EP2563937A1 - Verfahren zur bestimmung des vorhandenseins oder des fehlens verschiedener aneuploidien in einer probe - Google Patents

Verfahren zur bestimmung des vorhandenseins oder des fehlens verschiedener aneuploidien in einer probe

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Publication number
EP2563937A1
EP2563937A1 EP11744148A EP11744148A EP2563937A1 EP 2563937 A1 EP2563937 A1 EP 2563937A1 EP 11744148 A EP11744148 A EP 11744148A EP 11744148 A EP11744148 A EP 11744148A EP 2563937 A1 EP2563937 A1 EP 2563937A1
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EP
European Patent Office
Prior art keywords
chromosome
chromosomes
sequence
interest
normalizing
Prior art date
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EP11744148A
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English (en)
French (fr)
Inventor
Richard P. Rava
David A. COMSTOCK
Brian K. Rhees
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Verinata Health Inc
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Verinata Health Inc
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Publication of EP2563937A1 publication Critical patent/EP2563937A1/de
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6872—Methods for sequencing involving mass spectrometry
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B10/00—ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B45/00—ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B99/00—Subject matter not provided for in other groups of this subclass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • the invention relates generally to the field of diagnostics, and provides a method for determining variations in the amount of nucleic acid sequences in a mixture of nucleic acids derived from different genomes.
  • the method is applicable to the practice of noninvasive prenatal diagnostics, and to the diagnosis and monitoring of metastatic progression in cancer patients.
  • the present invention fulfills some of the above needs and in particular offers an advantage in providing a reliable method that is applicable at least to the practice of noninvasive prenatal diagnostics, and to the diagnosis and monitoring of metastatic progression in cancer patients.
  • the invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest.
  • the method comprises a statistical approach that accounts for accrued variability stemming from process-related,
  • CNV interchromosomal and inter-sequencing variability.
  • the method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions.
  • CNV that can be determined according to the present method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.
  • Any aneuploidy can be determined from sequencing information that is obtained by sequencing only once the nucleic acids of a test sample.
  • a method for determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acid molecules.
  • the steps of the method comprise (a) obtaining sequence information for the fetal and maternal nucleic acids in the maternal test sample; (b) using the sequence information to identify a number of sequence tags for each of any four or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing chromosome sequence for each of the any four or more chromosomes of interest; (c) using the number of sequence tags identified for each of the any four or more chromosomes of interest and the number of sequence tags identified for each normalizing chromosome to calculate a single chromosome dose for each of the any four or more chromosomes of interest; and (d) comparing each of the single chromosome doses for each of the any four or more chromosome
  • Step (a) can comprise sequencing at least a portion of the nucleic acid molecules of a test sample to obtain said sequence information for the fetal and maternal nucleic acid molecules of the test sample.
  • step (c) comprises calculating a single chromosome dose for each of the chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing chromosome sequence for each of the chromosomes of interest.
  • step (c) comprises (i) calculating a sequence tag density ratio for each of the chromosomes of interest, by relating the number of sequence tags identified for each of the chromosomes of interest in step (b) to the length of each of the chromosomes of interest; (ii) calculating a sequence tag density ratio for each normalizing chromosome sequence by relating the number of sequence tags identified for the sequence in step (b) to the length of each normalizing chromosome; and (iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a single chromosome dose for each of the chromosomes of interest, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for each of the chromosomes of interest and the sequence tag density ratio for the normalizing chromosome sequence for each of the chromosomes of interest.
  • a method for determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acid molecules.
  • the steps of the method comprise (a) obtaining sequence information for the fetal and maternal nucleic acids in the maternal test sample; (b) using the sequence information to identify a number of sequence tags for each of any four or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing chromosome sequence for each of the any four or more chromosomes of interest; (c) using the number of sequence tags identified for each of the any four or more chromosomes of interest and the number of sequence tags identified for each normalizing chromosome to calculate a single chromosome dose for each of the any four or more chromosomes of interest; and (d) comparing each of the single chromosome doses for each of the any four or more chromosome
  • Step (a) can comprise sequencing at least a portion of the nucleic acid molecules of a test sample to obtain said sequence information for the fetal and maternal nucleic acid molecules of the test sample.
  • step (c) comprises calculating a single chromosome dose for each of the chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing chromosome sequence for each of the chromosomes of interest.
  • step (c) comprises (i) calculating a sequence tag density ratio for each of the chromosomes of interest, by relating the number of sequence tags identified for each of the chromosomes of interest in step (b) to the length of each of the chromosomes of interest; (ii) calculating a sequence tag density ratio for each normalizing chromosome sequence by relating the number of sequence tags identified for the normalizing chromosome sequence in step (b) to the length of each normalizing chromosome; and (iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a single chromosome dose for each of the chromosomes of interest, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for each of the chromosomes of interest and the sequence tag density ratio for the normalizing chromosome sequence for each of the chromosomes of interest.
  • a method for determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acid molecules.
  • the steps of the method comprise (a) obtaining sequence information for the fetal and maternal nucleic acids in the maternal test sample; (b) using the sequence information to identify a number of sequence tags for each of any four or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing chromosome sequence for each of the any four or more chromosomes of interest; (c) using the number of sequence tags identified for each of the any four or more chromosomes of interest and the number of sequence tags identified for each normalizing chromosome sequence to calculate a single chromosome dose for each of the any four or more chromosomes of interest; and (d) comparing each of the single chromosome doses for each of the any four or more
  • Step (a) can comprise sequencing at least a portion of the nucleic acid molecules of a test sample to obtain said sequence information for the fetal and maternal nucleic acid molecules of the test sample.
  • step (c) comprises calculating a single chromosome dose for each of the chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing chromosome sequence for each of the chromosomes of interest.
  • step (c) comprises (i) calculating a sequence tag density ratio for each of the chromosomes of interest, by relating the number of sequence tags identified for each of the chromosomes of interest in step (b) to the length of each of the chromosomes of interest; (ii) calculating a sequence tag density ratio for each normalizing chromosome sequence by relating the number of sequence tags identified for the normalizing chromosome sequence in step (b) to the length of each normalizing chromosome; and (iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a single chromosome dose for each of the chromosomes of interest, wherein the chromosome dose is calculated as the ratio of the sequence tag density ratio for each of the chromosomes of interest and the sequence tag density ratio for the normalizing chromosome sequence for each of the chromosomes of interest.
  • the normalizing chromosome sequence may be a single chromosome selected from chromosomes 1-22, X, and Y.
  • the normalizing chromosome sequence may be a group of chromosomes selected from chromosomes 1-22, X, and Y.
  • a method is provided for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids.
  • the steps of the method comprise: (a) obtaining sequence information for the fetal and maternal nucleic acids in the sample; (b) using the sequence information to identify a number of sequence tags for each of any one or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing segment sequence for each of any one or more chromosomes of interest; (c) using the number of sequence tags identified for each of any one or more chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence to calculate a single chromosome dose for each of any one or more chromosomes of interest; and (d) comparing each of the single chromosome doses for each of any one or more chromosomes of interest to a threshold value for each of the one or more chromosomes of interest, and thereby determining the presence or absence of one or more different complete fetal chromosomal aneuploidies in the sample.
  • Step (a)
  • step (c) comprises calculating a single chromosome dose for each of the chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence for each of the chromosomes of interest.
  • step (c) comprises (i) calculating a sequence tag density ratio for each of chromosomes of interest, by relating the number of sequence tags identified for each chromosomes of interest in step (b) to the length of each of the chromosomes of interest; (ii) calculating a sequence tag density ratio for each normalizing segment sequence by relating the number of sequence tags identified for the normalizing segment sequence in step (b) to the length of each the normalizing chromosomes; and (iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a single chromosome dose for each of said chromosomes of interest, wherein said chromosome dose is calculated as the ratio of the sequence tag density ratio for each of the chromosomes of interest and the sequence tag density ratio for the normalizing segment sequence for each of the chromosomes of interest.
  • a method for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids.
  • the steps of the method comprise: (a) obtaining sequence information for the fetal and maternal nucleic acids in the sample; (b) using the sequence information to identify a number of sequence tags for each of any one or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing segment sequence for each of any one or more chromosomes of interest; (c) using the number of sequence tags identified for each of any one or more chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence to calculate a single chromosome dose for each of any one or more chromosomes of interest; and (d) comparing each of the single chromosome doses for each of any one or more chromosomes of interest to a threshold value
  • Step (a) can comprise sequencing at least a portion of the nucleic acid molecules of a test sample to obtain said sequence information for the fetal and maternal nucleic acid molecules of the test sample.
  • step (c) comprises calculating a single chromosome dose for each of the chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence for each of the chromosomes of interest.
  • step (c) comprises (i) calculating a sequence tag density ratio for each of chromosomes of interest, by relating the number of sequence tags identified for each chromosomes of interest in step (b) to the length of each of the chromosomes of interest; (ii) calculating a sequence tag density ratio for each normalizing segment sequence by relating the number of sequence tags identified for the normalizing segment sequence in step (b) to the length of each the normalizing chromosomes; and (iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a single chromosome dose for each of said chromosomes of interest, wherein said chromosome dose is calculated as the ratio of the sequence tag density ratio for each of the chromosomes of interest and the sequence tag density ratio for the normalizing segment sequence for each of the chromosomes of interest.
  • a method for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids.
  • the steps of the method comprise: (a) obtaining sequence information for the fetal and maternal nucleic acids in the sample; (b) using the sequence information to identify a number of sequence tags for each of any one or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing segment sequence for each of any one or more chromosomes of interest; (c) using the number of sequence tags identified for each of any one or more chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence to calculate a single chromosome dose for each of any one or more chromosomes of interest; and (d) comparing each of the single chromosome doses for each of any one or more chromosomes of interest to a threshold value
  • Step (a) can comprise sequencing at least a portion of the nucleic acid molecules of a test sample to obtain said sequence information for the fetal and maternal nucleic acid molecules of the test sample.
  • step (c) comprises calculating a single chromosome dose for each of the chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence for each of the chromosomes of interest.
  • step (c) comprises (i) calculating a sequence tag density ratio for each of chromosomes of interest, by relating the number of sequence tags identified for each chromosomes of interest in step (b) to the length of each of the chromosomes of interest; (ii) calculating a sequence tag density ratio for each normalizing segment sequence by relating the number of sequence tags identified for the normalizing segment sequence in step (b) to the length of each the normalizing chromosomes; and (iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a single chromosome dose for each of said chromosomes of interest, wherein said chromosome dose is calculated as the ratio of the sequence tag density ratio for each of the chromosomes of interest and the sequence tag density ratio for the normalizing segment sequence for each of the chromosomes of interest.
  • the different complete chromosomal aneuploidies are selected from complete chromosomal trisomies, complete chromosomal monosomies and complete chromosomal polysomies.
  • the different complete chromosomal aneuploidies are selected from complete aneuploidies of any one of chromosome 1-22, X, and Y.
  • the said different complete fetal chromosomal aneuploidies are selected from trisomy 2, trisomy 8, trisomy 9, trisomy 21, trisomy 13, trisomy 16, trisomy 18, trisomy 22, 47,XXY, 47.XXX, 47,XYY, and monosomy X.
  • steps (a)-(d) are repeated for test samples from different
  • the method comprises determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies in each of the test samples.
  • the method can further comprise calculating a normalized chromosome value (NCV), wherein the NCV relates the chromosome dose to the mean of the corresponding chromosome dose in a set of qualified samples as:
  • a method for determining the presence or absence of different partial fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acids.
  • the steps of the method comprise: (a) obtaining sequence information for the fetal and maternal nucleic acids in the sample; (b) using the sequence information to identify a number of sequence tags for each of any one or more segments of any one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and to identify a number of sequence tags for a normalizing segment sequence for each of any one or more segments of any one or more
  • Step (a) can comprise sequencing at least a portion of the nucleic acid molecules of a test sample to obtain said sequence information for the fetal and maternal nucleic acid molecules of the test sample.
  • step (c) comprises calculating a single segment dose for each of any one or more segments of any one or more chromosomes of interest as the ratio of the number of sequence tags identified for each of any one or more segments of any one or more chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence for each of the any one or more segments of any one or more chromosomes of interest.
  • step (c) comprises (i) calculating a sequence tag density ratio for each of segment of interest, by relating the number of sequence tags identified for each segment of interest in step (b) to the length of each of the segment of interest; (ii) calculating a sequence tag density ratio for each normalizing segment sequence by relating the number of sequence tags identified for the normalizing segment sequence in step (b) to the length of each the normalizing segment sequence; and (iii) using the sequence tag density ratios calculated in steps (i) and (ii) to calculate a single segment dose for each segment of interest, wherein the segment dose is calculated as the ratio of the sequence tag density ratio for each of the segments of interest and the sequence tag density ratio for the normalizing segment sequence for each of the segments of interest.
  • the method can further comprise calculating a normalized segment value (NSV), wherein the NSV relates said segment dose to the mean of the corresponding segment dose in a set of qualified samples as: where fij and Sj are the estimated mean and standard deviation, respectively, for the y ' -th segment dose in a set of qualified samples, and ⁇ ⁇ ⁇ is the observed y ' -th segment dose for test sample .
  • NSV normalized segment value
  • the normalizing segment sequence may be a single segment of any one or more of chromosomes 1-22, X, and Y.
  • the normalizing segment sequence may be a group of segments of any one or more of chromosomes 1-22, X, and Y.
  • Steps (a)-(d) of the method for determining the presence or absence of a partial fetal chromosomal aneuploidy are repeated for test samples from different maternal subjects, and the method comprises determining the presence or absence of different partial fetal chromosomal aneuploidies in each of said samples.
  • Partial fetal chromosomal aneuploidies that can be determined according to the method include partial aneuploidies of any segment of any chromosome.
  • the partial aneuploidies can be selected from partial duplications, partial multiplications, partial insertions and partial deletions.
  • partial aneuploidies examples include partial monosomy of chromosome 1 , partial monosomy of chromosome 4, partial monosomy of chromosome 5, partial monosomy of chromosome 7, partial monosomy of chromosome 11 , partial monosomy of chromosome 15, partial monosomy of chromosome 17, partial monosomy of chromosome 18, and partial monosomy of chromosome 22.
  • the test sample may be a maternal sample selected from blood, plasma, serum, urine and saliva samples.
  • the test sample is may be plasma sample.
  • the nucleic acid molecules of the maternal sample are a mixture of fetal and maternal cell-free DNA molecules. Sequencing of the nucleic acids can be performed using next generation sequencing (NGS).
  • NGS next generation sequencing
  • sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
  • sequencing is sequencing-by-ligation.
  • sequencing is single molecule sequencing.
  • an amplification step is performed prior to sequencing.
