Diagnosing fetal chromosomal aneuploidy using genomic sequencing

Abstract

Embodiments of the invention provide methods, systems and apparatuses for determining the presence or absence of a fetal chromosomal aneuploidy in a biological sample obtained from a pregnant woman. Nucleic acid molecules of the biological sample are sequenced so as to sequence a portion of the genome. Respective amounts of clinically relevant chromosomes and background chromosomes are determined from the sequencing results. A parameter (e.g., a ratio) obtained from these amounts is compared to one or more cutoff values, from which a determination is made as to whether a fetal chromosomal aneuploidy classification is present.

Description

[0001] Priority Statement

[0002] This application claims priority to U.S. Provisional Application No. 60 / 951438 (AttorneyDocket No. 016285-005200US), filed July 23, 2007, entitled “DETERMINING ANUCLEIC ACIDSEQUENCE IMBALANCE”, and is the formal application thereof, the entire contents of which are hereby incorporated by reference and used for various purposes.

[0003] Cross-references to related applications

[0004] This application also relates to the concurrently filed formal application entitled “DETERMINING A NUCLEICACID SEQUENCEIMBALANCE” (Attorney Docket No. 016285-005210US), the entire contents of which are hereby incorporated by reference for various purposes. Invention Field

[0005] This invention generally relates to diagnosing fetal chromosomal aneuploidy by identifying imbalances between different nucleic acid sequences, and more specifically, to identifying trisomy 21 (Down syndrome) and other chromosomal aneuploidy by testing maternal samples (such as blood). Background of the Invention

[0007] Fetal chromosomal aneuploidy is caused by the presence of an abnormal amount of chromosome or chromosomal region. The abnormal amount can be abnormally high, such as the presence of an extra chromosome 21 or chromosomal region in trisomy 21; or abnormally low, such as the lack of a copy of the X chromosome in Turner syndrome.

[0008] Routine prenatal diagnosis of fetal chromosomal aneuploidy, such as trisomy 21, involves invasive methods like amniocentesis or chorionic villus sampling to obtain material from the fetus, but this carries a limited risk of fetal loss. Non-invasive methods, such as ultrasound scans or screening with biochemical markers, have been used to risk-stratify pregnant women before resorting to invasive diagnostic methods. However, these screening methods typically measure side effects associated with chromosomal aneuploidy, such as trisomy 21, rather than the core chromosomal abnormality, thus their diagnostic accuracy is not optimal and they have other drawbacks, such as excessive influence from gestational age.

[0009] In 1997, circulating cell-free fetal DNA was discovered in maternal plasma, offering new possibilities for non-invasive prenatal diagnosis (Lo, YMD and Chiu, RWK 2007 Nat Rev Genet 8, 71-77). Although this method is readily applicable to the prenatal diagnosis of sex-linked disorders (Costa, JM et al. 2002 N Engl J Med 346, 1502) and certain monogenic disorders (Lo, YMD et al. 1998 N Engl J Med 339, 1734-1738), its application in prenatal detection of fetal chromosomal aneuploidy remains a significant challenge (Lo, YMD and Chiu, RWK 2007, ibid.). Firstly, fetal nucleic acids and high-background maternal nucleic acids coexist in maternal plasma, and these high-background maternal nucleic acids often interfere with the analysis of fetal nucleic acids (Lo, YMD et al. 1998 Am J Hum Genet 62, 768-775). Secondly, fetal nucleic acids mainly circulate in the maternal plasma in a cell-free form, making it difficult to obtain dosage information of genes or chromosomes in the fetal genome.

[0010] Significant progress has been made in recent years to overcome these challenges (Benachi, A & Costa, JM 2007, Lancet 369, 440-442). One approach is to detect fetal-specific nucleic acids in maternal plasma, thus overcoming the problem of maternal background interference (Lo, YMD and Chiu, RWK 2007, ibid.). The dosage of chromosome 21 is inferred from the ratio of polymorphic alleles in placental-derived DNA / RNA molecules. However, this method is less accurate when the sample contains low levels of the target nucleic acid and is only applicable to fetuses that are heterozygous for the target polymorphism; if only one polymorphism is used, the target nucleic acid is only a subset of the population.

[0011] Dhallan et al. (Dhallan, R, et al. 2007, ibid., Dhallan, R, et al. 2007 Lancet 369, 474-481) described an alternative strategy of enriching the proportion of circulating fetal DNA by adding formaldehyde to maternal plasma. The proportion of fetal chromosome 21 sequence in maternal plasma was determined by assessing the ratio of paternally inherited fetal-specific alleles to non-fetal-specific alleles of single nucleotide polymorphisms (SNPs) on chromosome 21. Similarly, the SNP ratio for a reference chromosome was calculated. Imbalances in fetal chromosome 21 were then inferred by detecting statistically significant differences between the SNP ratios of chromosome 21 and the reference chromosome, where a fixed p-value of less than or equal to 0.05 was used to define significance. To ensure high population coverage, more than 500 SNPs were targeted per chromosome. However, there is debate regarding the efficiency of formaldehyde in enriching fetal DNA to a high proportion (Chung, GTY, et al. 2005 Clin Chem 51, 655-658), therefore, the reproducibility of this method requires further evaluation. Furthermore, since each fetus and mother provides many different SNPs on each chromosome, the power of statistical tests for comparing SNP ratios will vary depending on the circumstances (Lo YMD & Chiu, RWK. 2007 Lancet 369, 1997). Moreover, because these methods rely on the detection of genetic polymorphisms, they are limited to fetuses that are heterozygous for these polymorphisms.

[0012] Using polymerase chain reaction (PCR) and DNA quantification of chromosome 21 loci and reference loci in amniotic fluid cell cultures obtained from trisomic and euploid fetuses, Zimmermann et al. (2002 Clin Chem 48, 362-363) demonstrated that a 1.5-fold increase in chromosome 21 DNA sequence from amniotic fluid cell cultures of trisomic fetuses could distinguish between the two groups of fetuses. Since a 2-fold difference in DNA template concentration constitutes only a difference in one threshold cycle (Ct), a 1.5-fold difference represents the limit of conventional real-time PCR. Alternative strategies are needed to achieve a higher degree of quantitative differentiation.

[0013] Digital PCR has been developed to detect allelic ratio skewing in nucleic acid samples (Chang, HW et al. 2002 J Natl Cancer Inst 94, 1697-1703). Digital PCR is an amplification-based nucleic acid analysis technique that requires distributing nucleic acid-containing samples across a large number of discrete samples, each containing, on average, no more than about one target sequence. Digital PCR generates specific amplicons by amplifying specific nucleic acid targets using sequence-specific primers. Before nucleic acid analysis, the target nucleic acid loci and the types or groups of sequence-specific primers to be included in the reaction are identified or selected.

[0014] Clinically, digital PCR has been demonstrated to detect loss of heterozygosity (LOH) in tumor DNA samples (Zhou, W. et al. 2002 Lancet 359, 219-225). To analyze digital PCR results, previous studies have used sequential probability ratio testing (SPRT) to classify experimental results as indicating the presence or absence of LOH in the sample (E1 Karoui et al. 2006 Stat Med 25, 3124-3133).

[0015] In previous studies, the amount of data collected by digital PCR was quite small. Therefore, the limited number of data points and typical statistical fluctuations compromised accuracy.

