference genomic sequences based on the sequence information. A reference genomic sequence is used to obtain the reference unique reads. As used therein, the term "reference unique reads" refers to all the unique polynucleotide fragments that have been assigned to a specific genomic location based on a reference genomic sequence. In some embodiments, the reference unique reads have the same length of, for example, about 10, 12, 15, 20, 25, 30, 35, 40, 50, 100, 200, 300, 500, or 1000 bp.

[0053] In some embodiments, the identity and/or level of one or more polynucleotides is assessed according to a reference genomic sequence from a foreign organism. In some embodiments, the foreign organism is selected from the group consisting of: a virus, a bacterium, a fungus and a parasite. In some embodiments, reference genomic sequences from multiple foreign organisms are used for the assessment of the identity and/or level of the one or more polynucleotides.

[0054] In some embodiments, the template DNA may be used to construct a DNA library. In some embodiments, the template DNA may be subject to amplification and/or enrichment steps. In some embodiments, the template DNA may be subject to a solution based sequence

enrichment method (Nimblegen, as described in U.S. Patent Publication No. 20090105081), and/or a sequence complexity reduction method (Agilent, as described in U.S. Patent No.

7,867,703).

[0055] In some embodiments, it may be desirable to initially assign the polynucleotide fragments to the host genome to facilitate the identification of foreign sequences. For example, when the host is a human being, the human genomic sequence may be used as a reference genomic sequence to assign the polynucleotide fragments. In some other embodiments, human genome builds hgl8 or hg 19 may be used as the reference genomic sequence. In some embodiments, the polynucleotide fragments that fail to assign to the host genome are assessed according to one or more reference genomic sequences from one or more foreign organisms.

[0056] In some embodiments, the method may use reagents for detecting presence of polypeptides. Such reagents may be antibodies or other binding molecules that specifically bind to a polypeptide. In some embodiments, such antibodies or binding molecules may be capable of distinguishing a structural variation to the polypeptide as a result of polymorphism, and thus may be used for genotyping. The antibodies or binding molecules may be labeled with a detectable marker, such as, for example, a radioisotope, a fluorescent compound, a bio luminescent compound, a chemiluminescent compound, a metal chelator, an enzyme, or a particle. Other reagents for performing binding assays, such as ELISA, may be used in the method.

[0057] Any health status of a fetus may be determined by the methods disclosed herein. In some embodiments, the status of health of the fetus is not HBV infection. In some embodiments, the status of health of the fetus is not an infection by the foreign organism. In some embodiments, the status of health is selected from the group consisting of: genetic abnormality, and

developmental abnormality. Genetic abnormality may refer to chromosomal abnormality, mutation, etc. Developmental abnormality may refer to stillbirth, abortion, intrauterine fetal death, intrauterine growth retardation, intrauterine infection, early neonatal death and/or congenital anomalies. See, e.g., Kumari et al., J. Health Popul. Nutr. (2011) 29:77-80; Ishaque et al., BMC Public Health (2011) l l(Suppl 3):S3; Zhang et al., World J Gastroenterol. (1998) 4:61- 63; Wylie et al., PLoS ONE (2012) 7:e27735.

[0058] The methods can be used to detect fetal chromosomal abnormalities, and is especially useful for the detection of aneuploidy, polyploidy, monosomy, trisomy, trisomy 21, trisomy 13, trisomy 14, trisomy 15, trisomy 16, trisomy 18, trisomy 22, triploidy, tetraploidy, and sex chromosome abnormalities including XO, XXY, XYY, and XXX. Partial trisomies of 13q, 8p (8p23.1), 7q, distal 6p, 5p, 3q (3q25.1), 2q, lq (lq42.1 and lq21-qter), partial Xpand monosomy 4q35.1 have been reported, among others. For example, partial duplications of the long arm of chromosome 18 can result in Edwards syndrome in the case of a duplication of 18q21.1-qter (Mewar, et al., Am J Hum Genet. (1993) 53:1269-78).

III. Identification of Bio markers for the Status of Health of a Fetus

[0059] Also provided herein is a method for identifying a bio marker for the status of health of a fetus, which method comprises: a) assessing the identity and/or level of a polynucleotide, polypeptide and/or small molecule from a foreign organism in a biological sample from one or more pregnant female; b) conducting statistical analysis of said identity and/or level of said foreign polynucleotide, polypeptide and/or small molecule; and c) identifying said non-human polynucleotide, polypeptide or small molecule as a bio marker for said status of health if statistically significant association is established, wherein the status of health of said fetus is not HBV infection, or the biological sample is obtained from at least 100 pregnant females. In some embodiments, the status of health of the fetus is not an infection by the foreign organism. In some embodiments, the small molecule may be related to host metabolism.

[0060] To assess the identity and/or level of one or more polynucleotide, sequence

information of polynucleotide fragments can be obtained by sequencing template DNA obtained from a biological sample. In some embodiments, the biological sample is obtained from at least 2, 5, 10, 20, 50, 100, 200, 500 or 1000 pregnant females. In some embodiments, the template DNA contains both maternal DNA and fetal DNA. In some embodiments, the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material. In some embodiments, template DNA is obtained from blood of a pregnant female. Blood may be collected using any standard technique for blood drawing including but not limited to venipuncture. For example, blood can be drawn from a vein from the inside of the elbow or the back of the hand. Blood samples can be collected from a pregnant female at any time during fetal gestation. For example, blood samples can be collected from human females at 1-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, or 40-44 weeks of fetal gestation, and preferably between 8-28 weeks of fetal gestation.