  • a method for determining the presence or absence of any twenty or more different complete fetal chromosomal aneuploidies in a maternal plasma test sample comprising a mixture of fetal and maternal cell-free DNA molecules.
  • the steps of the method comprise: (a) sequencing at least a portion of the cell-free DNA molecules to obtain sequence information for the fetal and maternal cell-free DNA molecules in the sample; (b) using the sequence information to identify a number of sequence tags for each of any twenty or more chromosomes of interest selected from chromosomes 1-22, X, and Y and to identify a number of sequence tags for a normalizing chromosome for each of said twenty or more chromosomes of interest; (c) using the number of sequence tags identified for each of the twenty or more chromosomes of interest and the number of sequence tags identified for each normalizing chromosome to calculate a single chromosome dose for each of the twenty or more chromosomes of interest; and (d) comparing each of the single
  • the invention provides a method for identifying copy number variation (CNV) of a sequence of interest e.g. a clinically relevant sequence, in a test sample comprising the steps of: (a) obtaining a test sample and a plurality of qualified samples, said test sample comprising test nucleic acid molecules and said plurality of qualified samples comprising qualified nucleic acid molecules; (b) obtaining sequence information for said fetal and maternal nucleic acids in said sample; (c) based on said sequencing of said qualified nucleic acid molecules, calculating a qualified sequence dose for said qualified sequence of interest in each of said plurality of qualified samples, wherein said calculating a qualified sequence dose comprises determining a parameter for said qualified sequence of interest and at least one qualified normalizing sequence; (d) based on said qualified sequence dose, identifying at least one qualified normalizing sequence, wherein said at least one qualified normalizing sequence has the smallest variability and/or the greatest differentiability in sequence dose in said plurality of qualified samples; (e) based on said sequencing of said nucleic
  • the parameter for said qualified sequence of interest and at least one qualified normalizing sequence relates the number of sequence tags mapped to said qualified sequence of interest to the number of tags mapped to said qualified normalizing sequence
  • said parameter for said test sequence of interest and at least one normalizing test sequence relates the number of sequence tags mapped to said test sequence of interest to the number of tags mapped to said normalizing test sequence
  • step (b) comprises sequencing at least a portion of the qualified and test nucleic acid molecules, wherein sequencing comprises providing a plurality of mapped sequence tags for a test and a qualified sequence of interest, and for at least one test and at least one qualified normalizing sequence; sequencing at least a portion of said nucleic acid molecules of the test sample to obtain the sequence information for the fetal and maternal nucleic acid molecules of the test sample.
  • the sequencing step is performed using next generation sequencing method.
  • the sequencing method may be a massively parallel sequencing method that uses sequencing-by-synthesis with reversible dye terminators.
  • the sequencing method is sequencing-by-ligation.
  • sequencing comprises an amplification.
  • sequencing is single molecule sequencing.
  • the CNV of a sequence of interest is an aneuploidy, which can be a chromosomal or a partial aneuploidy.
  • the chromosomal aneuploidy is selected from trisomy 2, trisomy 8, trisomy 9, trisomy 16, trisomy 21, trisomy 13, trisomy 18, trisomy 22, 47,XXY, 47.XXX, 47,XYY, and monosomy X.
  • the partial aneuploidy is a partial chromosomal deletion or a partial chromosomal insertion.
  • the CNV identified by the method is a chromosomal or partial aneuploidy associated with cancer.
  • test and qualified sample are biological fluid samples e.g. plasma samples, obtained from a pregnant subject such as a pregnant human subject.
  • a test and qualified biological fluid samples e.g. plasma samples, are obtained from a subject that is known or is suspected of having cancer.
  • Figure 1 is a flowchart of a method 100 for determining the presence or absence of a copy number variation in a test sample comprising a mixture of nucleic acids.
  • FIG 2 illustrates the distribution of the chromosome dose for chromosome 21 determined from sequencing cfDNA extracted from a set of 48 blood samples obtained from human subjects each pregnant with a male or a female fetus.
  • Chromosome 21 doses for qualified i.e. normal for chromosome 21 (O), and trisomy 21 test samples are shown ( ⁇ ) for chromosomes 1-12 and X ( Figure 2A), and for chromosomes 1-22 and X ( Figure 2B).
  • Figure 3 illustrates the distribution of the chromosome dose for chromosome 18 determined from sequencing cfDNA extracted from a set of 48 blood samples obtained from human subjects each pregnant with a male or a female fetus. Chromosome 18 doses for qualified i.e. normal for chromosome 18 (O), and trisomy 18 ( ⁇ ) test samples are shown for chromosomes 1-12 and X ( Figure 3A), and for chromosomes 1-22 and X ( Figure 3B).
  • Figure 4 illustrates the distribution of the chromosome dose for chromosome 13 determined from sequencing cfDNA extracted from a set of 48 blood samples obtained from human subjects each pregnant with a male or a female fetus. Chromosome 13 doses for qualified i.e. normal for chromosome 13 (O), and trisomy 13 ( ⁇ ) test samples are shown for chromosomes 1 -12 and X ( Figure 4A), and for chromosomes 1-22 and X ( Figure 4B).
  • Figure 5 illustrates the distribution of the chromosome doses for chromosome X determined from sequencing cfDNA extracted from a set of 48 test blood samples obtained from human subjects each pregnant with a male or a female fetus.
  • Chromosome X doses for males (46.XY; (O)), females (46,XX; ( ⁇ )); monosomy X (45,X; (+)), and complex karyotypes (Cplx (X)) samples are shown for chromosomes 1-12 and X ( Figure 5A), and for chromosomes 1-22 and X ( Figure 5B).
  • Figure 6 illustrates the distribution of the chromosome doses for chromosome Y determined from sequencing cfDNA extracted from a set of 48 test blood samples obtained from human subjects each pregnant with a male or a female fetus.
  • Chromosome Y doses for males (46,XY; ( ⁇ )), females (46,XX; (O)); monosomy X (45,X; (+)), and complex karyotypes (Cplx (X)) samples are shown for chromosomes 1-12 ( Figure 6A), and for chromosomes 1-22 ( Figure 6B).
  • Figure 7 shows the coefficient of variation (CV) for chromosomes 21 ( ⁇ ), 18 ( ⁇ ) and 13 (A) that was determined from the doses shown in Figures 2, 3, and 4, respectively.
  • Figure 8 shows the coefficient of variation (CV) for chromosomes X ( ⁇ ) and Y ( ⁇ ) that was determined from the doses shown in Figures 5 and 6, respectively.
  • Figure 9 shows the cumulative distribution of GC fraction by human chromosome.
  • the vertical axis represents the frequency of the chromosome with GC content below the value shown on the horizontal axis.
  • Figure 10 illustrates the sequence doses (Y-axis) for a segment of chromosome 11
  • Figure 11 illustrates the distribution of normalized chromosome doses for chromosome 21 (A), chromosome 18 (B), chromosome 13 (C), chromosome X (D) and chromosome Y (E) relative to the standard deviation of the mean (Y-axis) for the corresponding chromosomes in the unaffected samples.
  • Figure 12 shows normalized chromosome values for chromosomes 21 (O), 18 ( ⁇ ), and 13 ( ⁇ ) determined in samples from training set 1 using normalizing chromosomes as described in Example 6.
  • Figure 13 shows normalized chromosome values for chromosomes 21 (O), 18 ( ⁇ ), and 13
  • FIG. 14 shows normalized chromosome values for chromosomes 21 (O) and 18 ( ⁇ ) determined in samples from test set 1 using the normalizing method of Chiu et al. (normalizes the number of sequence tags identified for the chromosome of interest with the number of sequence tags obtained for the remaining chromosomes in the sample; see elsewhere herein Example 7).
  • Figure 15 shows normalized chromosome values for chromosomes 21 (O), 18 ( ⁇ ), and 13 ( ⁇ ) determined in samples from training set 1 using systematically determined normalizing chromosomes (as described in Example 7).
  • Figure 16 shows normalized chromosome values for chromosomes 21 (0), 18 ( ⁇ ), and 13 ( ⁇ ) determined in samples from test set 1 using systematically determined normalizing chromosomes (as described in Example 7).
  • Figure 17 shows normalized chromosome values for chromosome 9 (O) determined in samples from test set 1 using systematically determined normalizing chromosomes (as described in Example 7).
  • Figure 18 shows normalized chromosome values for chromosomes X (X-axis) and Y (Y- axis). The arrows point to the 5 ( Figure 18A) and 3 ( Figure 18B) monosomy X samples that were identified in the training and test sets, respectively, as described in Example 7.
  • Figure 19 shows normalized chromosome values for chromosomes 1-22 determined in samples from test set 1 using systematically determined normalizing chromosomes (as described in Example 7).
  • the invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest.
  • Sequences of interest include genomic sequences ranging from kilobases (kb) to megabases (Mb) to entire chromosomes that are known or are suspected to be associated with a genetic or a disease condition.
  • sequences of interest include chromosomes associated with well known aneuploidies e.g. trisomy 21, and segments of chromosomes that are multiplied in diseases such as cancer e.g. partial trisomy 8 in acute myeloid leukemia.
  • the method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal (intra-run), and inter-sequencing (inter-run) variability.
  • the method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions.
  • the practice of the present invention involves conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and recombinant DNA fields, which are within the skill of the art. Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Third Edition (Cold Spring Harbor), [2001]); and Ausubel et al, "Current Protocols in Molecular Biology” [1987]).
  • nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • assessing refers to characterizing the status of a chromosomal aneuploidy by one of three types of calls: "normal", “affected”, and “no-call".
  • the "normal” call is determined by the value of a parameter e.g. a test chromosome dose that is below a user-defined threshold of reliability
  • the "affected” call is determined by a parameter e.g. a test chromosome dose, that is above a user-defined threshold of reliability
  • the "no-call" result is determined by a parameter e.g. a test chromosome dose, that lies between the user-defined thresholds of reliability for making a "normal” or an "affected” call.
  • copy number variation herein refers to variation in the number of copies of a nucleic acid sequence that is 1 kb or larger present in a test sample in comparison with the copy number of the nucleic acid sequence present in a qualified sample.
  • a "copy number variant” refers to the 1 kb or larger sequence of nucleic acid in which copy-number differences are found by comparison of a sequence of interest in test sample with that present in a qualified sample.
  • Copy number variants/variations include deletions, including microdeletions, insertions, including microinsertions, duplications, multiplications, inversions, translocations and complex multi-site variants.
  • CNV encompass chromosomal aneuploidies and partial aneuploidies.
  • aneuploidy herein refers to an imbalance of genetic material caused by a loss or gain of a whole chromosome, or part of a chromosome.
  • chromosomal aneuploidy and “complete chromosomal aneuploidy” herein refer to an imbalance of genetic material caused by a loss or gain of a whole chromosome, and includes germline aneuploidy and mosaic aneuploidy.
  • partial aneuploidy and “partial chromosomal aneuploidy” herein refer to an imbalance of genetic material caused by a loss or gain of part of a chromosome e.g. partial monosomy and partial trisomy, and encompasses imbalances resulting from translocations, deletions and insertions.
  • aneuploid sample herein refers to a sample indicative of a subject whose chromosomal content is not euploid, i.e. the sample is indicative of a subject with an abnormal copy number of chromosomes.
  • aneuploid chromosome herein refers to a chromosome that is known or determined to be present in a sample in an abnormal copy number.
  • pluriality is used herein in reference to a number of nucleic acid molecules or sequence tags that is sufficient to identify significant differences in copy number variations (e.g. chromosome doses) in test samples and qualified samples using in the methods of the invention.
  • at least about 3 x 10 6 sequence tags, at least about 5 x 10 6 sequence tags, at least about 8 x 10 6 sequence tags, at least about 10 x 10 6 sequence tags, at least about 15 x 10 6 sequence tags, at least about 20 x 10 6 sequence tags, at least about 30 x 10 6 sequence tags, at least about 40 x 10 6 sequence tags, or at least about 50 x 10 6 sequence tags comprising between 20 and 40bp reads are obtained for each test sample.
  • polynucleotide refers to a covalently linked sequence of nucleotides (i.e., ribonucleotides for RNA and deoxyribonucleotides for DNA) in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next, include sequences of any form of nucleic acid, including, but not limited to RNA, DNA and cfDNA molecules.
  • polynucleotide includes, without limitation, single- and double-stranded polynucleotide.
  • portion is used herein in reference to the amount of sequence information of fetal and maternal nucleic acid molecules in a biological sample that in sum amount to less than the sequence information of ⁇ 1 human genome.
  • test sample refers to a sample comprising a mixture of nucleic acids comprising at least one nucleic acid sequence whose copy number is suspected of having undergone variation. Nucleic acids present in a test sample are referred to as "test nucleic acids”.
  • qualified sample refers to a sample comprising a mixture of nucleic acids that are present in a known copy number to which the nucleic acids in a test sample are compared, and it is a sample that is normal i.e. not aneuploid, for the sequence of interest e.g. a qualified sample used for identifying a normalizing chromosome for chromosome 21 is a sample that is not a trisomy 21 sample.
  • training set refers to a set of samples that can comprise affected and unaffected samples.
  • the unaffected samples in a training set are used as the qualified samples to identify normalizing sequences, e.g. normalizing chromosomes, and the chromosome doses of unaffected samples are used to set the thresholds for each of the sequences , e.g. chromosomes, of interest.
  • the affected samples in a training set can be used to verify that affected test samples can be easily differentiated from unaffected samples.
  • qualified nucleic acid is used interchangeably with “qualified sequence” is a sequence against which the amount of a test sequence or test nucleic acid is compared.
  • a qualified sequence is one present in a biological sample preferably at a known representation i.e. the amount of a qualified sequence is known.
  • a "qualified sequence of interest” is a qualified sequence for which the amount is known in a qualified sample, and is a sequence that is associated with a difference in sequence representation in an individual with a medical condition.
  • sequence of interest refers to a nucleic acid sequence that is associated with a difference in sequence representation in healthy versus diseased individuals.
  • a sequence of interest can be a sequence on a chromosome that is misrepresented i.e. over- or under-represented, in a disease or genetic condition.
  • a sequence of interest may also be a portion of a chromosome i.e. chromosome segment, or a chromosome.
  • a sequence of interest can be a chromosome that is over- represented in an aneuploidy condition, or a gene encoding a tumor-suppressor that is under- represented in a cancer.
  • Sequences of interest include sequences that are over- or under- represented in the total population, or a subpopulation of cells of a subject.
  • a "qualified sequence of interest” is a sequence of interest in a qualified sample.
  • a “test sequence of interest” is a sequence of interest in a test sample.
  • normalizing sequence refers to a sequence that displays a variability in the number of sequence tags that are mapped to it among samples and sequencing runs that best approximates that of the sequence of interest for which it is used as a normalizing parameter, and that can best differentiate an affected sample from one or more unaffected samples.
  • a "normalizing chromosome” or “normalizing chromosome sequence” is an example of a “normalizing sequence”.