[0016] Therefore, a non-invasive test with high sensitivity and specificity is desired to minimize false negatives and false positives, respectively. However, fetal DNA exists at a low absolute concentration and represents a small fraction of the total DNA sequence in maternal plasma and serum. Therefore, a non-invasive method is also desired to allow for the detection of fetal chromosomal aneuploidy by maximizing the amount of genetic information that can be inferred from a limited quantity of fetal nucleic acids present as a small fraction in a biological sample containing maternal background nucleic acids. Invention Overview

[0018] Embodiments of the present invention provide methods, systems, and apparatus for determining whether a nucleic acid sequence imbalance (such as chromosomal imbalance) exists in a biological sample obtained from a pregnant woman. This determination can be made using parameters relating to the amount of clinically relevant chromosomal regions (background regions) in relation to other non-clinically relevant chromosomal regions in the biological sample. On one hand, the amount of chromosomes is determined by sequencing nucleic acid molecules in maternal samples, such as urine, plasma, serum, and other suitable biological samples. The nucleic acid molecules in the biological sample are sequenced to sequence portions of the genome. To determine whether a change (i.e., imbalance) exists compared to a reference amount, one or more cutoff values ​​are selected, such as the ratio of the amounts of two chromosomal regions (or groups of chromosomal regions).

[0019] According to an exemplary embodiment, prenatal diagnosis of fetal chromosomal aneuploidy is performed by analyzing biological samples received from a pregnant woman. The biological samples include nucleic acid molecules. A portion of the nucleic acid molecules contained in the biological sample is sequenced. On the one hand, the amount of genetic information obtained is sufficient for diagnostic accuracy, but not excessive, in order to control costs and the required input amount of biological samples.

[0020] Based on sequencing, a first quantity of the first chromosome is determined from sequences identified as originating from the first chromosome. A second quantity of one or more second chromosomes is determined from sequences identified as originating from one of the second chromosomes. Subsequently, parameters of the first and second quantities are compared with one or more cutoff values. Based on the comparison, it is determined whether a classification of fetal chromosomal aneuploidy exists for the first chromosome. Sequencing helps to maximize the amount of genetic information that can be inferred from a limited number of fetal nucleic acids present as a small fraction in biological samples containing maternal background nucleic acids.

[0021] According to an exemplary implementation, prenatal diagnosis of fetal chromosomal aneuploidy is performed by analyzing a biological sample received from a pregnant woman. The biological sample includes nucleic acid molecules. The percentage of fetal DNA in the biological sample is determined. Based on this percentage, and based on the desired accuracy, the number N of sequences to be analyzed is calculated. At least N nucleic acid molecules contained in the biological sample are randomly sequenced.

[0022] Based on random sequencing, a first quantity of chromosome 1 is determined from sequences identified as originating from chromosome 1. A second quantity of one or more chromosome 2 is determined from sequences identified as originating from one of chromosome 2. The parameters of the first and second quantities are then compared to one or more cutoff values. Based on these comparisons, a classification of fetal chromosomal aneuploidy for chromosome 1 is determined. Random sequencing facilitates maximizing the amount of genetic information that can be inferred from a limited number of fetal nucleic acids present as a small fraction in samples containing maternal background nucleic acids.

[0023] Other embodiments of the present invention relate to systems and computer-readable media in connection with the methods described herein.

[0024] A better understanding of the features and advantages of the invention can be obtained by referring to the detailed description and accompanying drawings below. Brief description of the attached diagram

[0026] Figure 1 This is a flowchart of method 100 of an embodiment of the present invention, which is used for prenatal diagnosis of fetal chromosomal aneuploidy in biological samples obtained from an individual pregnant woman.

[0027] Figure 2 This is a flowchart of method 200 of an embodiment of the present invention, which is used for prenatal diagnosis of fetal chromosomal aneuploidy using random sequencing.

[0028] Figure 3A A graph illustrating the percentage representation of chromosome 21 sequence in maternal plasma samples associated with trisomy 21 or euploid fetuses, representing an embodiment of the present invention.

[0029] Figure 3B This illustrates an embodiment of the present invention, showing the correlation between fractional fetal DNA concentrations in maternal plasma, determined by massively parallel sequencing and microfluidics digital PCR.

[0030] Figure 4A A graph illustrating the percentage expression of aligned sequences for each chromosome, representing an embodiment of the present invention.

[0031] Figure 4B express Figure 4A The chart shows the difference (%) in percentage expression of each chromosome between the 21-somatic and euploid cases.

[0032] Figure 5 The embodiment of the present invention illustrates the correlation between the degree of over-representation of chromosome 21 in maternal plasma associated with trisomy 21 fetuses and the concentration of fetal DNA fraction.

[0033] Figure 6 The table represents a portion of the human genome analyzed according to an embodiment of the present invention. T21 represents a sample obtained from a pregnancy associated with a fetus with trisomy 21.

[0034] Figure 7A table illustrating the number of sequences required to distinguish euploidy from trisomy 21 fetuses, representing an embodiment of the present invention.

[0035] Figure 8A This table shows the first 10 start positions of the sequenced tag compared to chromosome 21, representing an embodiment of the present invention.

[0036] Figure 8B This table shows the first 10 start positions of the sequenced tag compared to chromosome 22, representing an embodiment of the present invention.

[0037] Figure 9 A block diagram illustrating an exemplary computer device that can be used with the systems and methods of embodiments of the present invention.

[0038] definition

[0039] As used in this article, “biological sample” refers to any sample containing one or more nucleic acid molecules of interest collected from an individual (such as a pregnant woman).

[0040] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and its polymers. Unless otherwise specified, the term includes nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (such as degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosin residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). The terms nucleic acid are used interchangeably with gene, cDNA, mRNA, small non-coding RNA, microRNA (miRNA), Piwi-interacting RNA, and short hairpin RNA (shRNA) encoded by a gene or locus.

[0041] The term "gene" refers to a segment of DNA involved in the production of polypeptide chains. It can include regions before and after the coding region (leader region and non-transcribed tail region), as well as intercalation sequences (introns) between individual coding segments (exons).

[0042] As used herein, the term "reaction" refers to any process involving a chemical, enzymatic, or physical action that indicates the presence or absence of a particular polynucleotide sequence of interest. An example of a "reaction" is an amplification reaction such as polymerase chain reaction (PCR). Another example of a "reaction" is a sequencing reaction, either synthetically or by ligation. An "information reaction" is a reaction that indicates the presence of one or more particular polynucleotide sequences of interest, and in one case, only one sequence of interest is present. As used herein, the term "well" refers to a reaction in a predetermined location and a finite structure, such as a well-drained flask, chamber, or chamber in a PCR array.

[0043] As used herein, the term "clinically relevant nucleic acid sequence" can refer to a polynucleotide sequence corresponding to a larger genomic sequence fragment being detected for a potential imbalance, or to the larger genomic sequence itself. One example is a sequence of chromosome 21. Other examples include chromosomes 18, 13, X, and Y. Other examples include mutated gene sequences or genetic polymorphisms or copy number variations inherited by a fetus from one or both parents. Other examples include sequences with mutations, deletions, or amplifications in malignant tumors, such as sequences with loss of heterozygosity or gene duplication. In some embodiments, multiple clinically relevant nucleic acid sequences, or multiple markers equivalent to clinically relevant nucleic acid sequences, can be used to provide data for detecting imbalances. For example, data from five discontinuous sequences from chromosome 21 can be used additively to identify possible chromosome 21 imbalances, thereby effectively reducing the required sample volume to 1 / 5.

[0044] As used in this article, the term "background nucleic acid sequence" refers to a known ratio of a background nucleic acid sequence to a clinically relevant nucleic acid sequence, such as a 1:1 ratio. As one example, the background nucleic acid sequence and the clinically relevant nucleic acid sequence are two different alleles from the same chromosome, differing due to heterozygosity. In another example, the background nucleic acid sequence is an allele heterozygous for another allele, which is the clinically relevant nucleic acid sequence. Furthermore, each of certain background and clinically relevant nucleic acid sequences can originate from different individuals.

[0045] The term "reference nucleic acid sequence" as used in this article refers to a nucleic acid sequence for which the average concentration of each reaction is known or has been equivalently measured.