[0061] The polynucleotide fragments are aligned to reference genomic sequences based on the sequence information. A reference genomic sequence is used to obtain the reference unique reads. As used therein, the term "reference unique reads" refers to all the unique polynucleotide fragments that have been assigned to a specific genomic location based on a reference genomic sequence. In some embodiments, the reference unique reads have the same length of, for example, about 10, 12, 15, 20, 25, 30, 35, 40, 50, 100, 200, 300, 500, or 1000 bp.

[0062] In some embodiments, the identity and/or level of one or more polynucleotides is assessed according to a reference genomic sequence from a foreign organism. In some

embodiments, the foreign organism is selected from the group consisting of: a virus, a bacterium, a fungus and a parasite. In some embodiments, reference genomic sequences from multiple foreign organisms are used for the assessment of the identity and/or level of the one or more polynucleotides.

[0063] In some embodiments, the template DNA may be used to construct a DNA library. In some embodiments, the template DNA may be subject to amplification and/or enrichment steps. In some embodiments, the template DNA may be subject to a solution based sequence

7,867,703).

[0064] In some embodiments, it may be desirable to initially assign the polynucleotide fragments to the host genome to facilitate the identification of foreign sequences. For example, when the host is a human being, the human genomic sequence may be used as a reference genomic sequence to assign the polynucleotide fragments. In some other embodiments, human genome builds hgl8 or hg 19 may be used as the reference genomic sequence. In some embodiments, the polynucleotide fragments that fail to assign to the host genome are assessed according to one or more reference genomic sequences from one or more foreign organisms.

[0065] In some embodiments, the method may use reagents for detecting presence of polypeptides. Such reagents may be antibodies or other binding molecules that specifically bind to a polypeptide. In some embodiments, such antibodies or binding molecules may be capable of distinguishing a structural variation to the polypeptide as a result of polymorphism, and thus may be used for genotyping. The antibodies or binding molecules may be labeled with a detectable marker, such as, for example, a radioisotope, a fluorescent compound, a bio luminescent compound, a chemiluminescent compound, a metal chelator, an enzyme, or a particle. Other reagents for performing binding assays, such as ELISA, may be used in the method.

[0066] In some embodiments, the comparison of the identity and/or level of the

polynucleotide, polypeptide, and/or small molecule between affected and normal control samples is conducted by a statistical test. Any suitable statistical tests known in the art can be used for performing the comparison. For example, Chi-square test of independence, Wilcoxon Mann Whitney U test (Wilcoxon rank sum test) and Unpaired samples t test may be used. Choosing the right statistics test is within the common knowledge of one of ordinary skill in the art.

[0067] In some embodiments, Fisher's exact test may be used to compare the difference between the samples having the health status and normal control samples. For example, the test formula may be as follows:

[0068] The read numbers may be calculated and compared between the samples having the health status and normal control samples for every virus in the database, followed by statistical analysis. For the virus named virus A, n denoted total sample number. n_*i denoted the sample having the health status number, ¾i denoted number of samples having the health status whose sequence reads uniquely mapped to virus A genome. Similarly, n_*2 denoted normal control samples number, ni₂ denoted number of normal control samples whose sequence reads can uniquely mapped to virus A genome. If the p-value<=0.05, this virus may be defined as being a biomarker for the health status.

IV. Determination of Fetal Health Status Using Biomarkers Identified Herein

[0069] Further provided herein is a method for determining the status of health of a fetus, which method comprises: a) assessing the identity and/or level of one or more biomarkers identified using the methods disclosed herein in a biological sample from a pregnant female; b) analyzing said identity and/or level of said biomarkers; and c) determining the status of health of said fetus.

[0070] In some embodiments, a cutoff value may be established for the biomarker. In some embodiments, a fetus having a level of the biomarker greater than the cutoff value may indicate that it has certain health status.

V. Biomarkers for Trisomy 21

[0071] In another aspect, provided here in is a panel of biomarkers for determining trisomy 21 of a human fetus, which panel comprises two or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedro virus, Pseudomonas phage F10,

Burkholderia phage Bcep22, Encephalo myocarditis virus, Hepatitis C virus genotype 1,

[0072] Any combinations of at least 2, 3, 4, 5, 10, or all 13 of the organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedro virus, Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalo myocarditis virus, Hepatitis C virus genotype 1,

Enterobacteria phage Ml 3, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6, are contemplated for the present disclosure. In some embodiments, the panel of biomarkers may comprise Agrotis ipsilon multiple nucleopolyhedro virus and one or more organisms selected from the group consisting of: Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalo myocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Pseudomonas phage F10, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Burkholderia phage Bcep22, Encephalomyocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Burkholderia phage Bcep22, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Pseudomonas phage F10, Encephalomyocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37,

Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Encephalomyocarditis virus, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Pseudomonas phage F10, Burkholderia phage Bcep22, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Hepatitis C virus genotype 1, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Pseudomonas phage F10,

Burkholderia phage Bcep22, Encephalomyocarditis virus, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37,

Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Enterobacteria phage M13, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Pseudomonas phage F10, Burkholderia phage Bcep22,

Encephalomyocarditis virus, Hepatitis C virus genotype 1, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Human herpesvirus 1, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalo myocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Human herpesvirus 7, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedro virus, Pseudomonas phage F10, Burkholderia phage Bcep22,

Encephalomyocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Cyprinid herpesvirus 3, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus,

Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalomyocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Staphylococcus phage 37, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalomyocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Choristoneura occidentalis granulovirus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Choristoneura occidentalis granulovirus, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalomyocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Helicoverpa zea nudivirus 2, and

Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise

Helicoverpa zea nudivirus 2, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalomyocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, and Enterobacteria phage Sf6. In some embodiments, the panel of biomarkers may comprise Enterobacteria phage Sf6, and one or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedrovirus, Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalo myocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulovirus, and Helicoverpa zea nudivirus 2.