  • a "normalizing chromosome sequence” can be composed of a single chromosome or of a group of chromosomes.
  • a "normalizing segment” is another example of a "normalizing sequence”.
  • normalizing segment sequence can be composed of a single segment of a chromosome or it can be composed of two or more segments of the same or of different chromosomes.
  • differentiateability refers to the characteristic of a normalizing chromosome that enables to distinguish one or more unaffected i.e. normal, samples from one or more affected i.e. aneuploid, samples.
  • sequence dose herein refers to a parameter that relates the sequence tag density of a sequence of interest to the tag density of a normalizing sequence.
  • a "test sequence dose” is a parameter that relates the sequence tag density of a sequence of interest e.g. chromosome 21, to that of a normalizing sequence e.g. chromosome 9, determined in a test sample.
  • a "qualified sequence dose” is a parameter that relates the sequence tag density of a sequence of interest to that of a normalizing sequence determined in a qualified sample.
  • sequence tag density refers to the number of sequence reads that are mapped to a reference genome sequence e.g. the sequence tag density for chromosome 21 is the number of sequence reads generated by the sequencing method that are mapped to chromosome 21 of the reference genome.
  • sequence tag density ratio refers to the ratio of the number of sequence tags that are mapped to a chromosome of the reference genome e.g. chromosome 21, to the length of the reference genome chromosome 21.
  • NGS Next Generation Sequencing
  • the term "parameter” herein refers to a numerical value that characterizes a quantitative data set and/or a numerical relationship between quantitative data sets. For example, a ratio (or function of a ratio) between the number of sequence tags mapped to a chromosome and the length of the chromosome to which the tags are mapped, is a parameter.
  • the terms "threshold value” and “qualified threshold value” herein refer to any number that is calculated using a qualifying data set and serves as a limit of diagnosis of a copy number variation e.g. an aneuploidy, in an organism. If a threshold is exceeded by results obtained from practicing the invention, a subject can be diagnosed with a copy number variation e.g. trisomy 21.
  • Appropriate threshold values for the methods described herein can be identified by analyzing normalizing values (e.g. chromosome doses, NCVs or NSVs) calculated for a training set of samples. Threshold values can be identified using qualified (i.e. unaffected) samples in a training set which comprises both qualified (i.e. unaffected) samples and affected samples. The samples in the training set known to have chromosomal aneuploidies (i.e. the affected samples) can be used to confirm that the chosen thresholds are useful in differentiating affected from unaffected samples in a test set (see the
  • the choice of a threshold is dependent on the level of confidence that the user wishes to have to make the classification.
  • the training set used to identify appropriate threshold values comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000 , at least 3000 , at least 4000, or more qualified samples. It may advantageous to use larger sets of qualified samples to improve the diagnostic utility of the threshold values.
  • normalizing value refers to a numerical value that relates the number of sequence tags identified for the sequence (e.g. chromosome or chromosome segment) of interest to the number of sequence tags identified for the normalizing sequence (e.g. normalizing chromosome or normalizing chromosome segment).
  • a "normalizing value” can be a chromosome dose as described elsewhere herein, or it can be an NCV (Normalized Chromosome Value) as described elsewhere herein, or it can be an NSV (Normalized Segment Value) as described elsewhere herein.
  • read refers to a DNA sequence of sufficient length (e.g., at least about 30 bp) that can be used to identify a larger sequence or region, e.g. that can be aligned and specifically assigned to a chromosome or genomic region or gene.
  • sequence tag is herein used interchangeably with the term "mapped sequence tag” to refer to a sequence read that has been specifically assigned i.e. mapped, to a larger sequence e.g. a reference genome, by alignment.
  • Mapped sequence tags are uniquely mapped to a reference genome i.e. they are assigned to a single location to the reference genome. Tags that can be mapped to more than one location on a reference genome i.e. tags that do not map uniquely, are not included in the analysis.
  • aligning refers to one or more sequences that are identified as a match in terms of the order of their nucleic acid molecules to a known sequence from a reference genome. Such alignment can be done manually or by a computer algorithm, examples including the Efficient Local Alignment of Nucleotide Data (ELAND) computer program distributed as part of the Illumina Genomics Analysis pipeline.
  • ELAND Efficient Local Alignment of Nucleotide Data
  • the matching of a sequence read in aligning can be a 100% sequence match or less than 100% (non-perfect match).
  • reference genome refers to any particular known genome sequence, whether partial or complete, of any organism or virus which may be used to reference identified sequences from a subject.
  • reference genome used for human subjects as well as many other organisms is found at the National Center for Biotechnology Information at www.ncbi.nlm.nih.gov.
  • a "genome” refers to the complete genetic information of an organism or virus, expressed in nucleic acid sequences.
  • clinically-relevant sequence refers to a nucleic acid sequence that is known or is suspected to be associated or implicated with a genetic or disease condition. Determining the absence or presence of a clinically-relevant sequence can be useful in determining a diagnosis or confirming a diagnosis of a medical condition, or providing a prognosis for the development of a disease.
  • nucleic acid when used in the context of a nucleic acid or a mixture of nucleic acids, herein refers to the means whereby the nucleic acid(s) are obtained from the source from which they originate.
  • a mixture of nucleic acids that is derived from two different genomes means that the nucleic acids e.g. cfDNA, were naturally released by cells through naturally occurring processes such as necrosis or apoptosis.
  • a mixture of nucleic acids that is derived from two different genomes means that the nucleic acids were extracted from two different types of cells from a subject.
  • mixed sample refers to a sample containing a mixture of nucleic acids, which are derived from different genomes.
  • maternal sample refers to a biological sample obtained from a pregnant subject e.g. a woman.
  • biological fluid refers to a liquid taken from a biological source and includes, for example, blood, serum, plasma, sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears, saliva, and the like.
  • blood serum
  • plasma sputum
  • lavage fluid cerebrospinal fluid
  • urine semen
  • sweat tears
  • saliva saliva
  • the terms "blood,” “plasma” and “serum” expressly encompass fractions or processed portions thereof.
  • sample expressly encompasses a processed fraction or portion derived from the biopsy, swab, smear, etc.
  • maternal nucleic acids and “fetal nucleic acids” herein refer to the nucleic acids of a pregnant female subject and the nucleic acids of the fetus being carried by the pregnant female, respectively.
  • corresponding to refers to a nucleic acid sequence e.g. a gene or a chromosome, that is present in the genome of different subjects, and which does not necessarily have the same sequence in all genomes, but serves to provide the identity rather than the genetic information of a sequence of interest e.g. a gene or chromosome.
  • substantially cell free encompasses preparations of the desired sample from which components that are normally associated with it are removed.
  • a plasma sample is rendered essentially cell free by removing blood cells e.g. red cells, which are normally associated with it.
  • substantially free samples are processed to remove cells that would otherwise contribute to the desired genetic material that is to be tested for a CNV.
  • fetal fraction refers to the fraction of fetal nucleic acids present in a sample comprising fetal and maternal nucleic acid.
  • chromosome refers to the heredity-bearing gene carrier of a living cell which is derived from chromatin and which comprises DNA and protein components (especially histones).
  • DNA and protein components especially histones.
  • the conventional internationally recognized individual human genome chromosome numbering system is employed herein.
  • polynucleotide length refers to the absolute number of nucleic acid molecules (nucleotides) in a sequence or in a region of a reference genome.
  • chromosome length refers to the known length of the chromosome given in base pairs e.g. provided in the
  • NCBI36/hgl8 assembly of the human chromosome found on the world wide web at
  • subject refers to a human subject as well as a non-human subject such as a mammal, an invertebrate, a vertebrate, a fungus, a yeast, a bacteria, and a virus.
  • a non-human subject such as a mammal, an invertebrate, a vertebrate, a fungus, a yeast, a bacteria, and a virus.
  • condition herein refers to "medical condition” as a broad term that includes all diseases and disorders, but can include [injuries] and normal health situations, such as pregnancy, that might affect a person's health, benefit from medical assistance, or have implications for medical treatments.
  • partial when used in reference to a chromosomal aneuploidy herein refers to a gain or loss of a portion of a chromosome.
  • mosaic herein refers to denote the presence of two populations of cells with different karyotypes in one individual who has developed from a single fertilized egg. Mosaicism may result from a mutation during development which is propagated to only a subset of the adult cells.
  • non-mosaic refers to an organism e.g. a human fetus, composed of cell of one karyotypes.
  • using a chromosome when used in reference to determining a chromosome dose, herein refers to using the sequence information obtained for a chromosome i.e. the number of sequence tags obtained for a chromosome.
  • sensitivity is equal to the number of true positives divided by the sum of true positives and false negatives.
  • patient sample refers to a biological sample obtained from a patient i.e. a recipient of medical attention, care or treatment.
  • the patient sample can be any of the samples described herein.
  • the patient sample is obtained by non-invasive procedures e.g.
  • peripheral blood sample or a stool sample
  • hypodiploid refers to a chromosome number that is one or more lower than the normal haploid number of chromosomes characteristic for the species.
  • the invention provides a method for determining copy number variations (CNV) of different sequences of interest in a test sample that comprises a mixture of nucleic acids derived from two different genomes, and which are known or are suspected to differ in the amount of one or more sequence of interest.
  • Copy number variations determined by the method of the invention include gains or losses of entire chromosomes, alterations involving very large chromosomal segments that are microscopically visible, and an abundance of sub-microscopic copy number variation of DNA segments ranging from kilobases (kb) to megabases (Mb) in size.
  • the method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability.
  • the method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions.
  • CNV that can be determined according to the present method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.
  • Any aneuploidy can be determined from sequencing information that is obtained by sequencing only once the nucleic acids of a test sample.
  • CNV in the human genome significantly influence human diversity and predisposition to disease (Redon et ai, Nature 23:444-454 [2006], Shaikh et al. Genome Res 19: 1682-1690 [2009]) .
  • CNVs have been known to contribute to genetic disease through different mechanisms, resulting in either imbalance of gene dosage or gene disruption in most cases. In addition to their direct correlation with genetic disorders, CNVs are known to mediate phenotypic changes that can be deleterious.
  • CNV arise from genomic rearrangements, primarily owing to deletion, duplication, insertion, and unbalanced translocation events.
  • NGS next generation sequencing technology
  • clonally amplified DNA templates or single DNA molecules are sequenced in a massively parallel fashion within a flow cell ⁇ e.g. as described in Volkerding et al. Clin Chem 55:641-658 [2009]; Metzker M Nature Rev 11:31-46 [2010]).
  • NGS provides quantitative information, in that each sequence read is a countable "sequence tag" representing an individual clonal DNA template or a single DNA molecule.
  • the sequencing technologies of NGS include pyrosequencing, sequencing-by-synthesis with reversible dye terminators, sequencing by oligonucleotide probe ligation and ion semiconductor sequencing.
  • DNA from individual samples can be sequenced individually ⁇ i.e. singleplex sequencing) or DNA from multiple samples can be pooled and sequenced as indexed genomic molecules ⁇ i.e. multiplex sequencing) on a single sequencing run, to generate up to several hundred million reads of DNA sequences. Examples of sequencing technologies that can be used to obtain the sequence information according to the present method are described below.
  • sequencing technologies are available commercially, such as the sequencing-by- hybridization platform from Affymetrix Inc. (Sunnyvale, CA) and the sequencing-by-synthesis platforms from 454 Life Sciences (Bradford, CT), Illumina/Solexa (Hayward, CA) and Helicos Biosciences (Cambridge, MA), and the sequencing-by-ligation platform from Applied Biosystems (Foster City, CA), as described below.
  • other single molecule sequencing technologies include the SMRTTM technology of Pacific Biosciences, the Ion TorrentTM technology, and nanopore sequencing being developed for example, by Oxford Nanopore Technologies.
  • Sanger sequencing including the automated Sanger sequencing, can also be employed by the method of the invention. Additional sequencing methods nucleic acid imaging technologies e.g. atomic force microscopy (AFM) or transmission electron microscopy (TEM). Exemplary sequencing technologies are described below.
  • AFM atomic force microscopy
  • TEM transmission electron microscopy
  • the present method comprises obtaining sequence information for the nucleic acids in a test sample e.g. cfDNA in a maternal sample, using single molecule sequencing technology of the Helicos True Single Molecule Sequencing (tSMS) technology ⁇ e.g. as described in Harris T.D. et al., Science 320: 106-109 [2008]).
  • tSMS Helicos True Single Molecule Sequencing
  • a DNA sample is cleaved into strands of approximately 100 to 200 nucleotides, and a polyA sequence is added to the 3' end of each DNA strand. Each strand is labeled by the addition of a fluorescently labeled adenosine nucleotide.
  • the DNA strands are then hybridized to a flow cell, which contains millions of oligo-T capture sites that are immobilized to the flow cell surface.
  • the templates can be at a density of about 100 million templates/cm 2 .
  • the flow cell is then loaded into an instrument, e.g., HeliScopeTM sequencer, and a laser illuminates the surface of the flow cell, revealing the position of each template.
  • a CCD camera can map the position of the templates on the flow cell surface.
  • the template fluorescent label is then cleaved and washed away.
  • the sequencing reaction begins by introducing a DNA polymerase and a fluorescently labeled nucleotide.
  • the oligo-T nucleic acid serves as a primer.
  • the polymerase incorporates the labeled nucleotides to the primer in a template directed manner.
  • the polymerase and unincorporated nucleotides are removed.
  • the templates that have directed incorporation of the fluorescently labeled nucleotide are discerned by imaging the flow cell surface.
  • a cleavage step removes the fluorescent label, and the process is repeated with other fluorescently labeled nucleotides until the desired read length is achieved.
  • Sequence information is collected with each nucleotide addition step.
  • Whole genome sequencing by single molecule sequencing technologies excludes PCR-based amplification in the preparation of the sequencing libraries, and the directness of sample preparation allows for direct measurement of the sample, rather than measurement of copies of that sample.
  • the present method comprises obtaining sequence information for the nucleic acids in the test sample e.g. cfDNA in a maternal test sample, using the 454 sequencing (Roche) (e.g. as described in Margulies, M. et al. Nature 437:376-380 [2005]).
  • 454 sequencing involves two steps. In the first step, DNA is sheared into fragments of approximately 300-800 base pairs, and the fragments are blunt-ended. Oligonucleotide adaptors are then ligated to the ends of the fragments. The adaptors serve as primers for amplification and sequencing of the fragments.
  • the fragments can be attached to DNA capture beads, e.g., streptavidin-coated beads using, e.g., Adaptor B, which contains 5'-biotin tag.
  • the fragments attached to the beads are PCR amplified within droplets of an oil-water emulsion. The result is multiple copies of clonally amplified DNA fragments on each bead.
  • the beads are captured in wells (pico-liter sized). Pyrosequencing is performed on each DNA fragment in parallel. Addition of one or more nucleotides generates a light signal that is recorded by a CCD camera in a sequencing instrument. The signal strength is proportional to the number of nucleotides incorporated.