[0046] The term “overrepresented nucleic acid sequence” as used in this article refers to a nucleic acid sequence in two sequences of interest (e.g., clinically relevant sequences and background sequences) that is more abundant than other sequences in a biological sample.

[0047] As used in this paper, the term "based on" means "at least partially based on" and refers to a value (or result) used to determine another value, such as a value existing in the relationship between the input and output of a method. The term "obtain" as used in this paper also refers to the relationship between the input and output of a method, such as the relationship existing when obtaining is the calculation of a formula.

[0048] As used in this article, “quantitative data” means data obtained from one or more reactions that provides one or more numerical values. For example, the number of wells containing fluorescently labeled sequences is quantitative data.

[0049] As used in this article, the term "parameter" refers to a numerical value that characterizes the numerical relationship between quantitative datasets and / or between quantitative datasets. For example, the ratio (or a function of the ratio) between a first quantity of a first nucleic acid sequence and a second quantity of a second nucleic acid sequence is a parameter.

[0050] As used in this article, the term "cutoff value" refers to a numerical value used to arbitrate between two or more classification states (e.g., diseased and non-diseased) of a biological sample. For example, if the parameter is greater than the cutoff value, quantitative data are classified into the first category (e.g., diseased state), or if the parameter is less than the cutoff value, quantitative data are classified into another category (e.g., non-diseased state).

[0051] As used in this article, “imbalance” means any significant deviation from a reference value, defined by at least one cutoff value in the quantity of clinically relevant nucleic acid sequences. For example, the reference value is 3 / 5, so if the measured ratio is 1:1, there is an imbalance.

[0052] As used in this article, "chromosomal aneuploidy" refers to the variation in the quantitative number of chromosomes compared to the number of chromosomes in a diploid genome. This variation can be an increase or a loss. It can include the entirety of a chromosome or a region of a chromosome.

[0053] As used herein, "random sequencing" refers to sequencing in which the nucleic acid fragments being sequenced are not specifically identified or targeted prior to the sequencing procedure. Sequence-specific primers targeting specific gene loci are not required. The pool of sequenced nucleic acids varies from sample to sample, and even for the same sample, it can differ between analyses. The characteristics of the sequenced nucleic acids are revealed solely by the resulting sequencing output. In some embodiments of the invention, a procedure for enriching biological samples with a specific population of nucleic acid molecules sharing certain common characteristics may precede random sequencing. In one embodiment, each fragment in the biological sample has an equal probability of being sequenced.

[0054] As used herein, the terms "fraction of the human genome" or "portion of the human genome" refer to less than 100% of the nucleotide sequence of the human genome, which consists of approximately 3 billion nucleotide base pairs. In the context of sequencing, the term refers to a human genome nucleotide sequence with less than 1x coverage. The term can be expressed as a percentage or absolute value of nucleotides / base pairs. As an example of use, the term can be used to represent the actual amount of sequencing performed. Implementation methods can determine the minimum amount of human genome sequenced required to obtain an accurate diagnosis. As another example of use, the term refers to the amount of sequencing data used to obtain parameters or quantities for disease classification.

[0055] As used in this article, the term "sequencing tag" refers to any part or all of a sequenced nucleotide string from a nucleic acid molecule. For example, a sequenced tag can be a short string of nucleotides from a nucleic acid fragment, a short string of nucleotides at both ends of a nucleic acid fragment, or the sequencing of a complete nucleic acid fragment present in a biological sample. A nucleic acid fragment is any part of a larger nucleic acid molecule. Fragments (such as genes) can exist separately from other parts of a larger nucleic acid molecule (i.e., not connected). Invention Details

[0057] Embodiments of the present invention provide methods, systems, and apparatus for determining whether the presence of clinically relevant chromosomes is increased or decreased (disease state) compared to a non-disease state. This determination can be made using parameters relating to the amount of clinically relevant chromosomal regions in relation to other non-clinically relevant chromosomal regions (background regions) in a biological sample. Nucleic acid molecules of the biological sample are sequenced to sequence a portion of the genome, and the amount can be determined from the sequencing results. One or more cutoff values ​​are selected to determine whether there is a change (i.e., imbalance) compared to a reference amount, for example, the ratio of the amounts of two chromosomal regions (or groups of chromosomal regions).

[0058] The change detected in the reference state can be any deviation (upward or downward) from a clinically relevant nucleic acid sequence compared to other non-clinically relevant sequences. Therefore, the reference state can be any ratio or other quantity (such as except for a 1-1 correspondence), and the measurement state representing the change, as determined by one or more cutoff values, can be any ratio or other quantity different from the reference state.

[0059] Clinically relevant chromosomal regions (also known as clinically relevant nucleic acid sequences) and background nucleic acid sequences can originate from a first type of cell and one or more second type of cell. For example, fetal nucleic acid sequences from fetal / placental cells are present in biological samples, such as maternal plasma containing background maternal nucleic acid sequences from maternal cells. In one embodiment, a cutoff value is determined at least in part based on the percentage of first-type cells in the biological sample. It should be noted that the percentage of fetal sequences in the sample can be determined from any fetal-derived locus and is not limited to measuring clinically relevant nucleic acid sequences. In another embodiment, a cutoff value is determined at least in part based on the percentage of tumor sequences in the biological sample, such as plasma, serum, saliva, or urine, containing background nucleic acid sequences from non-malignant cells in vivo.

[0060] I. General Method

[0061] Figure 1 This is a flowchart of method 100 of an embodiment of the present invention, which is used for prenatal diagnosis of fetal chromosomal aneuploidy in biological samples obtained from an individual pregnant woman.

[0062] In step 110, a biological sample is received from the pregnant woman. This biological sample can be plasma, urine, serum, or any other suitable sample. The sample contains nucleic acid molecules from both the fetus and the pregnant woman. For example, the nucleic acid molecules can be fragments of chromosomes.

[0063] In step 120, at least a portion of a plurality of nucleic acid molecules contained in the biological sample is sequenced. The sequenced portion represents a part of the human genome. In one embodiment, the nucleic acid molecules are fragments of their respective chromosomes. Sequencing can be performed on one end (e.g., 35 base pairs (bp)), both ends, or the entire fragment. All nucleic acid molecules in the sample can be sequenced, or only a subpopulation can be sequenced. As described in more detail below, the subpopulation can be randomly selected.

[0064] In one implementation, sequencing is performed using massively parallel sequencing. Massively parallel sequencing can be performed using platforms such as the 454 platform (Roche) (Margulies, M. et al. 2005 Nature 437, 376-380), the Illumina Genome Analyzer (or the Solexa platform), the SOLiD System (Applied Biosystems), the Helicos True Single Molecule DNA sequencing technology (Harris TD et al. 2008 Science, 320, 106-109), or Pacific Biosciences' single-molecule real-time (SMR) sequencing technology. TM This technology, including nanopore sequencing (Soni GV and Meller A. 2007 ClinChem 53: 1996-2001), allows for the parallel sequencing of numerous nucleic acid molecules isolated from a sample using high-order multiplexing (Dear Brief Funct Genomic Proteomic 2003; 1: 397-416). Each of these platforms can sequence single molecules of asexually amplified or even unamplified nucleic acid fragments.

[0065] Because each run generates hundreds of thousands, millions, or even hundreds of millions or billions of sequencing reads from each sample, the resulting sequencing reads represent a significant portion of the mixture of nucleic acid species in the original sample. For example, the haplotype, transcriptome, and methylation characteristics of the sequencing reads are similar to these representative characteristics of the original sample (Brenner et al. Nat Biotech 2000; 18: 630-634; Taylor et al. CancerRes 2007; 67: 8511-8518). Due to the large number of sequences sampled from each sample, the number of identical sequences, such as the number of identical sequences generated by sequencing the nucleic acid pool with several times the coverage or high redundancy, also provides a good quantitative representation of the specific nucleic acid species or loci count in the original sample.