VI. Detection of Fetal Health Status by Sequencing

[0073] In a further aspect, provided herein is a method for determining the status of health of a fetus, which method comprises: a) assessing the identity and/or level of one or more biomarkers from a foreign organism by sequencing, e.g., high- throughput sequencing, of a polynucleotide in a biological sample from a pregnant female; b) analyzing said identity and/or level of said one or more biomarkers; and c) determining the status of health of said fetus. In some embodiments, the foreign organism is selected from the group consisting of: HBV, HCV, HIV, TORCH

{Toxoplasma gondii, rubella virus, cytomegalovirus, and Herpes simplex virus), and syphilis. In some embodiments, the status of health is infection with one or more of the foreign organisms.

[0074] In some embodiments, the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material. In some embodiments, template DNA is obtained from blood of a pregnant female. Blood may be collected using any standard technique for blood drawing including but not limited to venipuncture. For example, blood can be drawn from a vein from the inside of the elbow or the back of the hand. Blood samples can be collected from a pregnant female at any time during fetal gestation. For example, blood samples can be collected from human females at 1-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, or 40-44 weeks of fetal gestation, and preferably between 8-28 weeks of fetal gestation.

[0075] To assess the identity and/or level of one or more biomarkers, sequence information of polynucleotide fragments is obtained by sequencing template DNA obtained from a biological sample. In some embodiments, the template DNA contains both maternal DNA and fetal DNA. The polynucleotide fragments are compared to the biomarkers based on the sequence information. A reference genomic sequence is used to obtain the reference unique reads.

[0076] In some embodiments, the biomarkers may comprise target DNA sequences from the genomic sequences of the foreign organisms. In some embodiments, the target DNA sequences are selected from the conserved regions of the genomes. In some embodiments, a DNA chip may be used to support probes that hybridize to the target DNA sequences. [0077] In some embodiments, the template DNA may be used to construct a DNA library. In some embodiments, the template DNA may be subject to amplification and/or enrichment steps. In some embodiments, the template DNA may be subject to a solution based sequence

7,867,703).

[0078] In some embodiments, it may be desirable to initially assign the polynucleotide fragments to the host genome to facilitate the identification of foreign sequences. For example, when the host is a human being, the human genomic sequence may be used as a reference genomic sequence to assign the polynucleotide fragments. In some other embodiments, human genome builds hgl8 or hg 19 may be used as the reference genomic sequence. In some embodiments, the polynucleotide fragments that fail to assign to the host genome are assessed according to one or more reference genomic sequences from one or more foreign organisms.

[0079] In some embodiments, a cutoff value may be established for the biomarker. In some embodiments, a fetus having a level of the biomarker greater than the cutoff value may indicate that it is infected with the foreign organism.

VII. Systems and Kits

[0080] In yet another aspect, provided herein is a system for determining the status of health of a fetus, which system comprises: a) means for assessing the identity and/or level of one or more polynucleotide, polypeptide and/or small molecule from a foreign organism in a biological sample from a pregnant female; and b) means for analyzing said identity and/or level of said foreign polynucleotide, polypeptide and/or small molecule. In some embodiments, the status of health of said fetus is not HBV infection. In some embodiments, the small molecule may be related to host metabolism.

[0081] Any suitable devices can be used, for example, gel electrophoresis, chromatography, spectrophotometry, etc., for assessing the identity and/or level of the polynucleotide, polypeptide and/or small molecule. In some embodiments, the means for assessing the identity and/or level of one or more polynucleotide, polypeptide and/or small molecule may be a sequencing device. In some embodiments, the means for analyzing said identity and/or level of said foreign

polynucleotide, polypeptide and/or small molecule may be a computer. In some embodiments, the computer may comprise a computer readable medium comprising a plurality of instructions for analyzing said identity and/or level of said foreign polynucleotide, polypeptide and/or small molecule, for example, sequence alignment with genomic sequences of the host organism, sequence alignment with genomic sequences of foreign organisms, etc.

[0082] Further provided herein is a kit for determining the status of health of a fetus comprising: a) a biomarker disclosed herein; and b) an instruction on how to use said biomarker.

[0083] In some embodiments, the invention provides compositions and kits comprising primers and primer pairs, which allow the specific amplification of the polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bio luminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers can be used to detect the presence of polynucleotides in a sample and as a means for detecting cell expressing proteins encoded by the polynucleotides. As will be understood by the skilled artisan, a great many different primers and probes may be prepared based on the sequences provided herein and used effectively to amplify, clone and/or determine the presence and/or levels of genomic DNAs.

[0084] In some embodiments, the kit may comprise reagents for detecting presence of polypeptides. Such reagents may be antibodies or other binding molecules that specifically bind to a polypeptide. In some embodiments, such antibodies or binding molecules may be capable of distinguishing a structural variation to the polypeptide as a result of polymorphism, and thus may be used for genotyping. The antibodies or binding molecules may be labeled with a detectable marker, such as, for example, a radioisotope, a fluorescent compound, a bio luminescent compound, a chemiluminescent compound, a metal chelator, an enzyme, or a particle. Other reagents for performing binding assays, such as ELISA, may be included in the kit.

[0085] In some embodiments, the kits comprise reagents for genotyping at least two, at least three, at least five, at least ten, or fifteen biomarkers. In some embodiments, the kits may further comprise a surface or substrate (such as a microarray) for capture probes for detecting of amplified nucleic acids.