  • Pyrosequencing makes use of pyrophosphate (PPi) which is released upon nucleotide addition.
  • PPi is converted to ATP by ATP sulfurylase in the presence of adenosine 5' phosphosulfate.
  • Lucif erase uses ATP to convert luciferin to oxyluciferin, and this reaction generates light that is measured and analyzed.
  • the present method comprises obtaining sequence information for the nucleic acids in the test sample e.g. cfDNA in a maternal test sample, using the SOLiDTM technology (Applied Biosystems).
  • SOLiDTM sequencing-by-ligation genomic DNA is sheared into fragments, and adaptors are attached to the 5' and 3' ends of the fragments to generate a fragment library.
  • internal adaptors can be introduced by ligating adaptors to the 5' and 3' ends of the fragments, circularizing the fragments, digesting the circularized fragment to generate an internal adaptor, and attaching adaptors to the 5' and 3' ends of the resulting fragments to generate a mate- paired library.
  • clonal bead populations are prepared in microreactors containing beads, primers, template, and PCR components. Following PCR, the templates are denatured and beads are enriched to separate the beads with extended templates. Templates on the selected beads are subjected to a 3' modification that permits bonding to a glass slide. The sequence can be determined by sequential hybridization and ligation of partially random oligonucleotides with a central determined base (or pair of bases) that is identified by a specific fluorophore. After a color is recorded, the ligated oligonucleotide is cleaved and removed and the process is then repeated.
  • the present method comprises obtaining sequence information for the nucleic acids in the test sample e.g. cfDNA in a maternal test sample, using the single molecule, realtime (SMRTTM) sequencing technology of Pacific Biosciences.
  • SMRTTM single molecule, realtime sequencing technology
  • Single DNA polymerase molecules are attached to the bottom surface of individual zero-mode wavelength detectors (ZMW detectors) that obtain sequence information while phospholinked nucleotides are being incorporated into the growing primer strand.
  • ZMW detectors zero-mode wavelength detectors
  • a ZMW is a confinement structure which enables observation of incorporation of a single nucleotide by DNA polymerase against the background of fluorescent nucleotides that rapidly diffuse in an out of the ZMW (in microseconds). It takes several milliseconds to incorporate a nucleotide into a growing strand. During this time, the fluorescent label is excited and produces a fluorescent signal, and the fluorescent tag is cleaved off. Measurement of the corresponding fluorescence of the dye indicates which base was incorporated. The process is repeated.
  • the present method comprises obtaining sequence information for the nucleic acids in the test sample e.g. cfDNA in a maternal test sample, using nanopore sequencing (e.g. as described in Soni GV and Meller A. Clin Chem 53: 1996-2001 [2007]).
  • Nanopore sequencing DNA analysis techniques are being industrially developed by a number of companies, including Oxford Nanopore Technologies (Oxford, United Kingdom).
  • Nanopore sequencing is a single- molecule sequencing technology whereby a single molecule of DNA is sequenced directly as it passes through a nanopore.
  • a nanopore is a small hole, of the order of 1 nanometer in diameter.
  • the present method comprises obtaining sequence information for the nucleic acids in the test sample e.g.
  • DNA molecules can be placed into reaction chambers, and the template molecules can be hybridized to a sequencing primer bound to a polymerase. Incorporation of one or more triphosphates into a new nucleic acid strand at the 3' end of the sequencing primer can be discerned by a change in current by a chemFET.
  • An array can have multiple chemFET sensors.
  • single nucleic acids can be attached to beads, and the nucleic acids can be amplified on the bead, and the individual beads can be transferred to individual reaction chambers on a chemFET array, with each chamber having a chemFET sensor, and the nucleic acids can be sequenced.
  • the present method comprises obtaining sequence information for the nucleic acids in the test sample e.g. cfDNA in a maternal test sample, using the Halcyon Molecular' s technology, which uses transmission electron microscopy (TEM).
  • the method termed Individual Molecule Placement Rapid Nano Transfer (IMPRNT), comprises utilizing single atom resolution transmission electron microscope imaging of high-molecular weight (150kb or greater) DNA selectively labeled with heavy atom markers and arranging these molecules on ultra-thin films in ultra-dense (3nm strand-to- strand) parallel arrays with consistent base-to-base spacing.
  • the electron microscope is used to image the molecules on the films to determine the position of the heavy atom markers and to extract base sequence information from the DNA.
  • the method is further described in PCT patent publication WO 2009/046445. The method allows for sequencing complete human genomes in less than ten minutes.
  • the DNA sequencing technology is the Ion Torrent single molecule sequencing, which pairs semiconductor technology with a simple sequencing chemistry to directly translate chemically encoded information (A, C, G, T) into digital information (0, 1) on a semiconductor chip.
  • Ion Torrent uses a high-density array of micro-machined wells to perform this biochemical process in a massively parallel way. Each well holds a different DNA molecule. Beneath the wells is an ion-sensitive layer and beneath that an ion sensor.
  • a nucleotide for example a C
  • a hydrogen ion will be released.
  • the charge from that ion will change the pH of the solution, which can be detected by Ion Torrent's ion sensor.
  • the sequencer essentially the world's smallest solid-state pH meter— calls the base, going directly from chemical information to digital information.
  • the Ion personal Genome Machine (PGMTM) sequencer then sequentially floods the chip with one nucleotide after another. If the next nucleotide that floods the chip is not a match. No voltage change will be recorded and no base will be called. If there are two identical bases on the DNA strand, the voltage will be double, and the chip will record two identical bases called. Direct detection allows recordation of nucleotide incorporation in seconds.
  • the present method comprises obtaining sequence information for the nucleic acids in the test sample e.g. cfDNA in a maternal test sample, using sequencing by hybridization.
  • Seqeuncing-by-hybridization comprises contacting the plurality of polynucleotide sequences with a plurality of polynucleotide probes, wherein each of the plurality of polynucleotide probes can be optionally tethered to a substrate.
  • the substrate might be flat surface comprising an array of known nucleotide sequences. The pattern of hybridization to the array can be used to determine the polynucleotide sequences present in the sample.
  • each probe is tethered to a bead, e.g., a magnetic bead or the like.
  • Hybridization to the beads can be determined and used to identify the plurality of polynucleotide sequences within the sample.
  • the present method comprises obtaining sequence information for the nucleic acids in the test sample e.g. cfDNA in a maternal test sample, by massively parallel sequencing of millions of DNA fragments using Illumina's sequencing-by- synthesis and reversible terminator-based sequencing chemistry (e.g. as described in Bentley et al., Nature 6:53-59 [2009]).
  • Template DNA can be genomic DNA e.g. cfDNA.
  • genomic DNA from isolated cells is used as the template, and it is fragmented into lengths of several hundred base pairs.
  • cfDNA is used as the template, and fragmentation is not required as cfDNA exists as short fragments.
  • fetal cfDNA circulates in the bloodstream as fragments approximately 170 base pairs (bp) in length (Fan et al., Clin Chem 56: 1279-1286 [2010]), and no fragmentation of the DNA is required prior to sequencing.
  • Illumina's sequencing technology relies on the attachment of fragmented genomic DNA to a planar, optically transparent surface on which oligonucleotide anchors are bound. Template DNA is end-repaired to generate 5'-phosphorylated blunt ends, and the polymerase activity of Klenow fragment is used to add a single A base to the 3' end of the blunt phosphorylated DNA fragments.
  • oligonucleotide adapters which have an overhang of a single T base at their 3' end to increase ligation efficiency.
  • the adapter oligonucleotides are complementary to the flow-cell anchors.
  • adapter-modified, single-stranded template DNA is added to the flow cell and immobilized by hybridization to the anchors.
  • Attached DNA fragments are extended and bridge amplified to create an ultra-high density sequencing flow cell with hundreds of millions of clusters, each containing ⁇ 1 ,000 copies of the same template.
  • the randomly fragmented genomic DNA e.g.
  • cfDNA is amplified using PCR before it is subjected to cluster amplification.
  • an amplification-free genomic library preparation is used, and the randomly fragmented genomic DNA e.g. cfDNA is enriched using the cluster amplification alone (Kozarewa et al., Nature Methods 6:291-295 [2009]).
  • the templates are sequenced using a robust four-color DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescent dyes. High-sensitivity fluorescence detection is achieved using laser excitation and total internal reflection optics. Short sequence reads of about 20-40 bp e.g.
  • the reference genome sequence is the GRCh37/hgl9, which is available on the world wide web at genome.ucsc.edu/cgi- bin/hgGateway.
  • Other sources of public sequence information include GenBank, dbEST, dbSTS, EMBL (the European Molecular Biology Laboratory), and the DDBJ (the DNA Databank of Japan).
  • BLAST Altschul et al., 1990
  • BLITZ MPsrch
  • FASTA Piererson & Lipman
  • BOWTffi Landmead et al, Genome Biology 10:R25.1-R25.10 [2009]
  • ELAND ELAND
  • one end of the clonally expanded copies of the plasma cfDNA molecules is sequenced and processed by bioinformatic alignment analysis for the Illumina Genome Analyzer, which uses the Efficient Large-Scale Alignment of Nucleotide Databases (ELAND) software.
  • ELAND ELAND
  • the mapped sequence tags comprise sequence reads of about 20bp, about 25bp, about 30bp, about 35bp, about 40bp, about 45bp, about 50bp, about 55bp, about 60bp, about 65bp, about 70bp, about 75bp, about 80bp, about 85bp, about90bp, about 95bp, about lOObp, about HObp, about 120bp, about 130, about 140bp, about 150bp, about 200bp, about 250bp, about 300bp, about 350bp, about 400bp, about 450bp, or about 500bp.
  • the mapped sequence tags comprise sequence reads that are 36bp. Mapping of the sequence tags is achieved by comparing the sequence of the tag with the sequence of the reference to determine the chromosomal origin of the sequenced nucleic acid (e.g. cfDNA) molecule, and specific genetic sequence information is not needed. A small degree of mismatch (0-2 mismatches per sequence tag) may be allowed to account for minor polymorphisms that may exist between the reference genome and the genomes in the mixed sample. A plurality of sequence tags are obtained per sample.
  • the CNV i.e. the over- or under-representation of a sequence of interest e.g. a chromosome or portion thereof, in the mixed DNA sample is determined.
  • the method does not require differentiation between the two genomes.
  • the accuracy required for correctly determining whether a CNV e.g. aneuploidy, is present or absent in a sample is predicated on the variation of the number of sequence tags that map to the reference genome among samples within a sequencing run (inter-chromosomal variability), and the variation of the number of sequence tags that map to the reference genome in different sequencing runs (inter-sequencing variability).
  • inter-chromosomal variability variability
  • inter-sequencing variability variation of the number of sequence tags that map to the reference genome in different sequencing runs.
  • the variations can be particularly pronounced for tags that map to GC-rich or GC-poor reference sequences.
  • Other variations can result from using different protocols for the extraction and purification of the nucleic acids, the preparation of the sequencing libraries, and the use of different sequencing platforms.
  • Chromosome doses are based on the knowledge of normalizing sequences (normalizing chromosome sequences or normalizing segment sequences), to intrinsically account for the accrued variability stemming from interchromosomal (intra-run), and inter-sequencing (inter-run) and platform-dependent variability.
  • Chromosome doses are based on the knowledge of a normalizing chromosome sequence, which can be composed of a single chromosome, or of two or more chromosomes selected from chromosomes 1-22, X, and Y.
  • normalizing chromosome sequences can be composed of a single chromosome segment, or of two or more segments of one chromosome or of two or more chromosomes. Segment doses are based on the knowledge of a normalizing segment sequence, which can be composed of a single segment of any one chromosome, or of two or more segments of any two or more of chromosomes 1-22, X, and Y.
  • Normalizing sequences are identified using sequence information from a set of qualified samples obtained from subjects known to comprise cells having a normal copy number for any one sequence of interest e.g. a chromosome or segment thereof. Determination of normalizing sequences is outlined in steps 100, 120, 130, 140, and 145 of the embodiment of the method depicted in Figure 1. The sequence information obtained from the qualified samples is also used for determining statistically meaningful identification of chromosomal aneuploidies in test samples (step 155 Figure 1, and Examples).
  • Figure 1 provides a flow diagram of an embodiment of the method of the invention 100 for determining a CNV of a sequence of interest e.g. a chromosome or segment thereof, in a biological sample.
  • a biological sample is obtained from a subject and comprises a mixture of nucleic acids contributed by different genomes.
  • the different genomes can be contributed to the sample by two individuals e.g. the different genomes are contributed by the fetus and the mother carrying the fetus.
  • the genomes are contributed to the sample by aneuploid cancerous cells and normal euploid cells from the same subject e.g. a plasma sample from a cancer patient.
  • a set of qualified samples is obtained to identify qualified normalizing sequences and to provide variance values for use in determining statistically meaningful identification of CNV in test samples.
  • a plurality of biological qualified samples are obtained from a plurality of subjects known to comprise cells having a normal copy number for any one sequence of interest.
  • the qualified samples are obtained from mothers pregnant with a fetus that has been confirmed using cytogenetic means to have a normal copy number of chromosomes.
  • the biological qualified samples may be a biological fluid e.g. plasma, or any suitable sample as described below.
  • a qualified sample contains a mixture of nucleic acid molecules e.g. cfDNA molecules.
  • the qualified sample is a maternal plasma sample that contains a mixture of fetal and maternal cfDNA molecules.
  • Sequence information for normalizing chromosomes and/or segments thereof is obtained by sequencing at least a portion of the nucleic acids e.g. fetal and maternal nucleic acids, using any known sequencing method.
  • any one of the Next Generation Sequencing (NGS) methods described elsewhere herein is used to sequence the fetal and maternal nucleic acids as single or clonally amplified molecules.
  • step 120 at least a portion of each of all the qualified nucleic acids contained in the qualified samples are sequenced to generate millions of sequence reads e.g. 36bp reads, which are aligned to a reference genome, e.g. hgl8.
  • the sequence reads comprise about 20bp, about 25bp, about 30bp, about 35bp, about 40bp, about 45bp, about 50bp, about 55bp, about 60bp, about 65bp, about 70bp, about 75bp, about 80bp, about 85bp, about90bp, about 95bp, about lOObp, about 1 lObp, about 120bp, about 130, about 140bp, about 150bp, about 200bp, about 250bp, about 300bp, . about 350bp, about 400bp, about 450bp, or about 500bp.
  • the mapped sequence reads comprise 36bp. Sequence reads are aligned to a reference genome, and the reads that are uniquely mapped to the reference genome are known as sequence tags.