[0066] In step 130, based on sequencing data (such as data from sequencing), a first quantity of the first chromosome (such as a clinically relevant chromosome) is determined. The first quantity is determined by sequences identified as originating from the first chromosome. For example, each of these DNA sequences can subsequently be mapped to the human genome using bioinformatics procedures. It is possible to discard some of these sequences from subsequent analyses because they exist in repetitive regions of the human genome or in regions that have undergone inter-individual variation, such as copy number variation. Therefore, the quantity of the chromosome of interest, or the quantity of one or more other chromosomes, can be determined.

[0067] In step 140, based on sequencing, a second quantity of one or more second chromosomes is determined from sequences identified as originating from one of the second chromosomes. In one embodiment, the second chromosome is all other chromosomes except the first chromosome (i.e., the chromosome being detected). In another embodiment, the second chromosome is a single other chromosome.

[0068] There are many ways to determine chromosome quantity, including but not limited to counting the number of sequenced tags, the number of sequenced nucleotides (base pairs), or the cumulative length of sequenced nucleotides (base pairs) from a particular chromosome or chromosomal region.

[0069] In another implementation, rules can be applied to the sequencing results to determine which data were counted. On one hand, the quantity can be obtained based on a subset of the sequencing output. For example, sequencing outputs corresponding to nucleic acid fragments within a specified size range can be selected after bioinformatics analysis. Examples of size ranges are approximately <300bp, <200bp, or <100bp.

[0070] In step 150, parameters are determined by the first and second quantities. Parameters can be, for example, a simple ratio of the first to the second quantity, or a ratio of the first to the second quantity plus the first quantity. On the one hand, each quantity can be a function or an independent variable of different functions, where the ratios of these different functions can subsequently be obtained. Those skilled in the art will understand the number of different suitable parameters.

[0071] In one implementation, parameters (such as fractional representation) of chromosomes potentially associated with chromosomal aneuploidy, such as aneuploidy of chromosome 21, 18, or 13, can subsequently be calculated from the results of a bioinformatics procedure. Fractional representation can be obtained based on the quantity of all sequences (such as certain measurements of all chromosomes, including clinically relevant chromosomes) or the quantity of a specific chromosomal subset (such as only one chromosome other than the one being tested).

[0072] In step 150, the parameter is compared to one or more cutoff values. Cutoff values ​​can be determined by any number of suitable methods. Such methods include Bayesian-type likelihood methods, sequential probability ratio tests, false discoveries, confidence intervals, and receiver operating characteristic (ROC). Examples of the application of these methods and sample-specific methods are described in the concurrently filed application "DETERMINING A NUCLEIC ACID SEQUENCE IMBALANCE" (Attorney Docket No. 016285-005210US), which is incorporated herein by reference.

[0073] In one implementation, parameters (such as the fractional representation of clinically relevant chromosomes) are then compared to a reference range established in pregnancies involving normal (i.e., euploid) fetuses. Possibly, in certain variations of the procedure, the reference range (i.e., the cutoff value) can be adjusted based on the fractional concentration (f) of fetal DNA in a specific maternal plasma sample. If the fetus is male, the f value can be determined from a sequencing dataset, for example, using a sequence that can be located on the Y chromosome. The f value can also be determined in a separate analysis, for example, using fetal exogenous markers (Chan KCA et al 2006 Clin Chem 52, 2211-8) or by analysis of single nucleotide polymorphisms.

[0074] In step 160, based on comparison, a classification is determined for the first chromosome to determine whether fetal chromosomal aneuploidy exists. In one embodiment, the classification is a definite yes or no. In another embodiment, the classification may be unclassifiable or indeterminate. In yet another embodiment, the classification may be, for example, a rating interpreted later by a physician.

[0075] II. Sequencing, alignment, and determination of quantities

[0076] As mentioned above, only a portion of the genome is sequenced. On the one hand, even when sequencing the nucleic acid pool in a sample with less than 100% genome coverage instead of several times that coverage, and in the nucleic acid molecules captured in a portion, most nucleic acid species are sequenced only once. Dosage imbalances of specific chromosomes or chromosomal regions can also be quantitatively determined. In other words, dosage imbalances of chromosomes or chromosomal regions are inferred from the percentage expression of said loci in other localizable sequenced tags of the sample.

[0077] This contrasts with the scenario where nucleic acids from the same pool are sequenced multiple times to achieve redundancy or several times the coverage, thus sequencing each nucleic acid species multiple times. In this case, the number of times a particular nucleic acid species has been sequenced relative to another nucleic acid species is related to its relative concentration in the original sample. As the coverage factor required to achieve accurate representation of nucleic acid species increases, the cost of sequencing increases.

[0078] In one instance, a portion of such sequences may originate from chromosomes associated with aneuploidy, such as chromosome 21 in this exemplary example. However, other sequences from such sequencing exercises may originate from other chromosomes. By considering the relative size of chromosome 21 compared to other chromosomes, the normalized frequency of chromosome 21-specific sequences for such sequencing exercises can be obtained within a reference range. If the fetus has trisomy 21, the normalized frequency obtained from chromosome 21 for such sequencing exercises will increase, thus allowing for the detection of trisomy 21. The extent of variation in normalized frequencies will depend on the fractional concentration of fetal nucleic acids in the analyzed sample.

[0079] In one implementation, we use an Illumina Genome Analyzer to perform single-end sequencing of human genomic DNA and human plasma DNA samples. The Illumina Genome Analyzer can sequence single DNA molecules asexually amplified and trapped on a solid surface called a flow cell. Each flow cell has eight lanes for sequencing eight individual samples or sample pools. Each lane produces approximately 200 Mb of sequence, which is only a fraction of the 3 billion base pairs in the human genome. Each genomic DNA or plasma DNA sample is sequenced using one lane of the flow cell. The resulting short sequence tags are aligned to a human reference genome sequence, indicating chromosome origin. The total number of individually sequenced tags aligned to each chromosome is tabulated and compared to the relative size of each chromosome expected from a reference human genome or non-disease-presenting sample. Chromosomal additions or loss are then determined.

[0080] The method described is merely one example of the gene / chromosome dosing strategy currently employed. Optionally, paired-end sequencing can be performed. The number of aligned sequenced tags is counted and categorized according to chromosomal location, rather than comparing the expected length of the sequenced fragment in the reference genome as described by Campbell et al. (Nat Genet 2008; 40: 722-729). Additions or losses of chromosomal regions or the entire chromosome are determined by comparing tag counts with the expected chromosome size in the reference genome or a non-disease-presenting sample. Because paired-end sequencing allows for the inference of the original nucleic acid fragment size, one example focuses on counting the number of paired sequenced tags corresponding to nucleic acid fragments of a specified size, such as <300 bp, <200 bp, or <100 bp.

[0081] In another implementation, prior to sequencing, a portion of the nucleic acid pool being sequenced during the run is further sub-selected. For example, hybridization-based techniques, such as oligonucleotide arrays, can be used to first sub-select nucleic acid sequences from certain chromosomes, such as potentially aneuploid chromosomes and other chromosomes unrelated to the detected aneuploidy. Another example is the sub-selection or enrichment of certain subpopulations of nucleic acid sequences in the sample pool prior to sequencing. For example, as discussed above, it has been reported that fetal DNA molecules in maternal plasma consist of fragments shorter than maternal background DNA molecules (Chan et al. Clin Chem 2004; 50: 88-92). Therefore, nucleic acid sequences in a sample can be graded according to molecular size using one or more methods known to those skilled in the art, for example, by gel electrophoresis or size exclusion columns or by a microfluidics-based approach. Alternatively, in instances of analyzing cell-free fetal DNA in maternal plasma, fetal nucleic acid fractions can be enriched by methods that suppress maternal background, such as by adding formaldehyde (Dhallan et al JAMA2004; 291: 1114-9). In one embodiment, a preselected pool or subpopulation of nucleic acids is randomly sequenced.