[0086] The kits may further comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a probe that is or can be detectably labeled. Such probe may be a polynucleotide specific for a biomarker. Where the kit utilizes nucleic acid

hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.

[0087] The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those described above.

[0088] The kit can further comprise a set of instructions and materials for preparing a tissue or cell sample and preparing nucleic acids (such as genomic DNA) from the sample.

[0089] The invention provides a variety of compositions suitable for use in performing methods of the invention, which may be used in kits. For example, the invention provides surfaces, such as arrays that can be used in such methods. In some embodiments, an array of the invention comprises individual or collections of nucleic acid molecules useful for detecting biomarkers of the invention. For instance, an array of the invention may comprises a series of discretely placed individual nucleic acid oligonucleotides or sets of nucleic acid oligonucleotide combinations that are hybridizable to a sample comprising target nucleic acids, whereby such hybridization is indicative of genotypes of the biomarkers of the invention.

[0090] Several techniques are well-known in the art for attaching nucleic acids to a solid substrate such as a glass slide. One method is to incorporate modified bases or analogs that contain a moiety that is capable of attachment to a solid substrate, such as an amine group, a derivative of an amine group or another group with a positive charge, into nucleic acid molecules that are synthesized. The synthesized product is then contacted with a solid substrate, such as a glass slide, which is coated with an aldehyde or another reactive group which will form a covalent link with the reactive group that is on the amplified product and become covalently attached to the glass slide. Other methods, such as those using amino propryl silica surface chemistry are also known in the art, as disclosed at world wide web at cmt.corning.com and cmgm. stanford.edu/pbrownl .

VIII. Examples

[0091] The following examples are offered to illustrate but not to limit the invention. [0092] In order to facilitate understanding, the specific embodiments are provided to help interpret the technical proposal, that is, these embodiments are only for illustrative purposes, but not in any way to limit the scope of the invention. Unless otherwise specified, embodiments do not indicate the specific conditions, are in accordance with the conventional conditions or the manufacturer's recommended conditions.

Example 1 Identification of viruses as bio markers for Down's syndrome

[0093] The current example illustrates non- invasive genetic analysis of fetal risk for chromosomal aneuploidy by obtaining peripheral blood (5 mL) from a pregnant female, extracting cell-free DNA, sequencing by new high-throughput techniques, followed by

bio informatics analysis. Foreign sequence is relative to sequences from the host organism. The present results show that foreign sequences are related to the health status of the host organism.

[0094] With the informed consent of the pregnant women, peripheral blood samples (12-24 weeks pregnant) were obtained and labeled by researchers by barcode for informative

categorization and management of the samples. Serum was separated from the peripheral blood and cell- free DNA extracted from the serum. DNA libraries were constructed followed by high- throughput sequencing. Sequencing results were compared to human genome sequence. Those that did not align with human genome sequence were categorized as non-human sequences. Non- human sequences were compared to known viral sequences to obtain uniquely aligned sequences. Figure 1 shows a flowchart for the experimental steps in this example.

Sample Collection

[0095] Samples were from the pregnant women who harbor normal fetuses and trisomy 21 fetuses. Under the guidance of obstetricians and gynecologists, five mL of maternal peripheral blood for each was collected into EDTA-containing tubes. Experimenter marked for each sample, and managed sample by category.

Sample preparation

[0096] Blood samples were centrifuged at 1600g in 4°C for 10 min, and the supernatants were collected into fresh tubes. Then, the treated blood were subjected to a second centrifugation at 1600g in 4°C for 10 min, and the supernatant were collected into fresh tubes again. The final supernatants were stored in -80°C until further processing. DNA extraction

[0097] This step was performed using a Nucleon DNA extraction kit. Under the premise of ensuring the results of DNA extraction, DNA Extraction Kit of magnetic beads method (DP327) was low cost and easy for automation (^80 samples), while TIANamp Micro DNA Kit (DP316) relatively was low cost and easy to perform sample for single. The method of operation, see the operating manual.

Sequencing

[0098] Extracted DNA was used to construct a DNA library according to the rules of the manufacturer's standard operation procedures of DNA library construction (see standard library construction manual http://www.illumina.com/), and then sequenced on the Illumina HiSeq 2000™.

[0099] Basic steps of the sequencing process were as follows:

1) DNA library construction: DNA was broken into fragments of a certain size, performed end-repaired, added "A" bases to the 3' end of the DNA fragments, ligated the adapters to the DNA and then performed polymerase chain reaction. End product was the DNA sequencing footage which already connected with joint on both ends.

2) Chip preparation and sequencing: Treated single- stranded DNA fragments and primers in the chip surface were anchored on a chip (flow cell) through complementary base. The non- labeled nucleotides and enzymes were added to the reaction system to, perform Bridge PCR reaction. Using adapter fixed on flow cell surface as a kind of template, dsDNA (double strand DNA) bridges were formed, which were amplified from ssDNA (single strand DNA) bridge. After amplification through 30 cycles, each single molecule was amplified

approximately 1000 times to become the Monoclonal DNA cluster, and then linearization.

3) Sequencing-By-Synthesis: DNA polymerase d, the adapter primer and four kinds of deoxy-ribonucleoside triphosphate (dNTP) with fluorescent dye labeled were added to the reaction system. Since all 3' end hydro xyl groups of the four dNTPs were protected using chemistry, a single dNTP was added during each cycle. All unused free dNTP and DNA polymerase were eluted after the dNTP was added to the synthesis chain. Buffer solution was added for fluorescence excitation, and laser was used to stimulate the fluorescent signal, and recorded with an optical device. Finally, the sequencing results were generated by computer analysis. Fluorescent signal was quenched by adding chemical reagents, and the dNTP 3'- hydroxyl protecting groups were removed to restore its stickiness, and then a second nucleotide was added. The process was repeated until each template sequence completely aggregated to be double- stranded. Analysis of the collected fluorescence signal results in each cycle showed the sequence of each template DNA fragments.