  • At least about 3 x 10 6 qualified sequence tags, at least about 5 x 10 6 qualified sequence tags, at least about 8 x 10 6 qualified sequence tags, at least about 10 x 10 6 qualified sequence tags, at least about 15 x 10 6 qualified sequence tags, at least about 20 x 10 6 qualified sequence tags, at least about 30 x 10 6 qualified sequence tags, at least about 40 x 10 6 qualified sequence tags, or at least about 50 x 10 6 qualified sequence tags comprising between 20 and 40bp reads are obtained from reads that map uniquely to a reference genome.
  • step 130 all the tags obtained from sequencing the nucleic acids in the qualified samples are counted to determine a qualified sequence tag density.
  • sequence tag density is determined as the number of qualified sequence tags mapped to the sequence of interest on the reference genome.
  • qualified sequence tag density is determined as the number of qualified sequence tags mapped to a sequence of interest normalized to the length of the qualified sequence of interest to which they are mapped.
  • Sequence tag densities that are determined as a ratio of the tag density relative to the length of the sequence of interest are herein referred to as tag density ratios. Normalization to the length of the sequence of interest is not required, and may be included as a step to reduce the number of digits in a number to simplify it for human interpretation.
  • sequence tag density for a sequence of interest e.g. a clinically-relevant sequence
  • sequence tag densities for additional sequences from which normalizing sequences are identified subsequently are determined, as are the sequence tag densities for additional sequences from which normalizing sequences are identified subsequently.
  • the sequence of interest is a chromosome that is associated with a complete chromosomal aneuploidy e.g. chromosome 21
  • the qualified normalizing sequence is a complete chromosome that is not associated with a chromosomal aneuploidy and whose variation in sequence tag density best approximates that of the sequence (i.e. chromosome) of interest e.g.
  • chromosome 21 Any one or more of chromosomes 1-22, X, and Y can be a sequence of interest, and one or more chromosomes can be identified as the normalizing sequence for each of the any one chromosomes 1-22, X and Y in the qualified samples.
  • the normalizing chromosome can be an individual chromosome or it can be a group of chromosomes as described elsewhere herein.
  • the sequence of interest is a segment of a chromosome associated with a partial aneuploidy, e.g. a chromosomal deletion or insertion, or unbalanced chromosomal translocation
  • the normalizing sequence is a chromosomal segment that is not associated with the partial aneuploidy and whose variation in sequence tag density best approximates that of the chromosome segment associated with the partial aneuploidy.
  • Any one or more segments of any one or more chromosomes 1-22, X, and Y can be a sequence of interest.
  • the qualified normalizing sequence has a variation in sequence tag density best approximates that of the sequence of interest as determined in the qualified samples.
  • a qualified normalizing sequence is a sequence that has the smallest variability i.e. the variability of the normalizing sequence is closest to that of the sequence of interest.
  • the normalizing sequence is a sequence that best distinguishes one or more qualified, samples from one or more affected samples, which implies that the normalizing sequence is a sequence that has the greatest differentiability i.e. the differentiability of the normalizing sequence is such that it provides optimal differentiation to a sequence of interest in an affected test sample to easily distinguish the affected test sample from other unaffected samples.
  • the normalizing sequence is a sequence that has the smallest variability and the greatest differentiability. The level of differentiability can be determined as a statistical difference between the sequence doses e.g. chromosome doses or segment doses, in a population of qualified samples and the chromosome dose(s) in one or more test samples as described below and shown in the Examples.
  • differentiability can be represented numerically as a T-test value, which represents the statistical difference between the chromosome doses in a population of qualified samples and the chromosome dose(s) in one or more test samples.
  • differentiability can be represented numerically as a Normalized Chromosome Value (NCV), which is a z-score for chromosome doses as long as the distribution for the NCV is normal.
  • NCV Normalized Chromosome Value
  • T-test value which represents the statistical difference between the segment doses in a population of qualified samples and the segment dose(s) in one or more test samples.
  • NSV Normalized Segment Value
  • the mean and standard deviation of chromosome or segment doses in a set of qualified samples can be used.
  • the mean and standard deviation of chromosome or segment doses in a training set comprising qualified samples and affected samples can be used.
  • the normalizing sequence is a sequence that has the smallest variability and the greatest differentiability.
  • the method identifies sequences that inherently have similar characteristics and that are prone to similar variations among samples and sequencing runs, and which are useful for determining sequence doses in test samples.
  • sequence doses i.e. chromosome doses or segment doses
  • a qualified sequence dose i.e. a chromosome dose or a segment dose, for a sequence of interest is determined as the ratio of the sequence tag density for the sequence of interest and the qualified sequence tag density for additional sequences from which normalizing sequences are identified subsequently in step 145.
  • the identified normalizing sequences are used subsequently to determine sequence doses in test samples.
  • the sequence dose in the qualified samples is a chromosome dose that is calculated as the ratio of the number of sequence tags for a chromosome of interest and the number of sequence tags for a normalizing chromosome sequence in a qualified sample.
  • the normalizing chromosome sequence can be a single chromosome, a group of chromosomes, a segment of one chromosome, or a group of segments from different chromosomes.
  • a chromosome dose for a chromosome of interest is determined in a qualified sample as (i) the ratio of the number of tags for a chromosome of interest and the number of tags for a normalizing chromosome sequence composed of a single chromosome, (ii) the ratio of the number of tags for a chromosome of interest and the number of tags for a normalizing chromosome sequence composed of two or more chromosomes, or (iii) the ratio of the number of tags for a chromosome of interest and the number of tags for a normalizing segment sequence composed of a single segment of a chromosome, (iv) the ratio of the number of tags for a chromosome of interest and the number of tags for a normalizing segment sequence composed of two or more segments form one chromosome, or (v) the ratio of the number of tags for a chromosome of interest and the number of tags for a normalizing segment sequence composed of two or more segments form one chromosome, or (
  • chromosome doses for chromosome of interest e.g. chromosome 21 are determined as a ratio of the sequence tag density of chromosome 21 and the sequence tag density for each of all the remaining chromosomes i.e. chromosomes 1-20, chromosome 22, chromosome X, and chromosome Y (i); chromosome doses for chromosome of interest e.g. chromosome 21, are determined as a ratio of the sequence tag density of chromosome 21 and the sequence tag density for all possible combinations of two or more remaining chromosomes (ii); chromosome doses for chromosome of interest e.g.
  • the sequence dose in the qualified samples is a segment dose that is calculated as the ratio of the number of sequence tags for a segment of interest and the number of sequence tags for a normalizing segment sequence in a qualified sample.
  • the normalizing segment sequence can be a segment of one chromosome, or a group of segments from different chromosomes.
  • a segment dose for a segment of interest is determined in a qualified sample as (i) the ratio of the number of tags for a segment of interest and the number of tags for a normalizing segment sequence composed of a single segment of a chromosome, (ii) the ratio of the number of tags for a segment of interest and the number of tags for a normalizing segment sequence composed of two or more segments of one chromosome, or (iii) the ratio of the number of tags for a segment of interest and the number of tags for a normalizing segment sequence composed of two or more segments of two or more different chromosomes.
  • Chromosome doses for one or more chromosomes of interest are determined in all qualified samples, and a normalizing chromosome sequence is identified in step 145.
  • segment doses for one or more segments of interest are determined in all qualified samples, and a normalizing segment sequence is identified in step 145.
  • a normalizing sequence is identified for a sequence of interest as the sequence based on the calculated sequence doses i.e. that results in the smallest variability in sequence dose for the sequence of interest across all qualified samples.
  • the method identifies sequences that inherently have similar characteristics and that are prone to similar variations among samples and sequencing runs, and which are useful for determining sequence doses in test samples.
  • Normalizing sequences for one or more sequences of interest can be identified in a set of qualified samples, and the sequences that are identified in the qualified samples are used subsequently to calculate sequence doses for one or more sequences of interest in each of the test samples (step 150) to determine the presence or absence of aneuploidy in each of the test samples.
  • the normalizing sequence identified for chromosomes or segments of interest may differ when different sequencing platforms are used and/or when differences exist in the purification of the nucleic acid that is to be sequenced and/or preparation of the sequencing library.
  • the use of normalizing sequences according to the method of the invention provides specific and sensitive measure of a variation in copy number of a chromosome or segment thereof irrespective of sample preparation and/or sequencing platform that is used.
  • more than one normalizing sequence is identified i.e. different normalizing sequences can be determined for one sequence of interest, and multiple sequence doses can be determined for one sequence of interest.
  • the variation e.g. coefficient of variation, in chromosome dose for chromosome of interest 21 is least when the sequence tag density of chromosome 14 is used.
  • two, three, four, five, six, seven, eight or more normalizing sequences can be identified for use in determining a sequence dose for a sequence of interest in a test sample.
  • a second dose for chromosome 21 in any one test sample can be determined using chromosome 7, chromosome 9, chromosome 11 or chromosome 12 as the normalizing chromosome sequence as these chromosomes all have CV close to that for chromosome 14 (see Example 2, Table 2).
  • the normalizing chromosome sequence will be a chromosome that results in chromosome doses for the chromosome of interest that has the smallest variability across all samples tested e.g. qualified samples.
  • Normalizing chromosome sequence as a normalizing sequence for chromosome(s)
  • a normalizing chromosome sequence can be a single sequence or it can be a group of sequences.
  • a normalizing sequence is a group of sequences e.g. a group of chromosomes, that is identified as the normalizing sequence for any or more of chromosomes 1-22, X and Y.
  • the group of chromosomes that compose the normalizing sequence for a chromosome of interest i.e.
  • a normalizing chromosome sequence can be a group of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, or twenty-two chromosomes, and including or excluding one or both of chromosomes X , and Y.
  • the group of chromosomes that is identified as the normalizing chromosome sequence is a group of chromosomes that results in chromosome doses for the chromosome of interest that has the smallest variability across all samples tested e.g. qualified samples.
  • individual and groups of chromosomes are tested together for their ability to best mimic the behavior of the sequence of interest for which they are chosen as normalizing chromosome sequences.
  • the normalizing sequence for chromosome 21 is selected from chromosome 9, chromosome 1 , chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 10, chromosome 11 , chromosome 12, chromosome 13, chromosome 14, chromosome 15, chromosome 16, and chromosome 17.
  • the normalizing sequence for chromosome 21 is selected from chromosome 9, chromosome 1, chromosome 2, chromosome 11 , chromosome 12, and chromosome 14.
  • the normalizing sequence for chromosome 21 is a group of chromosomes selected from chromosome 9, chromosome 1 , chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 10, chromosome 11 , chromosome 12, chromosome 13, chromosome 14, chromosome 15, chromosome 16, and chromosome 17.
  • the group of chromosomes is a group selected from chromosome 9, chromosome 1 , chromosome 2, chromosome 11 , chromosome 12, and chromosome 14.
  • the method is further improved by using a normalizing sequence that is determined by systematic calculation of all chromosome doses using each chromosome individually and in all possible combinations with all remaining chromosomes (see Example 7).
  • a systematically determined normalizing chromosome can be determined for each chromosome of interest by systematically calculating all possible chromosome doses using one of any of chromosomes 1-22, X, and Y, and combinations of two or more of chromosomes 1-22, X, and Y to determine which single or group of chromosomes is the normalizing chromosome that results in the least variability of the chromosome dose for a chromosome of interest across a set of qualified samples (see Example 7).
  • the systematically calculated normalizing chromosome sequence for chromosome 21 is a group of chromosomes consisting of chromosome 4, chromosome 14, chromosome 16, chromosome 20,-and chromosome 22. Single or groups of chromosomes can be determined for all chromosomes in the genome.
  • the normalizing sequence for chromosome 18 is selected chromosome 8, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 9, chromosome 10, chromosome 11 , chromosome 12, chromosome 13, and chromosome 14.
  • the normalizing sequence for chromosome 18 is selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14.
  • the normalizing sequence for chromosome 18 is a group of chromosomes selected from chromosome 8, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 9, chromosome 10, chromosome 11, chromosome 12, chromosome 13, and chromosome 14.
  • the group of chromosomes is a group selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14.
  • the normalizing sequence for chromosome 18 is determined by systematic calculation of all possible chromosome doses using each possible normalizing chromosome individually and all possible combinations of normalizing chromosomes (as explained elsewhere herein). Accordingly, in one embodiment, the normalizing sequence for chromosome 18 is a normalizing chromosome consisting of the group of chromosomes consisting of chromosome 2, chromosome 3, chromosome 5, and chromosome 7.
  • the normalizing sequence for chromosome X is selected from chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, chromosome 11 , chromosome 12, chromosome 13, chromosome 14, chromosome 15, and chromosome 16.
  • the normalizing sequence for chromosome X is selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6 and chromosome 8.
  • the normalizing sequence for chromosome X is a group of chromosomes selected from chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, chromosome 11 , chromosome 12, chromosome 13, chromosome 14, chromosome 15, and chromosome 16.
  • the group of chromosomes is a group selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.
  • the normalizing sequence for chromosome X is determined by systematic calculation of all possible chromosome doses using each possible normalizing chromosome individually and all possible combinations of normalizing chromosomes (as explained elsewhere herein). Accordingly, in one embodiment, the normalizing sequence for chromosome X is a normalizing chromosome consisting of the group of chromosome 4 and chromosome 8.
  • the normalizing sequence for chromosome 13 is a chromosome selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, chromosome 11, chromosome 12, chromosome 14, chromosome 18, and chromosome 21.
  • the normalizing sequence for chromosome 13 is a chromosome selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.
  • the normalizing sequence for chromosome 13 is a group of chromosomes selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, chromosome 11, chromosome 12, chromosome 14, chromosome 18, and chromosome 21.
  • the group of chromosomes is a group selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.
  • the normalizing sequence for chromosome 13 is determined by systematic calculation of all possible chromosome doses using each possible normalizing chromosome individually and all possible combinations of normalizing chromosomes (as explained elsewhere herein). Accordingly, in one embodiment, the normalizing sequence for chromosome 13 is a normalizing chromosome comprising the group of chromosome 4 and chromosome 5. In another embodiment, the normalizing sequence for chromosome 13 is a normalizing chromosome consisting of the group of chromosome 4 and chromosome 5.
  • the variation in chromosome dose for chromosome Y is greater than 30 independently of which normalizing chromosome is used in determining the chromosome Y dose. Therefore, any one chromosome, or a group of two or more chromosomes selected from chromosomes 1-22 and chromosome X can be used as the normalizing sequence for chromosome Y.
  • the at least one normalizing chromosome is a group of chromosomes consisting of chromosomes 1-22, and chromosome X.
  • the group of chromosomes consists of chromosome 2, chromosome 3, chromosome 4, chromosome 5, and chromosome 6.
  • the normalizing sequence for chromosome Y is determined by systematic calculation of all possible chromosome doses using each possible normalizing chromosome individually and all possible combinations of normalizing chromosomes (as explained elsewhere herein). Accordingly, in one embodiment, the normalizing sequence for chromosome Y is a normalizing chromosome comprising the group of chromosomes consisting of chromosome 4 and chromosome 6. In another embodiment, the normalizing sequence for chromosome Y is a normalizing chromosome consisting of the group of chromosomes consisting of chromosome 4 and chromosome 6.