[0082] Similarly, other single-molecule sequencing strategies can also be used in this application, such as the Roche 454 platform, the Applied Biosystems SOLiD platform, Helicos real single-molecule DNA sequencing technology, and Pacific Biosciences' single-molecule real-time technology (SMRT). TM ) and nanopore sequencing.

[0083] III. Determining the amount of chromosomes from the sequencing output.

[0084] Following massively parallel sequencing, bioinformatics analysis was performed to pinpoint the chromosomal origin of the sequenced tags. This procedure then quantitatively compared tags identified as originating from a potential aneuploid chromosome, specifically chromosome 21 in this study, with all sequenced tags or tags from one or more chromosomes unrelated to aneuploidy. The correlation between the sequencing outputs of chromosome 21 and other non-chromosomes in the tested sample was compared to a cutoff value obtained using the methods described in the previous section to determine whether the sample was obtained from a pregnancy associated with euploidy or trisomy 21.

[0085] Many different quantities, including but not limited to those that can be obtained from the sequenced tags, can be obtained. For example, the absolute count of the number of sequenced tags aligned to a specific chromosome can be compared with the absolute count of sequenced tags aligned to other chromosomes. Alternatively, the fractional count of the number of sequenced tags on chromosome 21 can be compared with the fractional counts of other non-aneploid chromosomes, referring to all or some of the other sequenced tags. In this experiment, because 36 bp of each DNA fragment was sequenced, the number of sequenced nucleotides on a specific chromosome can be easily obtained by multiplying the count of the sequenced tags by 36 bp.

[0086] Furthermore, because a flow cell capable of sequencing only portions of the human genome is used, and sequencing is performed on only each maternal plasma sample, statistically, most maternal plasma DNA fragment types are sequenced only once, resulting in a single count of sequenced tags. In other words, nucleic acid fragments present in the maternal plasma sample are sequenced with less than 1-fold coverage. Therefore, for any given chromosome, the total number of sequenced nucleotides typically corresponds to the amount, proportion, or length of the partially sequenced chromosome. Consequently, the quantitative determination of the phenotype of a potential aneuploid chromosome can be obtained by referring to similarly obtained quantities for other chromosomes, from the partial number or equivalent length of sequenced nucleotides of the potential aneuploid chromosome.

[0087] IV. Enrichment of nucleic acid pools for sequencing

[0088] As mentioned above and established in the examples in the following section, only a portion of the human genome needs to be sequenced to distinguish trisomy 21 from euploidy. Therefore, it is possible and cost-effective to enrich the pool of nucleic acids to be sequenced before random sequencing of a portion of the enriched pool. For example, fetal DNA molecules in maternal plasma consist of fragments shorter than maternal background DNA molecules (Chan et al., Clin Chem 2004; 50: 88-92). Therefore, nucleic acid sequences in a sample can be graded according to molecular size, for example, by gel electrophoresis, size exclusion columns, or microfluidic-based methods, using one or more methods known to those skilled in the art.

[0089] Alternatively, in instances where cell-free fetal DNA is analyzed from maternal plasma, fetal nucleic acid fractions can be enriched using methods such as the addition of formaldehyde to suppress the maternal background (Dhallan et al., JAMA 2004; 291: 1114-9). The proportion of sequences obtained from the fetus will be enriched in a pool of nucleic acid fragments composed of shorter segments. Figure 7 The number of sequenced tags required to distinguish between euploidy and trisomy 21 will decrease as the concentration of fetal DNA fraction increases.

[0090] Optionally, sequences from potentially aneuploid chromosomes and one or more chromosomes unrelated to aneuploidy can be enriched using hybridization techniques, such as oligonucleotide microarrays. The enriched pool of nucleic acids is then randomly sequenced. This reduces sequencing costs.

[0091] V. Random sequencing

[0092] Figure 2 This is a flowchart of a method 200 for prenatal diagnosis of fetal chromosomal aneuploidy using random sequencing, according to an embodiment of the present invention. In one aspect of massively parallel sequencing methods, representative data for all chromosomes can be generated simultaneously. The source of specific fragments is not pre-selected. Sequencing is performed randomly, followed by a database search to determine the origin of specific fragments. This is in contrast to the case of amplifying a specific fragment of chromosome 21 and another specific fragment of chromosome 1.

[0093] In step 210, a biological sample from the pregnant woman is received. In step 220, the number N of sequences to be analyzed is calculated for the desired accuracy. In one embodiment, the percentage of fetal DNA in the biological sample is first determined. This can be done by any suitable method known to those skilled in the art. The determination can be simply reading a value measured by another entity. In this embodiment, the calculation of the number N of sequences to be analyzed is based on a percentage. For example, when the percentage of fetal DNA decreases, the number of sequences to be analyzed will increase, while when the percentage of fetal DNA increases, the number of sequences to be analyzed can decrease. The number N can be a fixed number or a relative number, such as a percentage. In another embodiment, a number N known to be sufficient for accurate disease diagnosis can be sequenced. Even in pregnancies with fetal DNA concentrations at the lower end of the normal range, the number N can be sufficient.

[0094] In step 230, at least N of a plurality of nucleic acid molecules contained in the biological sample are randomly sequenced. The method is characterized in that the nucleic acids to be sequenced are not specifically identified or targeted prior to sample analysis, i.e., sequencing. Sequencing does not require sequence-specific primers targeting specific loci. The pool of nucleic acids to be sequenced varies from sample to sample, and even for the same sample, it varies depending on the analysis. Furthermore, according to the description below ( Figure 6 The amount of sequencing output required for diagnostic analysis can vary between the tested sample and the reference population. These aspects differ significantly from most molecular diagnostic methods, such as fluorescence-based methods in situ hybridization, quantitative fluorescent PCR, quantitative real-time PCR, digital PCR, comparative genomic hybridization, and microarray comparative genomic hybridization. In these methods, the target locus needs to be pre-determined, thus requiring the use of locus-specific primers or locus-specific probe pairs or panels.

[0095] In one embodiment, DNA fragments present in the plasma of a pregnant woman are randomly sequenced to obtain a genome sequence originally derived from the fetus or mother. Random sequencing involves sampling (sequencing) random portions of nucleic acid molecules present in a biological sample. Because sequencing is random, different subpopulations (parts) of the nucleic acid molecules (and therefore the genome) can be sequenced in each analysis. This embodiment remains effective even when the subpopulation varies with the sample or analysis. Examples of portions are approximately 0.1%, 0.5%, 1%, 5%, 10%, 20%, or 30% of the genome. In another embodiment, a portion is at least any of these values.

[0096] The remaining steps 240-270 can be performed in a manner similar to that of method 100.

[0097] VI. Post-sequencing selection of the tag pool to be sequenced

[0098] As described in Examples II and III below, the subpopulations of sequencing data are sufficient to distinguish between trisomy 21 and aneuploidy. The subpopulations of sequencing data can be a proportion of sequenced tags that convey certain property parameters. For example, in Example II, sequenced tags aligned to a unique and repeat-masked reference human genome were used. Alternatively, a representative pool of nucleic acid fragments from all chromosomes can be sequenced, but the focus is on comparing data concerning potential aneuploid chromosomes with data concerning numerous non-aneneuploid chromosomes.