Sequence alignment and statistical analysis

[00100] Illumina HiSeq 2000™ was used to sequence samples and generated single-end, 35bp or 49bp reads for each individual with sequencing depth less than IX. The human reference genome was downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/), version hgl9 (build37). Alignment of the sequences to human genome was performed using SOAPaligner, resulting in unmapped sequences, which also means non-human sequences. Viral genomes were also downloaded from the NCBI database, and the non-human sequences were aligned to viral genomes using SOAPaligner and resulting in unique map reads.

[0100] The read numbers were calculated and compared between the Down's syndrome samples and normal control samples for every virus in the database, followed by statistical analysis. For the virus named virus A, n denoted total sample number. n_*i denoted Down's syndrome sample number, ¾i denoted number of Down's syndrome samples whose sequence reads uniquely mapped to virus A genome. Similarly, n_*2 denoted normal control samples number, ni₂ denoted number of normal control samples whose sequence reads can uniquely mapped to virus A genome.

Table 1 Tables of statistics see below.

[0101] Fisher's exact test was used to compare the difference between Down's syndrome samples and normal control samples. Test formula is as follows:

p - value x 2

[0102] If the p-value<=0.05, this virus was defined as being nominally significant, and it may have some effect on fetal health.

Statistical results

[0103] 110 Down's syndrome samples and 300 normal control samples were analyzed using the above Fisher's exact test to find 4 significant viruses, which were then validated in 153 Down's syndrome samples and 551 normal control samples using the same method. Finally 2 candidate viruses were identified which show significant association to Down's syndrome fetuses (Table 2).

Table 2 Viruses Significantly Related to Down's Syndrome

[0104] All viruses that may be related to Down's syndrome fetuses identified by the statistical analysis using the combined 263 Down' s syndrome samples and 851 normal control samples as having a p- value <= 0.05 are listed in Table 3.

Table 3 Viruses Significantly Related to Down's Syndrome

Example 2 Sequence analysis of fetal pathogens

[0105] In situations where pathogen infection occurs in pregnant women, there would be trace amount of the pathogen DNA in the peripheral blood of the pregnant women. For example, we have detected HBV DNA in the peripheral blood through a DNA sequencing technique. As one of ordinary skill in the art would appreciate, the more DNA of the pathogen origin exists in peripheral blood of the pregnant women, the more likely it is for fetal infection by the pathogen. Therefore, provided herein is a method for detecting fetal infection by a pathogen, wherein a chip comprising probes for the pathogen is used. Pathogen- specific sequences are detected through target sequence capture followed by sequencing. A cutoff value is set according to the case-control group statistical analysis, which is used to determine the infection status of the fetus.

[0106] Also contemplated by this disclosure is designing probes that hybridize to pathogen- specific conserved sequences to be made onto a single chip, which will capture corresponding pathogen target sequences. The obtained sequences go through high-throughput sequencing to generate the raw data. Then, the raw data is removed of contaminated fragments, and the remaining data will be aligned with target reference sequences. Finally, unique reads number aligned to the corresponding pathogens is compared to case-control group reads number to determine whether the fetus is infected or not. Zhang et al., World J Gastroenterol. (1998) 4(l):61-63; Jiang, Contemporary Medicine (2012) 18(7). This method is used for the detection of fetal infection of pathogens, and has several advantages. Firstly, noninvasive, so pregnant women and fetuses are safe; secondly, multiple outputs, one chip can detect multiple pathogens, and can capture multiple samples in one test; thirdly, low cost, due to the small size of the capture region, the cost of sequencing high depth is greatly reduced; finally, accurate, because the sequencing depth is higher, detection signal is stronger, which greatly improves detection accuracy.

[0107] The present embodiment involves reported and unreported pathogens, which relate to pregnancy infection, such as viruses, bacteria, fungi, parasites and other pathogens. The specific, conserved sequences of the causative agent are used as reference sequences. The biological sample used may be blood, cells and tissues, saliva, tears, milk, urine, feces or combinations thereof. The sequencing reagents and equipment include not only the second generation sequencing platforms by Illumia^®, 454^® and AB^® SOLiD™, but also the third generation, fourth generation of high- throughput sequencing platforms. Experimental design

[0108] The experiment includes the following steps:

1, designing capture probes according to the pathogen reference genome;

2, preparing the genomic DNA from maternal serum;

3, obtaining pathogen fragment using a solution based sequence enrichment method

(Nimblegen, as described in U.S. Patent Publication No. 20090105081), and/or a sequence complexity reduction method (Agilent, as described in U.S. Patent No. 7,867,703);

4, sequencing;

5, filtering raw reads through;

6, obtaining high quality reads mapped onto target pathogens uniquely and calculate the total number of unique reads number on each target pathogens genome; and

7, determining the cutoff according to the control group, if the ratio exceeds the cutoff, the sample is found positive, which means it has pathogen infection.

[0109] The present embodiment uses the following information analysis models:

1. Sequencing

[0110] Sequencing libraries with 140-800 bp insert size were constructed following the instruction of Illumina. Then pair-end sequencing was performed.