  • the normalizing sequence used to calculate the dose of different chromosomes of interest, or of different segments of interest can be the same or it can be a different normalizing sequence for different chromosomes or segments of interest, respectively.
  • the normalizing sequence e.g. a normalizing chromosome (one or a group) for chromosome of interest A can be the same or it can be different from the normalizing sequence e.g. a normalizing chromosome (one or a group) for chromosome of interest B.
  • the normalizing sequence for a complete chromosome may be a complete chromosome or a group of complete chromosomes, or it may be a segment of a chromosome, or a group of segments of one or more chromosomes.
  • Normalizing segment sequence as a normalizing sequence for chromosome(s)
  • the normalizing sequence for a chromosome can be a normalizing segment sequence.
  • the normalizing segment sequence can be a single segment or it can be a group of segments of one chromosome, or they can be segments from two or more different chromosomes.
  • a normalizing segment sequence can be determined by systematic calculation of all combinations of segment sequences in the genome.
  • a normalizing segment sequence for chromosome 21 can be a single segment that is bigger or smaller than the size of chromosome 2, which is approximately 47Mbp (million base pairs) from chromosome 9, which is approximately 140 Mbp.
  • a normalizing sequence for chromosome 21 can be a combination of a sequence form chromosome 1, and a sequence from chromosome 12.
  • the normalizing sequence for chromosome 21 is a normalizing segment sequence of one segment or of a group of two or more segments of chromosomes 1-20, 22, X, and Y.
  • the normalizing sequence for chromosome 18 is a segment or groups segments of chromosomes 1-17, 19-22, X, and Y.
  • the normalizing sequence for chromosome 13 is a segment or groups of segments of chromosomes 1-12, 14-22, X, and Y.
  • the normalizing sequence for chromosome X is a segment or groups segments of chromosomes 1-22, and Y.
  • the normalizing sequence for chromosome Y is a segment or group of segments of chromosomes 1-22, and X. Normalizing segment sequences of single or groups of segments can be determined for all chromosomes in the genome.
  • the two or more segments of a normalizing segment sequence can be segments from one chromosome, or the two or more segments can be segments of two or more different chromosomes.
  • a normalizing segment sequence can be the same for two or more different chromosomes.
  • Normalizin segment sequence as a normalizing sequence for chromosome segment(s)
  • the presence or absence of CNV of a sequence of interest can be determined when the sequence of interest is a segment of a chromosome. Variation in the copy number of a chromosome segment allows for determining the presence or absence of a partial chromosomal aneuploidy.
  • the segment of the chromosome can be of any length. For example, it can range from a kilobase to hundreds of megabases.
  • the human genome occupies just over 3 billion DNA bases, which can be divided into tens, thousands, hundreds of thousands and millions of segments of different sizes of which the copy number can be determined according to the present method.
  • the normalizing sequence for a segment of a chromosome is a normalizing segment sequence, which can be a single segment from any one of the chromosomes 1-22, X and Y, or it can be a group of segments from any one or more of chromosomes 1-22, X, and Y.
  • the normalizing sequence for a segment of interest is a sequence that has a variability across chromosomes and across samples that is closest to that of the segment of interest. Determination of a normalizing sequence can be performed as described for determining the normalizing sequence for a chromosome of interest when the normalizing sequence is a group of segments of any one or more of chromosomes 1-22, X and Y.
  • a normalizing segment sequence of one or a group of segments can be identified by calculating segment doses using one, and all possible combinations of two or more segments as normalizing sequences for the segment of interest in each sample of a set of qualified samples i.e. samples known to be diploid for the segment of interest, and the normalizing sequence is determined as that providing a segment dose having the lowest variability for the segment of interest across all qualified samples, as is described above for normalizing chromosome sequences.
  • the remaining 3 million segments (minus the 1 mg segment of interest) of the approximately 3Gb human genome can be used individually or in combination with each other to calculate segment doses for a segment of interest in a qualified set of sample to determine which one or group of segments would serve as the normalizing segment sequence for qualified and test samples.
  • Segments of interest can vary from about 1000 bases to tens of megabases.
  • Normalizing segment sequences can be composed of one or more segments of the same size as that of the sequence of interest. In other embodiment, the normalizing segment sequence can be composed of segments that differ from that of the sequence of interest, and/or from each other.
  • a normalizing segment sequence for a 10,0000 base long sequence can be 20,000 bases long, and comprise a combination of sequences of different lengths e.g. a 7,000+8,000+5,000 bases.
  • normalizing segment sequences can be determined by systematic calculation of all possible chromosome and/or segment doses using each possible normalizing chromosome segment individually and all possible combinations of normalizing segments (as explained elsewhere herein). Single or groups of segments can be determined for all segments and/or chromosomes in the genome.
  • the normalizing sequence used to calculate the dose of different chromosome segments of interest can be the same or it can be a different normalizing sequence for different chromosome segments of interest.
  • the normalizing sequence e.g. a normalizing segment (one or a group) for chromosome segment of interest A can be the same or it can be different from the normalizing sequence e.g. a normalizing segment (one or a group) for chromosome segment of interest B. Determination of aneuploidies in test samples
  • a sequence dose is determined for a sequence of interest in a test sample comprising a mixture of nucleic acids derived from genomes that differ in one or more sequences of interest.
  • a test sample is obtained from a subject suspected or known to carry a clinically-relevant CNV of a sequence of interest.
  • the test sample may be a biological fluid e.g. plasma, or any suitable sample as described below.
  • a test sample contains a mixture of nucleic acid molecules e.g. cfDNA molecules.
  • the test sample is a maternal plasma sample that contains a mixture of fetal and maternal cfDNA molecules.
  • step 125 at least a portion of the test nucleic acids in the test sample is sequenced as described for the qualified samples to generate millions of sequence reads e.g. 36bp reads.
  • the reads generated from sequencing the nucleic acids in the test sample are uniquely mapped to a reference genome.
  • step 135 all the tags obtained from sequencing the nucleic acids in the test samples are counted to determine a test sequence tag density.
  • the number of test sequence tags mapped to a sequence of interest is normalized to the known length of a sequence of interest to which they are mapped to provide a test sequence tag density ratio.
  • normalization to the known length of a sequence of interest is not required, and may be included as a step to reduce the number of digits in a number to simplify it for human interpretation.
  • the sequence tag density for a sequence of interest e.g. a clinically-relevant sequence
  • the test samples is determined, as are the sequence tag densities for additional sequences that correspond to at least one normalizing sequence identified in the qualified samples.
  • a test sequence dose is determined for a sequence of interest in the test sample.
  • the at least one normalizing sequence can be a single sequence or a group of sequences.
  • the sequence dose for a sequence of interest in a test sample is a ratio of the sequence tag density determined for the sequence of interest in the test sample and the sequence tag density of at least one normalizing sequence determined in the test sample, wherein the normalizing sequence in the test sample corresponds to the normalizing sequence identified in the qualified samples for the particular sequence of interest. For example, if the normalizing sequence identified for chromosome 21 in the qualified samples is determined to be a chromosome e.g.
  • test sequence dose for chromosome 21 is determined as the ratio of the sequence tag density for chromosome 21 in and the sequence tag density for chromosome 14 each determined in the test sample.
  • chromosome doses for chromosomes 13, 18, X, Y, and other chromosomes associated with chromosomal aneuploidies are determined.
  • a normalizing sequence for a chromosome of interest can be one or a group of chromosomes, or one or a group of chromosome segments. As described previously, a sequence of interest can be part of a chromosome e.g. a chromosome segment.
  • the dose for a chromosome segment can be determined as the ratio of the sequence tag density determined for the segment in the test sample and the sequence tag density for the normalizing chromosome segment in the test sample, wherein the normalizing segment in the test sample corresponds to the normalizing segment (single or a group of segments) identified in the qualified samples for the particular segment of interest.
  • Chromosome segments can range from kilobases (kb) to megabases (Mb) in size.
  • threshold values are derived from standard deviation values established for qualified sequence doses determined in a plurality of qualified samples and sequence doses determined for samples known to be aneuploid for a sequence of interest. Accurate classification depends on the differences between probability distributions for the different classes i.e. type of aneuploidy. Preferably, thresholds are chosen from empirical distribution for each type of aneuploidy e.g. trisomy 21.
  • the threshold value that is determined to distinguish samples affected for an aneuploidy of a chromosome can be the same or can be different from the threshold that is determined to distinguish samples affected for a different aneuploidy.
  • the threshold value for each chromosome of interest is determined from the variability in the dose of the chromosome of interest across samples and sequencing runs. The less variable the chromosome dose for any chromosome of interest, the narrower the spread in the dose for the chromosome of interest across all the unaffected samples, which are used to set the threshold for determining different aneuploidies.
  • step 160 the copy number variation of the sequence of interest is determined in the test sample by comparing the test sequence dose for the sequence of interest to at least one threshold value established from the qualified sequence doses.
  • step 165 the calculated dose for a test sequence of interest is compared to that set as the threshold values that are chosen according to a user-defined threshold of reliability to classify the sample as a "normal” an "affected” or a "no call".
  • the "no call" samples are samples for which a definitive diagnosis cannot be made with reliability.
  • Another embodiment of the invention provides a method for providing prenatal diagnosis of a fetal chromosomal aneuploidy in a biological sample comprising fetal and maternal nucleic acid molecules. The diagnosis is made based on obtaining sequence information sequencing at least a portion of the mixture of the fetal and maternal nucleic acid molecules derived from a biological test sample e.g.
  • a maternal plasma sample computing from the sequencing data a normalizing chromosome dose for one or more chromosomes of interest, and/or a normalizing segment dose for one or more segments of interest, and determining a statistically significant difference between the chromosome dose for the chromosome of interest and/or the segment dose for the segment of interest, respectively, in the test sample and a threshold value established in a plurality of qualified (normal) samples, and providing the prenatal diagnosis based on the statistical difference.
  • a diagnosis of normal or affected is made. A "no call" is provided in the event that the diagnosis for normal or affected cannot be made with confidence.
  • Samples that are used for determining a CNV comprise nucleic acids that are present in cells or that are "cell-free".
  • cell-free nucleic acids e.g. cell-free DNA (cfDNA).
  • Cell-free nucleic acids, including cell-free DNA can be obtained by various methods known in the art from biological samples including but not limited to plasma and serum (Chen et at, Nature Med. 2: 1033- 1035 [1996]; Lo et al, Lancet 350: 485-487 [1997]).
  • fractionation, centrifugation e.g., density gradient centrifugation
  • DNA-specific precipitation e.g., DNA-specific precipitation, or high-throughput cell sorting and/or separation methods can be used.
  • the sample comprising the mixture of nucleic acids to which the methods described herein are applied is a biological sample such as a tissue sample, a biological fluid sample, or a cell sample.
  • the mixture of nucleic acids is purified or isolated from the biological sample by any one of the known methods.
  • a sample can consist of purified or isolated polynucleotide, or it can comprise a biological sample such as a tissue sample, a biological fluid sample, or a cell sample.
  • a biological fluid includes, as non-limiting examples, blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, lymph, saliva, cerebrospinal fluid, ravages, bone marrow suspension, vaginal flow, transcervical lavage, brain fluid, ascites, milk, secretions of the respiratory, intestinal and genitourinary tracts, amniotic fluid and leukophoresis samples.
  • the sample is a sample that is easily obtainable by non-invasive procedures e.g. blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, saliva or feces.
  • the biological sample is a peripheral blood sample, or the plasma and serum fractions.
  • the biological sample is a swab or smear, a biopsy specimen, or a cell culture.
  • the sample is a mixture of two or more biological samples e.g. a biological sample can comprise two or more of a biological fluid sample, a tissue sample, and a cell culture sample.
  • a biological sample can comprise two or more of a biological fluid sample, a tissue sample, and a cell culture sample.
  • samples can be obtained from sources, including, but not limited to, samples from different individuals, different developmental stages of the same or different individuals, different diseased individuals (e.g., individuals with cancer or suspected of having a genetic disorder), normal individuals, samples obtained at different stages of a disease in an individual, samples obtained from an individual subjected to different treatments for a disease, samples from individuals subjected to different environmental factors, or individuals with predisposition to a pathology, or individuals with exposure to an infectious disease agent (e.g., HIV).
  • sources including, but not limited to, samples from different individuals, different developmental stages of the same or different individuals, different diseased individuals (e.g., individuals with cancer or suspected of having a genetic disorder), normal individuals, samples obtained at different stages of a disease in an individual, samples obtained from an individual subjected to different treatments for a disease, samples from individuals subjected to different environmental factors, or individuals with predisposition to a pathology, or individuals with exposure to an infectious disease agent (e.g., HIV).
  • infectious disease agent e.g.
  • the sample is a maternal sample that is obtained from a pregnant female, for example a pregnant woman.
  • the sample can be analyzed using the methods described herein to provide a prenatal diagnosis of potential chromosomal abnormalities in the fetus.
  • the maternal sample can be a tissue sample, a biological fluid sample, or a cell sample.
  • a biological fluid includes, as non-limiting examples, blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, lymph, saliva, cerebrospinal fluid, ravages, bone marrow suspension, vaginal flow, transcervical lavage, brain fluid, ascites, milk, secretions of the respiratory, intestinal and genitourinary tracts, and leukophoresis samples.
  • the maternal sample is a mixture of two or more biological samples e.g. a biological sample can comprise two or more of a biological fluid sample, a tissue sample, and a cell culture sample.
  • the sample is a sample that is easily obtainable by non-invasive procedures e.g.
  • the biological sample is a peripheral blood sample, or the plasma and serum fractions.
  • the biological sample is a swab or smear, a biopsy specimen, or a cell culture.
  • the terms "blood,” “plasma” and “serum” expressly encompass fractions or processed portions thereof.
  • the "sample” expressly encompasses a processed fraction or portion derived from the biopsy, swab, smear, etc.
  • Samples can also be obtained from in vitro cultured tissues, cells, or other polynucleotide- containing sources.
  • the cultured samples can be taken from sources including, but not limited to, cultures (e.g., tissue or cells) maintained in different media and conditions (e.g., pH, pressure, or temperature), cultures (e.g., tissue or cells) maintained for different periods of length, cultures (e.g., tissue or cells) treated with different factors or reagents (e.g., a drug candidate, or a modulator), or cultures of different types of tissue or cells.
  • nucleic acid samples are well known and will differ depending upon the nature of the source.
  • One of skill in the art can readily isolate nucleic acid from a source as needed for the method described herein.
  • sample nucleic acids are obtained from as cfDNA, which is not subjected to fragmentation.
  • sample nucleic acids are obtained as genomic DNA, which is subjected to fragmentation into fragments of approximately 500 or more base pairs, and to which NGS methods can be readily applied.