[0099] Additionally, alternatively, during post-sequencing analysis, secondary selection can be performed on the sequencing output subgroups, which include sequenced tags generated corresponding to nucleic acid fragments of a specified size window in the original sample. For example, using an Illumina genome analyzer, paired-end sequencing involving sequencing both ends of the nucleic acid fragment can be used. The sequencing data for each paired end is then compared to a reference human genome sequence. The distance or number of nucleotides spanning the two ends can then be deduced. The full-length of the original nucleic acid fragment can also be deduced. Optionally, sequencing platforms such as the 454 platform, and possibly certain single-molecule sequencing technologies, can sequence short nucleic acid fragments of full length, such as 20 bp. In this way, the actual length of the nucleic acid fragment can be directly determined from the sequencing data.

[0100] Such paired-end analysis is also possible using other sequencing platforms, such as the Applied Biosystems SOLiD system. For the Roche 454 platform, because of its increased read length compared to other massively parallel sequencing systems, determining the fragment length of the full sequence is also possible.

[0101] Focusing data analysis on subgroups of sequenced tags corresponding to short nucleic acid fragments in raw maternal plasma samples has advantages because fetal DNA sequences effectively enrich the dataset. This is because fetal DNA molecules in maternal plasma consist of fragments shorter than maternal background DNA molecules (Chan et al., Clin Chem 2004; 50: 88-92). Figure 7 The number of sequenced tags required to distinguish between euploidy and trisomy 21 will decrease as the concentration of fetal DNA fraction increases.

[0102] Post-sequencing selection of silico pool subsets differs from other nucleic acid enrichment strategies implemented before sample analysis, such as gel electrophoresis or size exclusion columns for selecting nucleic acid molecules of specific sizes, and these strategies require physical separation of the enriched pool from the background nucleic acid pool. Physical procedures can introduce more experimental steps and thus introduce problems such as contamination. Post-sequencing in silico selection of sequencing output subsets can also allow for alteration of selection, depending on the sensitivity and specificity required for disease identification.

[0103] Bioinformatics, computational, and statistical methods used to determine whether a maternal plasma sample was obtained from a pregnant woman carrying a fetus with trisomy 21 or euploidy can be compiled into a computer program product to determine the parameters of the sequencing output. The execution of the computer program involves determining the quantitative number of potential aneuploid chromosomes and the amount of one or more other chromosomes. Parameters are determined and compared with appropriate cutoff values ​​to determine whether fetal chromosomal aneuploidy exists for the potential aneuploid chromosomes. Example

[0104] The following embodiments are provided for illustrative purposes and not for limiting the claimed invention.

[0105] I. Prenatal diagnosis of fetal trisomy 21

[0106] This study recruited eight pregnant women. All women were in the first or second trimester of pregnancy and had singleton pregnancies. Four of them carried fetuses with trisomy 21, and the other four carried fetuses with euploidy. 20 ml of peripheral venous blood was collected from each individual. After centrifugation at 1600×g for 10 minutes, maternal plasma was harvested and further centrifuged at 16000×g for 10 minutes. DNA was then extracted from 5–10 ml of each plasma sample. The maternal plasma DNA was used for massively parallel sequencing using an Illumina genome analyzer, according to the manufacturer's instructions. During sequencing and sequence data analysis, the sequencing technicians were unaware of the fetal diagnostic status.

[0107] In short, approximately 50 ng of maternal plasma DNA is used to prepare the DNA library. A smaller amount, such as 15 ng or 10 ng of maternal plasma DNA, can be started. The maternal plasma DNA fragments are blunt-ended, ligated with Solexa adapters, and selected for fragments of 150–300 bp via gel purification. Optionally, the blunt-ended and adapter-ligated maternal plasma DNA fragments can be passed through a column (e.g., AMPure, Agencourt) to remove unligated adapters without requiring size selection prior to cluster genearation. The adapter-ligated DNA is hybridized to the surface of a flow cell, and DNA clusters are generated using an Illumina cluster station, followed by 36 cycles of sequencing on an Illumina genome analyzer. DNA from each maternal plasma sample is sequenced using a single flow cell. Sequencing reads are edited using the Solexa Analysis Pipeline. Then, using Eland application software, all read and duplicate-masked reference human genome sequences, namely NCBI 36 assembly (GenBank accession numbers: NC_000001 to NC_000024), were compared.

[0108] In this study, to reduce the complexity of data analysis, only sequences already located at unique positions to the duplicate-masked human genome reference were considered. Optionally, other subgroups or the entire sequencing data could be used. The total number of uniquely mappable sequences for each sample was counted. The number of sequences uniquely aligned to chromosome 21 was expressed as the proportion of the total count of sequences aligned to each sample. Because maternal plasma contains fetal DNA in the background DNA from the mother, trisomy 21 fetuses provide additional sequencing tags from chromosome 21 due to the presence of an extra copy of chromosome 21 in the fetal genome. Therefore, the percentage of chromosome 21 sequences in maternal plasma from pregnancies carrying trisomy 21 fetuses is higher than the percentage of chromosome 21 from pregnancies carrying euploid fetuses. The analysis did not require targeting fetal-specific sequences. The analysis also did not require prior physical isolation of fetal nucleic acids from maternal nucleic acids. The analysis also did not require differentiation or identification of fetal sequences from maternal sequences after sequencing.

[0109] Figure 3AThis represents the percentage of chromosome 21 sequences located in each of the eight maternal plasma DNA samples (percentage expression of chromosome 21). The percentage expression of chromosome 21 in maternal plasma from trisomy 21 pregnancies is significantly higher than that from euploid pregnancies. These data suggest that non-invasive prenatal diagnosis of fetal aneuploidy can be achieved by determining the percentage expression of the aneuploid chromosome compared to the percentage expression in a reference population. Optionally, excessive expression of chromosome 21 can be detected by comparing the experimentally obtained percentage expression of chromosome 21 with the percentage expression of the expected chromosome 21 sequence in the euploid human genome. This can be done with or without masking repetitive regions in the human genome.

[0110] Five of the eight pregnant women carried male fetuses. The sequences located on the Y chromosome were fetal-specific. The percentage of sequences located on the Y chromosome was used to calculate the fractional concentration of fetal DNA in the original maternal plasma samples. Furthermore, the fractional concentration of fetal DNA was also determined using microfluidic digital PCR involving zinc finger protein, X-linked (ZFX), and zinc finger protein, Y-linked (ZFY) co-homologs.

[0111] Figure 3B This represents the correlation between the fetal DNA fractional concentration inferred from the percentage phenotype of the sequenced Y chromosome and the fetal DNA fractional concentration determined by ZFY / ZFX microfluidic digital PCR. A positive correlation exists between the fetal DNA fractional concentrations in maternal plasma determined by these two methods. The positive correlation coefficient (r) is 0.917 in Pearson correlation analysis.

[0112] For two representative cases, the percentage of maternal plasma DNA sequences compared to each of the 24 chromosomes (22 autosomes, the X chromosome, and the Y chromosome) is shown in [data missing]. Figure 4A In this study, one pregnant woman was carrying a fetus with trisomy 21, while the other women were carrying euploid fetuses. The percentage phenotype of the sequence located on chromosome 21 was higher in women carrying fetuses with trisomy 21 compared to women carrying normal fetuses.

[0113] The difference (%) in percentage expression of each chromosome between the maternal plasma DNA samples in the two scenarios described above is shown in... Figure 4B The percentage difference for a specific chromosome is calculated using the following formula:

[0114] Percentage difference (%) = (P) 21 -P E ) / P E ×100%, of which

[0115] P 21 = The percentage of plasma DNA sequences that match a specific chromosome in pregnant women carrying fetuses with trisomy 21; and

[0116] P E = The percentage of plasma DNA sequences that match a specific chromosome in pregnant women carrying euploid fetuses.