2. Removing pollution in sequencing data

[0111] The PCR duplication, low quality sequencing reads and reads containing sequencing adapter were removed from sequencing data. Clean reads were obtained through this step.

[0112] The strategy of removing PCR duplication: when two or more sequencing reads are completely identical, only one was reserved.

3. Filtering the reads which are able to map on to human genome

[0113] After getting clean reads, the clean reads were aligned on to human genome hgl9 (UCSC Build hgl9) using SOAP2. SOAP2 is a member of the SOAP (Short Oligonucleotide Analysis Package). It is an updated version of SOAP software for short oligonucleotide alignment. The reads which were able to map on to hgl9 were filtered. 4. Aligning the filtered reads on to Fundamental Dataset.

[0114] Next, the remaining reads after filtering were aligned onto Fundamental Dataset using SOAP2. Fundamental Dataset here means dataset which contains genome sequences of various species including target species we wanted to detect.

5. Calculating the relative abundance of reads uniquely mapping reads

[0115] Upon finishing of the above alignment, the reads that mapped onto target pathogens uniquely were picked and the total number of uniquely mapping reads number on each target pathogens genome was calculated. The proportion of uniquely mapping reads was obtained by dividing the total number of uniquely mapping reads number to total clean reads number. The proportion was defined as the relative abundance of the sample on the target pathogen.

[0116] If a sequencing read only had one unique matching location on one reference sequence, the read was called as mapping onto the reference sequence uniquely and the read was called uniquely mapping reads.

6. Obtaining of a cutoff value

[0117] The cutoff value was obtained from control group which can be defined as negative samples. The first 5 steps of obtaining the cutoff were the same as step 1 to step 5 described above. However, the treating objects here were samples from control group. The 6th step of obtaining the cutoff value was calculating the average value of relative abundances of all samples on certain target pathogen. The formula of cutoff value was as follows: cutoff = / m + 2 t

[0118] In the formula: n means the total number of control samples, r; means the relative abundance of the first i control sample on a certain target pathogen, and sd means standard deviation of the data set ri~r_n.

7. Determining infection

[0119] In this step, the calculated relative abundance obtained in step 5 was compared to cutoff value obtained from the control group in step 6. If the relative abundance was bigger than the cutoff value, the result was confirmed as positive, which means the sample tested was infected by the target pathogens. Results of embodiment samples

HBV

[0120] 10 clinical HBV antigen positive pregnant women cases were selected, 5 cases confirmed as fetal HBV antigen positive and the other 5 cases as negative. At the same time 5 HBV antigen negative pregnant women and fetus also negative cases were selected. About 5 ml of peripheral blood from pregnant women were collected, followed by low-speed centrifugation or static natural precipitation supernatant, aspirate supernatant to new pipe pieces. Maternal serum free DNA was extracted using TIANGEN virus genomic DNA/RNA extraction kit according to requirements of Illumina^® HiSeq2000™ sequencing construct library. End- repairing of obtained DNA fragments was performed using T4 DNA polymerase, Klenow™ polymerase, and T4 polynucleotide kinase. Commercially available adapters (Illumina) were ligated to the DNA fragments after addition of terminal A-residues. The adapter-ligated DNA was then additionally amplified using a 14-cycle PCR with standard multiplex primers.

Agencourt AMPure™ XP magnetic beads were used for the purification of PCR products.

[0121] Samples were purified using Agencourt AMPure XP magnetic beads, and dissolved in 25μ1 of EB buffer.

[0122] Constructed library was determined to meet the requirement of fragment distribution range by Agilent^® Bioanalyzer 2100™. Then it was quantified through a Q-PCR method. The library was hybridized with a customized chip (Agilent), and hybridization products were sequenced with Illumina^® HiSeq2000™. The sequencing strategy was PE91+8+91 (Paired-End sequencing, 91-bp reads and 8-bp index sequence), the parameters of the apparatus setting and operation method were in accordance with the Illumina^® operation manual (which can be obtained by the http://www.illumina.com/support/documentation.ilmn).

[0123] Result of the sequencing data is shown in Table 4.

Table 4 Result of sequencing data

Reads Reads

Sample Q20(%) GC% Production

Length Number

PDB10AA00158 49 96.81% 39.59% 6.7M 326.0M

PDB11AA00346 49 97.00% 39.38% 6.5M 318.8M

PDB11AA02150 49 97.59% 41.24% 5.2M 255.6M

PDB11AD00038 49 96.87% 40.22% 6.0M 292.4M

PDB11AG00022 35 99.10% 39.40% 6.4M 223.3M

PDB11AJ00097 49 97.94% 40.72% 8.1M 399.3M

PDB11AJ00469 49 97.48% 40.95% 7.2M 353.8M

PDB11AM00005 49 98.01% 40.93% 5.8M 282.7M PDB11AM00011 49 97.71% 40.87% 6.2M 305.6M

PDB11AR00001 49 97.22% 39.82% 7.6M 373.6M

PDB11AR00002 49 97.05% 39.57% 9.9M 483.5M

PDB11AR00291 49 98.27% 40.59% 5.1M 249.8M

PDB11AR00342 35 99.24% 40.01% 8.2M 288.6M

PDB11AS00026 49 98.63% 40.32% 6.9M 338.9M

PDB12AE00013 35 99.01% 40.70% 7.6M 266.3M

PDB11AA00416 49 96.92% 39.19% 7.1M 346.3M

PDB11AD00909 35 98.49% 40.33% 8.4M 293.9M

PDB11BC00028 35 98.89% 40.38% 8.8M 306.4M

PDB12AJ00147 35 98.47% 41.41% 4.5M 158.3M

PDP110000122 49 98.88% 39.15% 7.6M 374.8M

[0124] The reads were aligned to HBV reference sequence (gi:3582357). Cutoff value was set according to the positive samples and negative samples Unique Reads statistical results. The cutoff value was used to determine which samples have HBV infection. The method in this embodiment can detect HBV positive samples from pregnant women with HBV infection, and the cutoff value can be used to determine whether the fetus has HBV infection or not. The results are shown in Table 5.