  • Cell-free fetal DNA and RNA circulating in maternal blood can be used for the early noninvasive prenatal diagnosis (NIPD) of an increasing number of genetic conditions, both for pregnancy management and to aid reproductive decision-making.
  • NIPD noninvasive prenatal diagnosis
  • the presence of cell-free DNA circulating in the bloodstream has been known for over 50 years. More recently, presence of small amounts of circulating fetal DNA was discovered in the maternal bloodstream during pregnancy (Lo et al., Lancet 350:485-487 [1997]).
  • cfDNA cell-free fetal DNA
  • cfRNA cell-free fetal RNA
  • the present method is a polymorphism-independent method that for use in NIPD and that does not require that the fetal cfDNA be distinguished from the maternal cfDNA to enable the determination of a fetal aneuploidy.
  • the aneuploidy is a complete chromosomal trisomy or monosomy, or a partial trisomy or monosomy. Partial aneuploidies are caused by loss or gain of part of a chromosome, and encompass chromosomal imbalances resulting from unbalanced translocations, unbalanced inversions, deletions and insertions. By far, the most common known aneuploidy compatible with life is trisomy 21 i.e.
  • Down Syndrome which is caused by the presence of part or all of chromosome 21. Rarely, DS can be cause by an inherited or sporadic defect whereby an extra copy of all or part of chromosome 21 becomes attached to another chromosome (usually chromosome 14) to form a single aberrant chromosome. DS is associated with intellectual impairment, severe learning difficulties and excess mortality caused by long-term health problems such as heart disease. Other aneuploidies with known clinical significance include Edward syndrome (trisomy 18) and Patau Syndrome (trisomy 13), which are frequently fatal within the first few months of life. Abnormalities associated with the number of sex chromosomes are also known and include monosomy X e.g.
  • Turner syndrome and triple X syndrome (XXX) in female births and Klinefelter syndrome (XXY) and XYY syndrome in male births, which are all associated with various phenotypes including sterility and reduction in intellectual skills.
  • the method of the invention can be used to diagnose these and other chromosomal abnormalities prenatally.
  • the trisomy determined by the present invention include without limitation trisomy 21 (T21; Down Syndrome), trisomy 18 (T18; Edward's Syndrome), trisomy 16 (T16), trisomy 22 (T22; Cat Eye Syndrome), trisomy 15 (T15; Prader Willi Syndrome), trisomy 13 (T13; Patau Syndrome), trisomy 8 (T8; Warkany Syndrome) and the XXY (Klinefelter Syndrome), XYY, or XXX trisomies. It will be appreciated that various other complete trisomies and partial trisomies can be determined in fetal cfDNA according to the teachings of the present invention.
  • partial trisomies include, but are not limited to, partial trisomy lq32-44, trisomy 9 p with trisomy, trisomy 4 mosaicism, trisomy 17p, partial trisomy 4q26-qter, trisomy 9, partial 2p trisomy, partial trisomy lq, and/or partial trisomy 6p/monosomy 6q.
  • the method of the present invention can be also used to determine chromosomal monosomy X, and partial monosomies such as, monosomy 13, monosomy 15, monosomy 16, monosomy 21, and monosomy 22, which are known to be involved in pregnancy miscarriage. Partial monosomy of chromosomes typically involved in complete aneuploidy can also be determined by the method of the invention.
  • Monosomy 18p is a rare chromosomal disorder in which all or part of the short arm (p) of chromosome 18 is deleted (monosomic). The disorder is typically characterized by short stature, variable degrees of mental retardation, speech delays, malformations of the skull and facial
  • craniofacial region region, and/or additional physical abnormalities.
  • Associated craniofacial defects may vary greatly in range and severity from case to case.
  • Conditions caused by changes in the structure or number of copies of chromosome 15 include Angelman Syndrome and Prader-Willi Syndrome, which involve a loss of gene activity in the same part of chromosome 15, the 15ql l-ql3 region.
  • Angelman Syndrome and Prader-Willi Syndrome which involve a loss of gene activity in the same part of chromosome 15, the 15ql l-ql3 region.
  • several translocations and microdeletions can be asymptomatic in the carrier parent, yet can cause a major genetic disease in the offspring.
  • a healthy mother who carries the 15ql l-ql3 microdeletion can give birth to a child with Angelman syndrome, a severe
  • Partial monosomy 13q is a rare chromosomal disorder that results when a piece of the long arm (q) of chromosome 13 is missing (monosomic). Infants born with partial monosomy 13q may exhibit low birth weight, malformations of the head and face (craniofacial region), skeletal abnormalities (especially of the hands and feet), and other physical abnormalities. Mental retardation is characteristic of this condition. The mortality rate during infancy is high among individuals born with this disorder. Almost all cases of partial monosomy 13q occur randomly for no apparent reason (sporadic).
  • 22ql l .2 deletion syndrome also known as DiGeorge syndrome, is a syndrome caused by the deletion of a small piece of chromosome 22.
  • the deletion (22 ql 1.2 ) occurs near the middle of the chromosome on the long arm of one of the pair of chromosome.
  • Characteristic signs and symptoms may include birth defects such as congenital heart disease, defects in the palate, most commonly related to neuromuscular problems with closure (velopharyngeal insufficiency), learning disabilities, mild differences in facial features, and recurrent infections.
  • Microdeletions in chromosomal region 22ql 1.2 are associated with a 20 to 30-fold increased risk of schizophrenia.
  • the method of the invention is used to determine partial monosomies including but not limited to monosomy 18p, partial monosomy of chromosome 15 (15ql l-ql3), partial monosomy 13q, and partial monosomy of chromosome 22 can also be determined using the method.
  • the method of the invention can be also used to determine any aneuploidy if one of the parents is a known carrier of such abnormality.
  • These include, but not limited to, mosaic for a small supernumerary marker chromosome (SMC); t(l l ;14)(pl5;pl3) translocation; unbalanced translocation t(8;l l)(p23.2;pl5.5); l lq23 microdeletion; Smith-Magenis syndrome 17pl l .2 deletion; 22ql3.3 deletion; Xp22.3 microdeletion; 10pl4 deletion; 20p microdeletion, DiGeorge syndrome [del(22)(ql 1.2ql 1.23)], Williams syndrome (7ql 1.23 and 7q36 deletions); lp36 deletion; 2p microdeletion; neurofibromatosis type 1 (17ql 1.2 microdeletion), Yq deletion ; Wolf-Hirschhorn syndrome (WHS, 4pl6.3 microdeletion);
  • the present invention provides a method for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acid molecules.
  • the method determines the presence or absence of any four or more different complete chromosomal aneuploidies.
  • the steps of the method comprise (a) obtaining sequence information for the fetal and maternal nucleic acids in the maternal test sample; and (b) using the sequence information to identify a number of sequence tags for each of any one or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing chromosome sequence for each of the any one or more chromosomes of interest.
  • the normalizing chromosome sequence can be a single chromosome, or it can be a group of chromosomes selected from chromosomes 1-22, X, and Y.
  • the method further uses in step (c) the number of sequence tags identified for each of the any one or more chromosomes of interest and the number of sequence tags identified for each normalizing chromosome sequence to calculate a single chromosome dose for each of the any one or more chromosomes of interest; and (d) compares each of the single chromosome doses for each of the any one or more chromosomes of interest to a threshold value for each of the one or more chromosomes of interest, thereby determining the presence or absence of any one or more complete different fetal chromosomal aneuploidies in the maternal test sample.
  • step (c) comprises calculating a single chromosome dose for each chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing chromosome for each of the chromosomes of interest.
  • step (c) comprises calculating a single chromosome dose for each of the chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing chromosome for each of the chromosomes of interest.
  • step (c) comprises calculating a sequence tag ratio for a chromosome of interest by relating the number of sequence tags obtained for the chromosome of interest to the length of the chromosome of interest, and relating the number of tags for the corresponding normalizing chromosome sequence for the chromosome of interest to the length of the normalizing chromosome sequence, and calculating a chromosome dose for the chromosome of interest as a ratio of the sequence tags density of the chromosome of interest and the sequence tag density for the normalizing sequence. The calculation is repeated for each of all chromosomes of interest. Steps (a)-(d) can be repeated for test samples from different maternal subjects.
  • An example of the embodiment whereby four or more complete fetal chromosomal aneuploidies are determined in a maternal test sample comprising a mixture of fetal and maternal cell- free DNA molecules comprises: (a) sequencing at least a portion of cell-free DNA molecules to obtain sequence information for the fetal and maternal cell-free DNA molecules in the test sample; (b) using the sequence information to identify a number of sequence tags for each of any twenty or more chromosomes of interest selected from chromosomes 1-22, X, and Y and to identify a number of sequence tags for a normalizing chromosome for each of the twenty or more chromosomes of interest; (c) using the number of sequence tags identified for each of the twenty or more chromosomes of interest and the number of sequence tags identified for each the normalizing chromosome to calculate a single chromosome dose for each of the twenty or more chromosomes of interest; and (d) comparing each of the single chromosome doses for each of the twenty or more chro
  • the method for determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample as described above uses a normalizing segment sequence for determining the dose of the chromosome of interest.
  • the method comprises (a)obtaining sequence information for said fetal and maternal nucleic acids in said sample; (b) using said sequence information to identify a number of sequence tags for each of any one or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing segment sequence for each of said any one or more chromosomes of interest.
  • the normalizing segment sequence can be a single segment of a chromosome or it can be a group of segments form one or more different chromosomes.
  • the method further uses in step (c) the number of sequence tags identified for each of said any one or more chromosomes of interest and said number of sequence tags identified for said normalizing segment sequence to calculate a single chromosome dose for each of said any one or more chromosomes of interest; and (d) comparing each of said single chromosome doses for each of said any one or more chromosomes of interest to a threshold value for each of said one or more chromosomes of interest, and thereby determining the presence or absence of one or more different complete fetal chromosomal aneuploidies in said sample.
  • step (c) comprises calculating a single chromosome dose for each of said chromosomes of interest as the ratio of the number of sequence tags identified for each of said chromosomes of interest and the number of sequence tags identified for said normalizing segment sequence for each of said chromosomes of interest.
  • step (c) comprises calculating a sequence tag ratio for a chromosome of interest by relating the number of sequence tags obtained for the chromosome of interest to the length of the chromosome of interest, and relating the number of tags for the corresponding normalizing segment sequence for the chromosome of interest to the length of the normalizing segment sequence, and calculating a chromosome dose for the chromosome of interest as a ratio of the sequence tags density of the chromosome of interest and the sequence tag density for the normalizing segment sequence. The calculation is repeated for each of all chromosomes of interest. Steps (a)-(d) can be repeated for test samples from different maternal subjects.
  • NCV normalized chromosome value
  • flj and Sj are the estimated mean and standard deviation, respectively, for the j ' -th chromosome dose in a set of qualified samples, and is the observed y ' -th chromosome dose for test sample i.
  • aneuploidies of sex chromosomes can comprise tetrasomies, pentasomies and other polysomies
  • the number of different complete chromosomal aneuploidies that can be determined according to the present method may be at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 complete chromosomal aneuploidies.
  • the number of different complete fetal chromosomal aneuploidies that are determined is related to the number of chromosomes of interest that are selected for analysis.
  • determining the presence or absence of any one or more different complete fetal chromosomal aneuploidies in a maternal test sample as described above uses a normalizing segment sequence for one chromosome of interest, which is selected from chromosomes 1-22, X, and Y.
  • two or more chromosomes of interest are selected from any two or more of chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, X, or Y.
  • any one or more chromosomes of interest are selected from
  • chromosomes 1-22, X, and Y comprise at least twenty chromosomes selected from chromosomes 1- 22, X, and Y, and wherein the presence or absence of at least twenty different complete fetal chromosomal aneuploidies is determined.
  • any one or more chromosomes of interest selected from chromosomes 1-22, X, and Y is all of chromosomes 1-22, X, and Y, and wherein the presence or absence of complete fetal chromosomal aneuploidies of all of chromosomes 1-22, X, and Y is determined.
  • Complete different fetal chromosomal aneuploidies that can be determined include complete chromosomal trisomies, complete chromosomal monosomies and complete chromosomal polysomies.
  • Examples of complete fetal chromosomal aneuploidies include without limitation trisomies of any one or more of the autosomes e.g. trisomy 2, trisomy 8, trisomy 9, trisomy 21 , trisomy 13, trisomy 16, trisomy 18, trisomy 22; trisomies of the sex chromosomes e.g. 47,XXY, 47 XXX, and 47 XYY; tetrasomies of sex chromosomes e.g. 48,XXYY, 48,XXXY, 48XXXX, and 48.XYYY; pentasomies of sex chromosomes e.g. 49.XXXYY 49,XXXY,
  • the invention provides a method for determining the presence or absence of any one or more different partial fetal chromosomal aneuploidies in a maternal test sample comprising fetal and maternal nucleic acid molecules.
  • the steps of the method comprise (a) obtaining sequence information for the fetal and maternal nucleic acids in said sample; and (b) using the sequence information to identify a number of sequence tags for each of any one or more segments of any one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and to identify a number of sequence tags for a normalizing segment sequence for each of said any one or more segments of any one or more chromosomes of interest.
  • the normalizing segment sequence can be a single segment of a chromosome or it can be a group of segments form one or more different chromosomes.
  • the method further uses in step (c) the number of sequence tags identified for each of any one or more segments of any one or more chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence to calculate a single segment dose for each of any one or more segments of any one or more chromosome of interest; and (d) comparing each of the single chromosome doses for each of any one or more segments of any one or more chromosomes of interest to a threshold value for each of said any one or more chromosomal segments of any one or more chromosome of interest, and thereby determining the presence or absence of one or more different partial fetal chromosomal aneuploidies in said sample.
  • step (c) comprises calculating a single segment dose for each of any one or more segments of any one or more chromosomes of interest as the ratio of the number of sequence tags identified for each of any one or more segments of any one or more chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence for each of any one or more segments of any one or more chromosomes of interest.
  • step (c) comprises calculating a sequence tag ratio for a segment of interest by relating the number of sequence tags obtained for the segment of interest to the length of the segment of interest, and relating the number of tags for the corresponding normalizing segment sequence for the segment of interest to the length of the normalizing segment sequence, and calculating a segment dose for the segment of interest as a ratio of the sequence tags density of the segment of interest and the sequence tag density for the normalizing segment sequence. The calculation is repeated for each of all chromosomes of interest. Steps (a)-(d) can be repeated for test samples from different maternal subjects.
  • a means for comparing segment doses of different sample sets is provided by determining a normalized segment value (NSV), which relates the segment dose in a test sample to the mean of the of the corresponding segment dose in a set of qualified samples.
  • NSV normalized segment value
  • flj and ⁇ ; ⁇ are the estimated mean and standard deviation, respectively, for the j-t segment dose in a set of qualified samples, and is the observed j-th segment dose for test sample i.