[0117] like Figure 4B As shown, compared with pregnant women carrying euploid fetuses, pregnant women carrying trisomy 21 fetuses had 11% overexpression of chromosome 21 in their plasma. For sequences compared with other chromosomes, the difference between the two cases was within 5%. Because the percentage expression of chromosome 21 is increased in trisomy 21 compared to euploid maternal plasma samples, the difference (%) can optionally be referred to as the degree of chromosome 21 overexpression. In addition to the difference (%) and absolute difference in chromosome 21 percentage expression, the ratio of the test sample count to the reference sample count can also be calculated, and this ratio represents the degree of chromosome 21 overexpression in trisomy 21 compared to euploid samples.

[0118] For each of the four pregnant women carrying euploid fetuses, their average 1.345% plasma DNA sequence was compared with chromosome 21. Of the four pregnant women carrying fetuses with trisomy 21, three of their fetuses were male. The percentage phenotype of chromosome 21 was calculated for each of the three cases. As described above, the difference (%) in the percentage phenotype of chromosome 21 among the three trisomy 21 cases was determined based on the average percentage phenotype of chromosome 21 obtained from the values ​​of the four euploid cases. In other words, in this calculation, the average of the four cases carrying euploid fetuses was used as a reference. The fetal DNA fraction concentration in these three male trisomy 21 cases was inferred from the percentage phenotype of their respective Y chromosome sequences.

[0119] The correlation between the degree of overexpression of chromosome 21 sequence and the concentration of fetal DNA fraction was shown in Figure 5 A significant positive correlation exists between the two parameters. The correlation coefficient (r) is 0.898 in the Pearson correlation analysis. These results indicate that the degree of overexpression of chromosome 21 sequence in maternal plasma is related to the fractional concentration of fetal DNA in maternal plasma samples. Therefore, a cutoff value for the degree of chromosome 21 sequence overexpression in relation to the fractional concentration of fetal DNA can be determined to identify pregnancies associated with trisomy 21.

[0120] The determination of the concentration of fetal DNA fraction in maternal plasma can also be performed independently of sequencing runs. For example, the concentration of Y chromosome DNA can be predetermined using real-time PCR, microfluidic PCR, or mass spectrometry. For instance, we have already... Figure 3B This study demonstrated a strong correlation between fetal DNA concentration estimated based on Y chromosome counts generated during sequencing runs and the ZFY / ZFX ratio generated outside of sequencing runs. In fact, fetal DNA concentration can be determined using loci other than the Y chromosome and is applicable to female fetuses. For example, Chan et al. demonstrated that fetal-derived methylated RASSF1A sequences can be detected in maternal plasma against a background of maternally unmethylated RASSF1A sequences (Chan et al., ClinChem 2006; 52: 2211-8). Therefore, the fractional concentration of fetal DNA can be determined by dividing the amount of methylated RASSF1A sequence by the amount of all RASSF1A sequences (methylated and unmethylated).

[0121] For the implementation of our invention, maternal plasma is expected to be preferred over maternal serum because maternal blood cells release DNA during blood clotting. Therefore, if serum is used, the fractional concentration of fetal DNA is expected to be lower in maternal plasma than in maternal serum. In other words, if maternal serum is used, more sequences are expected to be generated for the diagnosis of fetal chromosomal aneuploidy compared to plasma samples obtained simultaneously from the same pregnant woman.

[0122] Another alternative approach to determining the fractional concentration of fetal DNA is by quantifying polymorphisms between the mother and fetus (Dhallan R, et al. 2007 Lancet, 369, 474-481). An example of this method is targeting polymorphic sites where the mother is homozygous and the fetus is heterozygous. The amount of fetal-specific alleles is compared to the amount of common alleles to determine the fractional concentration of fetal DNA.

[0123] In contrast to existing techniques for detecting chromosomal aberrations, which include comparative genomic hybridization (CGH), microarray comparative genomic hybridization (MGE), quantitative real-time polymerase chain reaction (PCR), and massively parallel sequencing (MSS) that do not rely on a predetermined or predefined set of DNA sequences, this method involves sequencing a randomly representative portion of DNA molecules in a sample pool. The number of different sequenced tags aligned to various chromosomal regions is compared between samples containing or not containing the DNA species of interest. Chromosomal aberrations will be revealed by the difference in the number (or percentage) of sequences aligned to any given chromosomal region in the sample.

[0124] In another embodiment, cell-free plasma DNA sequencing technology can be used to detect chromosomal aberrations in plasma DNA to detect specific cancers. Different cancers have a typical set of chromosomal aberrations. Variations (amplifications and deletions) in multiple chromosomal regions can be used. Therefore, the proportion of sequences aligned to amplified regions will increase, while the proportion of sequences aligned to depleted regions will decrease. The percentage expression of each chromosome can be compared to the size of each corresponding chromosome in a reference genome, which is expressed as a percentage of genomic expression relative to any given chromosome in the whole genome. Direct comparison or comparison with a reference chromosome can also be used.

[0125] II. Sequencing only a portion of the human genome.

[0126] In the experiment described in Example I above, only one flow cell was used to sequence the maternal plasma DNA of each individual sample. The number of sequenced tags generated from each tested sample after the sequencing run is shown in [data missing]. Figure 6 In the middle, T21 indicates a sample obtained from a pregnancy associated with a fetus with trisomy 21.

[0127] Because 36 bp of each sequenced maternal plasma DNA fragment is sequenced, the number of sequenced nucleotide / base pairs in each sample can be determined by multiplying the number of sequenced tags by 36 bp, and is also shown in... Figure 6 Because the human genome contains approximately 3 billion base pairs, the amount of sequencing data generated from each maternal plasma sample represents only about 10% to 13% of the total genome.

[0128] Furthermore, in this study, as described in Example I above, only the uniquely locatable sequenced tag, referred to as U0 in the Eland software nomenclature, was used to demonstrate the presence of an overexpression of the chromosome 21 sequence in maternal plasma samples from every pregnancy carrying a fetus with trisomy 21. Figure 6 As shown, the U0 sequence represents only a subset of all sequenced tags generated from each sample, and also represents an even smaller proportion, approximately 2%, of the human genome. These data suggest that sequencing only a portion of the human genome sequence present in the tested samples is sufficient for the diagnosis of fetal aneuploidy.

[0129] III. Determining the number of sequences required

[0130] This analysis used sequencing results from plasma DNA from pregnant women carrying euploid male fetuses. The number of sequenced tags that could be mismatch-free located to the reference human genome sequence was 1,990,000. Subclusters of sequences were randomly selected from these 1,990,000 tags, and the percentage of sequences aligned to chromosome 21 was calculated within each subcluster. The number of sequences in each subcluster varied from 60,000 to 540,000. For each subcluster size, multiple subclusters of the same number of sequenced tags were edited by randomly selecting sequenced tags from the total pool until no other possible combinations were found. Subsequently, within each subcluster size, the mean percentage of sequences aligned to chromosome 21 and its standard deviation (SD) were calculated for multiple subclusters. These data were compared across different subcluster sizes to determine the effect of subcluster size on the distribution of the percentage of sequences aligned to chromosome 21. The 5th and 95th percentage points were then calculated based on the mean and SD.

[0131] When a pregnant woman carries a fetus with trisomy 21, the sequenced tag aligned to chromosome 21 should be overexpressed in the maternal plasma due to the additional dose from the fetus's chromosome 21. The degree of overexpression depends on the percentage of fetal DNA in the maternal plasma DNA sample and is calculated using the following equation:

[0132] Per T21 =Per Eu ×(1+f / 2), where,

[0133] Per T21 This indicates the percentage of sequences aligned to chromosome 21 in women carrying fetuses with trisomy 21; and

[0134] Per Eu This indicates the percentage of sequences aligned to chromosome 21 in women carrying euploid fetuses; and

[0135] f represents the percentage of fetal DNA in maternal plasma DNA.