Table 5 Result of HBV infection

relative Clinic Test Successful or

Sample

abundance (10-3) Data Result not ?

PDB11AJ00469 0.382 ++ ++ Y

PDB11AD00038 0.233 ++ ++ Y

PDB11AM00005 0.179 ++ ++ Y

PDB11AA02150 0.163 ++ ++ Y

PDB10AA00158 0.160 ++ ++ Y

PDB12AE00013 0.111 +- +- Y

PDB11AR00291 0.074 ++ +- N

PDB11AS00026 0.067 +- +- Y

PDB11AG00022 0.061 +- +- Y

PDB11AM00011 0.060 +- +- Y

PDB11AA00416 0.004 — — Y

PDP110000122 0.002 — — Y

PDB11BC00028 0.002 — — Y

PDB11AD00909 0.002 — — Y

PDB12AJ00147 0.001 — — Y

"++" means the mother and the fetus are both infected; "+-" means the mother is infected but the fetus is not infected; "— " means the mother and the fetus are both uninfected.

[0125] In all 15 samples we tested, only 1 sample was not consistent with the clinical data, which proved powerful performance of the disclosed method. TORCH

[0126] 10 clinical TORCH IgM positive pregnant women cases are selected, 5 cases are fetal TORCH IgM positive and the other 5 cases are negative. At the same time 5 TORCH IgM negative pregnant women and fetus also negative cases are selected. Pregnant women are collected about 5 ml of peripheral blood. Blood sample processing, structure library, target region capture, sequencing methods and data analysis are similar to the HBV embodiment detailed above. TORCH IgM samples from pregnant women with TORCH infection can be detected, and the cutoff value about whether fetus had TORCH infection or not can also be given.

[0127] All publications, including patent documents and scientific articles, referred to in this application and the bibliography and attachments are incorporated by reference in their entireties for all purposes to the same extent as if each individual publication were individually incorporated by reference.

[0128] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0129] Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0130] Although the present invention has been fully described in connection with embodiments thereof with reference to the accompanying drawings, it is to be noted that various changes and modifications will become apparent to those skilled in the art. Such changes and modifications are to be understood as being included within the scope of the present invention. The various embodiments of the invention should be understood that they have been presented by way of example only, and not by way of limitation. Likewise, the various diagrams may depict an example architectural or other configuration for the invention, which is done to aid in understanding the features and functionality that can be included in the invention. The invention is not restricted to the illustrated example architectures or configurations, but can be implemented using a variety of alternative architectures and configurations. Additionally, although the invention is described above in terms of various exemplary embodiments and implementations, it should be understood that the various features and functionality described in one or more of the individual embodiments are not limited in their applicability to the particular embodiment with which they are described. They instead can, be applied, alone or in some combination, to one or more of the other embodiments of the invention, whether or not such embodiments are described, and whether or not such features are presented as being a part of a described embodiment. Thus the breadth and scope of the invention should not be limited by any of the above-described exemplary embodiments.

[0131] Terms and phrases used in this document, and embodiments thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing: the term "including" should be read as meaning "including, without limitation" or the like; the term "example" is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; and adjectives such as "conventional," "traditional," "normal," "standard," "known", and terms of similar meaning, should not be construed as limiting the item described to a given time period, or to an item available as of a given time. But instead these terms should be read to encompass conventional, traditional, normal, or standard technologies that may be available, known now, or at any time in the future. Likewise, a group of items linked with the conjunction "and" should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as "and/or" unless expressly stated otherwise. Similarly, a group of items linked with the conjunction "or" should not be read as requiring mutual exclusivity among that group, but rather should also be read as "and/or" unless expressly stated otherwise. Furthermore, although items, elements or components of the invention may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated. For example, "at least one" may refer to a single or plural and is not limited to either. The presence of broadening words and phrases such as "one or more," "at least," "but not limited to", or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent.

Claims

Claims We claim:

1. A method for determining the status of health of a fetus, which method comprises:

a) assessing the identity and/or level of one or more polynucleotide, polypeptide and/or small molecule related to host metabolism from a foreign organism in a biological sample from a pregnant female;

b) analyzing said identity and/or level of said foreign polynucleotide, polypeptide and/or small molecule related to host metabolism; and

c) determining the status of health of said fetus,

wherein the status of health of said fetus is not HBV infection.

2. The method of claim 1, wherein the status of health of the fetus is not an infection by the foreign organism.

3. The method according to claim 1 or 2, wherein the fetus is a human fetus.

4. The method according to any one of claims 1-3, wherein the analysis is conducted using one or more normal controls.

5. The method according to any one of claims 1-4, wherein the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material.

6. The method according to any one of claims 1-5, wherein the foreign organism is selected from the group consisting of: a virus, a bacterium, a fungus and a parasite.

7. The method according to any one of claims 1-6, wherein the status of health is selected from the group consisting of: genetic abnormality, and developmental abnormality.

8. The method of claim 7, wherein the genetic abnormality is a fetal aneuploidy.

9. The method of claim 8, wherein the fetal aneuploidy is a disorder for an autosome selected from the group consisting of trisomy 13, 18 and 21, or a disorder for a sex chromosome selected from the group consisting of XO, XXX, XXY and XYY.