[0136] like Figure 7 As shown, the SD of the percentage of sequences aligned to chromosome 21 decreases with increasing sequence count in each subpopulation. Therefore, the interval between the 5th and 95th percentage points decreases as the sequence count in each subpopulation increases. Distinguishing between the 5%–95% intervals for euploidy and trisomy 21 cases is possible with greater than 95% accuracy when the intervals do not overlap.

[0137] like Figure 7As shown, the minimum subgroup size for distinguishing between trisomy 21 and euploidy depends on the percentage of fetal DNA. For fetal DNA percentages of 20%, 10%, and 5%, the minimum subgroup sizes for distinguishing between trisomy 21 and euploidy are 120,000, 180,000, and 540,000 sequences, respectively. In other words, when a maternal plasma DNA sample contains 20% fetal DNA, 120,000 sequences need to be analyzed to determine whether the fetus has trisomy 21. When the percentage of fetal DNA decreases to 5%, the number of sequences that need to be analyzed increases to 540,000.

[0138] Because data is generated using 36-base-pair sequencing, 120,000, 180,000, and 540,000 sequences correspond to 0.14%, 0.22%, and 0.65% of the human genome, respectively. Since the concentration of fetal DNA in maternal plasma obtained from early pregnancy is reported to be in the low range of approximately 5% (Lo, YMD et al. 1998 Am J Hum Genet 62, 768-775), sequencing approximately 0.6% of the human genome can represent the minimum amount of sequencing required for a diagnosis with at least 95% accuracy in detecting fetal chromosomal aneuploidy in any pregnancy.

[0139] IV. Random sequencing

[0140] To illustrate that the sequenced DNA fragments were randomly selected during the sequencing run, we obtained sequenced tags from eight maternal plasma samples analyzed in Example I. For each maternal plasma sample, we determined the start position of each 36 bp sequenced tag relative to the reference human genome sequence, NCBI Compilation 36, which uniquely aligned to chromosome 21 without mismatch. We then sorted the start positions of the sequenced tag pool from each sample in ascending order based on the number of alignments. We performed a similar analysis on chromosome 22. For illustrative purposes, the first 10 start positions of chromosomes 21 and 22 from each maternal plasma sample are shown below. Figure 8A and Figure 8B As can be seen from these tables, the sequencing pools of DNA fragments differ between samples.

[0141] Using any suitable computer language, such as Java, C++, or Perl using conventional or object-oriented technologies, any software component or function described in this application can be executed as software code run by a processor. The software code can be stored as a series of instructions or commands on a computer-readable medium for storage and / or transmission, suitable media including random access memory (RAM), read-only memory (ROM), magnetic media such as hard disks or floppy disks, or optical media such as optical discs (CDs) or DVDs (Digital Optical Discs), flash memory, etc. The computer-readable medium can be any combination of such storage or transmission devices.

[0142] Such programs can also be encoded and transmitted using carrier signals suitable for propagation via wired, optical, and / or wireless networks that conform to various protocols, including the Internet. Therefore, computer-readable media according to embodiments of the present invention can generate data signals encoded using such programs. Computer-readable media encoded with program code can be assembled with compatible devices or provided independently by other devices (e.g., downloaded via the Internet). Any such computer-readable medium can be located on or within a computer program product (e.g., a hard disk or an entire computer system) and can exist on or within different computer program products within a system or network. The computer system may include a display screen, printer, or other suitable display that provides the user with any of the results mentioned herein.

[0143] Examples of computer systems are shown in Figure 9 middle. Figure 9 The subsystems shown are interconnected via system bus 975. Figure 9 Other subsystems are shown, such as printer 974, keyboard 978, hard disk 979, and display screen 976 connected to display adapter 982. Peripheral devices and input / output (I / O) devices connected to I / O controller 971 can be connected to the computer system in any number of ways known in the art, such as serial port 977. For example, serial port 977 or external interface 981 can be used to connect the computer device to a wide area network such as the Internet, a mouse input device, or a scanner. Interconnection via system bus allows central processing unit 973 to communicate with each subsystem and control the execution of instructions in system memory 972 or hard disk 979, as well as the exchange of information between subsystems. System memory 972 and / or hard disk 979 are concrete manifestations of computer-readable media.

[0144] For purposes of illustration and description, the foregoing presents a description of exemplary embodiments of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms described, and many modifications and variations can be made in accordance with the teachings above. Embodiments have been chosen and described to best explain the principles of the invention and its practical application, thereby enabling those skilled in the art to best utilize the invention in various embodiments and with various modifications suited to the particular use contemplated.

[0145] For various purposes, all publications, patents and patent applications cited in this article are incorporated herein by reference in their entirety.

Claims

1. A computer system for analyzing cell-free nucleic acid molecules in biological samples obtained from an individual pregnant woman, said computer system comprising: An apparatus for receiving sequenced tag data, wherein the sequenced tags are obtained from random sequencing of at least a portion of a plurality of cell-free nucleic acids contained in a biological sample of a pregnant woman, from the individual genome of the pregnant woman and from at least one fetal genome, wherein the sequenced tags include sequenced tags corresponding to cell-free nucleic acids from the individual genome of the pregnant woman and sequenced tags corresponding to cell-free nucleic acids from at least one fetal genome, wherein the portion represents a portion of the human genome, and wherein the biological sample is plasma or serum; An apparatus for comparing the sequenced tag with a human reference genome to determine the chromosome origin of the sequenced tag; A device for determining a first quantity of a first chromosome based on the random sequencing data, the first quantity being determined by a sequenced tag identified as originating from the first chromosome; A device for determining a second quantity of one or more second chromosomes based on the random sequencing data, the second quantity being determined by a sequenced tag identified as one of the second chromosomes; A means for determining a parameter from the first quantity and the second quantity, wherein the parameter represents a relative quantity between the first quantity and the second quantity; as well as A means for comparing the parameter with one or more cutoff values ​​and, based on the comparison, determining whether a classification of fetal chromosomal aneuploidy exists for the first chromosome.

2. The computer system of claim 1, wherein at least one of the cutoff values ​​is related to the percentage of fetal DNA in the biological sample.

3. The computer system of claim 2, wherein the percentage of fetal DNA in the biological sample is determined by any one or more of the proportion of the Y chromosome sequence, fetal exogenous markers, or single nucleotide polymorphism analysis.

4. The computer system of claim 1, wherein the cutoff value is a reference value established in normal biological samples.

5. The computer system of claim 1, further comprising: A device for identifying the amount of fetal DNA in the biological sample; as well as A device for calculating the number N of sequences to be analyzed, based on the desired accuracy.

6. The computer system of claim 1, wherein the first chromosome is chromosome 21, chromosome 18, chromosome 13, X chromosome, or Y chromosome.

7. The computer system of claim 1, wherein the sequence from the first chromosome is selected to be less than a specified number of base pairs.

8. The computer system of claim 7, wherein the specified number of base pairs is 300 bp, 200 bp, or 100 bp.

9. The computer system of claim 1, wherein the nucleic acid molecules of the biological sample have been enriched for sequences from at least one specific chromosome.

10. The computer system of claim 1, wherein for sequences smaller than 300 bp, the nucleic acid molecules of the biological sample have been enriched.

11. The computer system of claim 1, wherein for sequences smaller than 200 bp, the nucleic acid molecules of the biological sample have been enriched.

12. The computer system of claim 1, wherein the nucleic acid molecules of the biological sample have been amplified using polymerase chain reaction.

13. The computer system of claim 1, wherein the sequenced portion represents at least a predetermined portion of the human genome.

14. The computer system of claim 1, wherein the portion represents at least 0.1% of the human genome.

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