10. A method for identifying a biomarker for the status of health of a fetus, which method comprises:

a) assessing the identity and/or level of a polynucleotide, polypeptide and/or small molecule related to host metabolism from a foreign organism in a biological sample from one or more pregnant female;

b) conducting statistical analysis of said identity and/or level of said foreign polynucleotide, polypeptide and/or small molecule related to host metabolism; and

c) identifying said non-human polynucleotide, polypeptide or small molecule related to host metabolism as a biomarker for said status of health if statistically significant association is established,

wherein the status of health of said fetus is not HBV infection, or the biological sample is obtained from at least 100 pregnant females.

11. The method of claim 10, wherein the fetus is a human fetus.

12. The method according to claim 10 or 11, wherein the biological sample is obtained from at least 2, 5, 10, 20, 50, 100, 200, 500 or 1000 pregnant females.

13. The method according to any one of claims 10-12, wherein the foreign organism is selected from the group consisting of: a virus, a bacterium, a fungus and a parasite.

14. The method according to any one of claims 10-13, wherein the status of health is selected from the group consisting of: genetic abnormality, and developmental abnormality.

15. The method of claim 14, wherein the genetic abnormality is a fetal aneuploidy.

16. The method of claim 15, wherein the fetal aneuploidy is trisomy 21.

17. The method of claim 16, wherein the biomarker is selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedro virus, Pseudomonas phage F10,

18. The method of claim 15, wherein the fetal aneuploidy is a disorder for a sex chromosome selected from the group consisting of XO, XXX, XXY and XYY.

19. The method according to any one of claims 11-18, wherein the biological sample is obtained from at least 2, 5, 10, 20, 50, 100, 200, 500 or 1000 pregnant women.

20. A method for determining the status of health of a fetus, which method comprises:

a) assessing the identity and/or level of one or more biomarkers identified using the method according to any one of claims 10-19 in a biological sample from a pregnant female; b) analyzing said identity and/or level of said biomarkers; and

c) determining the status of health of said fetus.

21. The method of claim 20, wherein the fetus is a human fetus.

22. The method according to claim 20 or 21, wherein the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material.

23. The method according to any one of claims 20-22, wherein the biomarker is a polynucleotide.

24. The method of claim 23, wherein step a) comprises purifying cell-free DNA from the biological sample.

25. The method of claim 24, wherein the DNA is amplified.

26. The method according to claim 24 or 25, wherein the DNA is constructed into a library.

27. The method according to any one of claims 24-26, wherein the DNA is sequenced.

28. The method of claim 27, wherein the DNA is sequenced by high- throughput sequencing.

29. The method according to any of claims 23-28, wherein step b) comprises comparing the sequences obtained from step a) to the sequences of the biomarkers.

30. The method of claim 29, wherein the sequences obtained from step a) that match human genomic sequences are removed before step b).

31. A panel of biomarkers for determining trisomy 21 of a human fetus, which panel comprises two or more organisms selected from the group consisting of: Agrotis ipsilon multiple nucleopolyhedro virus, Pseudomonas phage F10, Burkholderia phage Bcep22, Encephalomyocarditis virus, Hepatitis C virus genotype 1, Enterobacteria phage M13, Human herpesvirus 1, Human herpesvirus 7, Cyprinid herpesvirus 3, Staphylococcus phage 37, Choristoneura occidentalis granulo virus, Helicoverpa zea nudivirus 2, and Enterobacteria phage Sf6.

32. A method for determining the status of health of a fetus, which method comprises:

a) assessing the identity and/or level of one or more biomarkers from a foreign organism by sequencing of a polynucleotide in a biological sample from a pregnant female; b) analyzing said identity and/or level of said one or more biomarkers; and c) determining the status of health of said fetus.

33. The method of claim 32, wherein the fetus is a human fetus.

34. The method according to claim 32 or 33, wherein the biological sample is selected from the group consisting of: serum, blood, effusion, urine, bone marrow, ascities fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, mucus, sputum, saliva, occular fluid, extract of nasal, throat swab, genital swab, cell suspension from digested tissue, and extract of fecal material.

35. The method according to any one of claims 32-34, wherein step a) comprises purifying cell- free DNA from the biological sample.

36. The method of claim 35, wherein the DNA is amplified.

37. The method according to claim 35 or 36, wherein the DNA is constructed into a library.

38. The method according to any one of claims 32-37, wherein step b) comprises comparing the sequences obtained from step a) to the biomarkers.

39. The method of claim 38, wherein the sequences obtained from step a) that match human genomic sequences are removed before step b).

40. The method according to any one of claims 32-39, wherein the foreign organism is selected from the group consisting of: HBV, HCV, HIV, TORCH {Toxoplasma gondii, rubella virus, cytomegalovirus, and Herpes simplex virus), and syphilis.

41. The method according to any one of claims 32-40, wherein a cutoff value is established for the biomarker.

42. The method of claim 41, wherein a fetus having a level of the biomarker greater that the cutoff value indicates that it is infected by the foreign organism.

43. A system for determining the status of health of a fetus, which system comprises: a) means for assessing the identity and/or level of one or more polynucleotide, polypeptide and/or small molecule related to host metabolism from a foreign organism in a biological sample from a pregnant female; and b) means for analyzing said identity and/or level of said foreign polynucleotide, polypeptide and/or small molecule related to host metabolism,

wherein the status of health of said fetus is not HBV infection.

44. A kit for determining the status of health of a fetus comprising:

a) a biomarker identified by the method according to any one of claims 10-19; and b) an instruction on how to use said biomarker.