  • the presence or absence of one partial fetal chromosomal aneuploidy is determined. In other embodiments, the presence or absence of two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, or more partial fetal chromosomal aneuplodies are determined in a sample. In one embodiment, one segment of interest selected from any one of chromosomes 1- 22, X, and Y is selected from chromosomes 1-22, X, and Y.
  • two or more segments of interest selected from chromosomes 1-22, X, and Y are selected from any two or more of chromosomes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,17, 18, 19, 20, 21 , 22, X, or Y.
  • any one or more segments of interest are selected from chromosomes 1-22, X, and Y comprise at least one, five, ten, 15, 20, 25 or more segments selected from chromosomes 1-22, X, and Y, and wherein the presence or absence of at least one, five, ten, 15, 20, 25 different partial fetal chromosomal aneuploidies is determined.
  • fetal chromosomal aneuploidies include partial duplications, partial multiplications, partial insertions and partial deletions.
  • Examples of partial fetal chromosomal aneuploidies include partial monosomies and partial trisomies of autosomes.
  • Partial monosomies of autosomes include partial monosomy of chromosome 1 , partial monosomy of chromosome 4, partial monosomy of chromosome 5, partial monosomy of chromosome 7, partial monosomy of chromosome 11 , partial monosomy of chromosome 15, partial monosomy of chromosome 17, partial monosomy of chromosome 18, and partial monosomy of chromosome 22.
  • Other partial fetal chromosomal aneuploidies that can be determined according to the present method are described below.
  • the test sample is a maternal sample selected from blood, plasma, serum, urine and saliva samples.
  • the maternal test sample is a plasma sample.
  • the nucleic acid molecules of the maternal sample are a mixture of fetal and maternal cell-free DNA molecules. Sequencing of the nucleic acids can be performed using next generation sequencing (NGS) as described elsewhere herein.
  • NGS next generation sequencing
  • sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
  • sequencing is sequencing-by-ligation.
  • sequencing is single molecule sequencing.
  • an amplification step is performed prior to sequencing. Determination of CNV of clinical disorders
  • the methods described herein can be applied to the determination of any abnormality in the representation of genetic sequences within the genome.
  • tumor DNA which can be recovered and used as surrogate source of tumor DNA, and tumors are characterized by aneuploidy, or inappropriate numbers of gene sequences or even entire chromosomes.
  • the determination of a difference in the amount of a given sequence i.e. a sequence of interest, in a sample from an individual can thus be used in the diagnosis of a medical condition.
  • the method can be used to determine the presence or absence of a chromosomal aneuploidy in a patient suspected or known to be suffering from cancer.
  • the method can also be applied to determining the presence or absence of the status of a disease; to determining the presence or absence of nucleic acids of a pathogen e.g. virus; to determining chromosomal abnormalities associated with graft versus host disease (GVHD), and to determining the contribution of individuals in forensic analyses.
  • a pathogen e.g. virus
  • GVHD graft versus host disease
  • Embodiments of the invention provide for a method to assess copy number variation of a sequence of interest e.g. a clinically-relevant sequence, in a test sample that comprises a mixture of nucleic acids derived from two different genomes, and which are known or are suspected to differ in the amount of one or more sequence of interest.
  • the mixture of nucleic acids is derived from two or more types of cells.
  • the mixture of nucleic acids is derived from normal and cancerous cells derived from a subject suffering from a medical condition e.g. cancer.
  • CIN chromosome instability
  • cfDNA has been found in the circulation of patients diagnosed with malignancies including but not limited to lung cancer (Pathak et al. Clin Chem 52: 1833-1842 [2006]), prostate cancer
  • the method of the invention assesses CNV of a sequence of interest in a sample comprising a mixture of nucleic acids derived from a subject that is suspected or is known to have cancer e.g. carcinoma, sarcoma, lymphoma, leukemia, germ cell tumors and blastoma.
  • the sample is a plasma sample derived (processes) from peripheral blood and that comprises a mixture of cfDNA derived from normal and cancerous cells.
  • the biological sample that is needed to determine whether a CNV is present is derived from a mixture of cancerous and non-cancerous cells from other biological fluids including but not limited to serum, sweat, tears, sputum, urine, sputum, ear flow, lymph, saliva, cerebrospinal fluid, ravages, bone marrow suspension, vaginal flow, transcervical lavage, brain fluid, ascites, milk, secretions of the respiratory, intestinal and genitourinary tracts, and leukophoresis samples, or in tissue biopsies, swabs, or smears.
  • the biological sample is a stool (fecal) sample.
  • sequence of interest is a nucleic acid sequence that is known or is suspected to play a role in the development and/or progression of the cancer.
  • a sequence of interest include nucleic acids sequences i.e. complete chromosomes and/or segments of chromosomes, that are amplified or deleted in cancerous cells as described in the following.
  • the present method can be used to determine the presence or absence of a chromosomal amplification.
  • the chromosomal amplification is the gain of one or more entire chromosomes.
  • the chromosomal amplification is the gain of one or more segments of a chromosome.
  • the chromosomal amplification is the gain of two or more segments of two or more chromosomes.
  • the chromosomal amplification can involve the gain of one or more oncogenes.
  • Examples of the amplification of cellular oncogenes in human tumors include amplifications of: c-myc in promyelocytic leukemia cell line HL60, and in small-cell lung carcinoma cell lines, N-myc in primary neuroblastomas (stages III and IV), neuroblastoma cell lines, retinoblastoma cell line and primary tumors, and small-cell lung carcinoma lines and tumors, L-myc in small-cell lung carcinoma cell lines and tumors, c-myb in acute myeloid leukemia and in colon carcinoma cell lines, c-erbb in epidermoid carcinoma cell, and primary gliomas, c-K-ras-2 in primary carcinomas of lung, colon, bladder, and rectum, N-ras in mammary carcinoma cell line (Varmus H., Ann Rev Genetics 18: 553-612 (1984) [cited in Watson et al., Molecular Biology of the Gene (4th ed.; Benjamin/Cummings Publishing Co. 1987)].
  • the present method can be used to determine the presence or absence of a chromosomal deletion.
  • the chromosomal deletion is the loss of one or more entire chromosomes.
  • the chromosomal deletion is the loss of one or more segments of a chromosome.
  • the chromosomal deletion is the loss of two or more segments of two or more chromosomes.
  • the chromosomal deletion can involve the loss of one or more tumor suppressor genes.
  • retinoblastoma tumor suppressor gene located in chromosome 13ql4
  • the Rb- 1 gene product a 105 kDa nuclear phosphoprotein, apparently plays an important role in cell cycle regulation (Howe et al., Proc Natl Acad Sci (USA) 87:5883-5887 [1990]).
  • Altered or lost expression of the Rb protein is caused by inactivation of both gene alleles either through a point mutation or a chromosomal deletion.
  • Rb-i gene alterations have been found to be present not only in
  • retinoblastomas but also in other malignancies such as osteosarcomas, small cell lung cancer
  • Chromosome 1 abnormalities including duplications, deletions and unbalanced translocations involving chromosome 6 and other partner chromosomes indicate that regions of chromosome 1, in particular Iq21-lq32 and lpl 1-13, might harbor oncogenes or tumor suppressor genes that are pathogenetically relevant to both chronic and advanced phases of myeloproliferative neoplasms (Caramazza et al, Eur J
  • Myeloproliferative neoplasms are also associated with deletions of chromosome 5.
  • Complete loss or interstitial deletions of chromosome 5 are the most common karyotypic abnormality in myelodysplastic syndromes (MDSs). Isolated del(5q)/5q- MDS patients have a more favorable prognosis than those with additional karyotypic defects, who tend to develop myeloproliferative neoplasms (MPNs) and acute myeloid leukemia.
  • MDSs myelodysplastic syndromes
  • allelic loss from several distinct regions on chromosome 3p including 3p25, 3p21-22, 3p21.3, 3pl2— 13 and 3pl4, are the earliest and most frequent genomic abnormalities involved in a wide spectrum of major epithelial cancers of lung, breast, kidney, head and neck, ovary, cervix, colon, pancreas, esophagus, bladder and other organs.
  • Chromosome 21 harboring about 300 genes, may be involved in numerous structural aberrations, e.g., translocations, deletions, and amplifications, in leukemias, lymphomas, and solid tumors.
  • genes located on chromosome 21 have been identified that play an important role in tumorigenesis. Somatic numerical as well as structural chromosome 21 aberrations are associated with leukemias, and specific genes including RUNXl, TMPRSS2, and TFF, which are located in 21q, play a role in tumorigenesis (Fonatsch C Gene Chromosomes Cancer 49:497-508 [2010]).
  • the method provides a means to assess the association between gene amplification and the extent of tumor evolution. Correlation between amplification and/or deletion and stage or grade of a cancer may be prognostically important because such information may contribute to the definition of a genetically based tumor grade that would better predict the future course of disease with more advanced tumors having the worst prognosis. In addition, information about early amplification and/or deletion events may be useful in associating those events as predictors of subsequent disease progression. Gene amplification and deletions as identified by the method can be associated with other known parameters such as tumor grade, histology, Brd/Urd labeling index, hormonal status, nodal involvement, tumor size, survival duration and other tumor properties available from epidemiological and biostatistical studies. For example, tumor DNA to be tested by the method could include atypical hyperplasia, ductal carcinoma in situ, stage I-III cancer and metastatic lymph nodes in order to permit the identification of associations between
  • consistently amplified regions may contain an overexpressed gene, the product of which may be able to be attacked therapeutically (for example, the growth factor receptor tyrosine kinase, pl85 HER2 ).
  • the method can be used to identify amplification and/or deletion events that are associated with drug resistance by determining the copy number variation of nucleic acid sequences from primary cancers to those of cells that have metastasized to other sites. If gene amplification and/or deletion is a manifestation of karyotypic instability that allows rapid development of drug resistance, more amplification and/or deletion in primary tumors from chemoresistant patients than in tumors in chemosensitive patients would be expected. For example, if amplification of specific genes is responsible for the development of drug resistance, regions surrounding those genes would be expected to be amplified consistently in tumor cells from pleural effusions of chemoresistant patients but not in the primary tumors. Discovery of associations between gene amplification and/or deletion and the development of drug resistance may allow the identification of patients that will or will not benefit from adjuvant therapy. )
  • the method of the invention can be used to determine the presence or absence of complete and/or partial chromosomal aneuploidies in any patient sample comprising nucleic acids e.g. DNA or cfDNA (including patient samples that are not maternal samples).
  • the patient sample can be any biological sample type as described elsewhere herein.
  • the sample is obtained by non-invasive procedures.
  • the sample can be a blood sample, or the serum and plasma fractions thereof.
  • the sample can be a urine sample or a fecal sample.
  • the sample is a tissue biopsy sample.
  • the sample comprises nucleic acids e.g. cfDNA or genomic DNA, which is purified, and sequenced using any of the NGS sequencing methods described previously.
  • the present invention provides a method for determining the presence or absence of any one or more different complete chromosomal aneuploidies in a patient test sample comprising nucleic acid molecules. In some embodiments, the method determines the presence or absence of any one or more different complete chromosomal aneuploidies.
  • the steps of the method comprise (a) obtaining sequence information for the patient nucleic acids in the patient test sample; and (b) using the sequence information to identify a number of sequence tags for each of any one or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing chromosome sequence for each of the any one or more chromosomes of interest.
  • the normalizing chromosome sequence can be a single chromosome, or it can be a group of chromosomes selected from chromosomes 1-22, X, and Y.
  • the method further uses in step (c) the number of sequence tags identified for each of the any one or more chromosomes of interest and the number of sequence tags identified for each normalizing chromosome sequence to calculate a single chromosome dose for each of the any one or more chromosomes of interest; and (d) compares each of the single chromosome doses for each of the any one or more chromosomes of interest to a threshold value for each of the one or more chromosomes of interest, thereby determining the presence or absence of any one or more different complete patient chromosomal aneuploidies in the patient test sample.
  • step (c) comprises calculating a single chromosome dose for each chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing chromosome for each of the chromosomes of interest.
  • step (c) comprises calculating a single chromosome dose for each of the chromosomes of interest as the ratio of the number of sequence tags identified for each of the chromosomes of interest and the number of sequence tags identified for the normalizing chromosome for each of the chromosomes of interest.
  • step (c) comprises calculating a sequence tag ratio for a chromosome of interest by relating the number of sequence tags obtained for the chromosome of interest to the length of the chromosome of interest, and relating the number of tags for the corresponding normalizing chromosome sequence for the chromosome of interest to the length of the normalizing chromosome sequence, and calculating a chromosome dose for the chromosome of interest as a ratio of the sequence tags density of the chromosome of interest and the sequence tag density for the normalizing sequence. The calculation is repeated for each of all chromosomes of interest. Steps (a)-(d) can be repeated for test samples from different patients.
  • An example of the embodiment whereby one or more complete chromosomal aneuploidies are determined in a cancer patient test sample comprising cell-free DNA molecules comprises: (a) sequencing at least a portion of cell-free DNA molecules to obtain sequence information for the patient cell-free DNA molecules in the test sample; (b) using the sequence information to identify a number of sequence tags for each of any twenty or more chromosomes of interest selected from chromosomes 1-22, X, and Y and to identify a number of sequence tags for a normalizing chromosome for each of the twenty or more chromosomes of interest; (c) using the number of sequence tags identified for each of the twenty or more chromosomes of interest and the number of sequence tags identified for each the normalizing chromosome to calculate a single chromosome dose for each of the twenty or more chromosomes of interest; and (d) comparing each of the single chromosome doses for each of the twenty or more chromosomes of interest to a threshold value for each of the twenty
  • the method for determining the presence or absence of any one or more different complete chromosomal aneuploidies in a patient test sample as described above uses a normalizing segment sequence for determining the dose of the chromosome of interest.
  • the method comprises (a) obtaining sequence information for the nucleic acids in the sample; (b) using the sequence information to identify a number of sequence tags for each of any one or more chromosomes of interest selected from chromosomes 1-22, X and Y and to identify a number of sequence tags for a normalizing segment sequence for each of any one or more chromosomes of interest.
  • the normalizing segment sequence can be a single segment of a chromosome or it can be a group of segments form one or more different chromosomes.
  • the method further uses in step (c) the number of sequence tags identified for each of said any one or more chromosomes of interest and said number of sequence tags identified for said normalizing segment sequence to calculate a single chromosome dose for each of said any one or more chromosomes of interest; and (d) comparing each of said single chromosome doses for each of said any one or more chromosomes of interest to a threshold value for each of said one or more chromosomes of interest, and thereby determining the presence or absence of one or more different complete chromosomal aneuploidies in the patient sample.
  • step (c) comprises calculating a single chromosome dose for each of said chromosomes of interest as the ratio of the number of sequence tags identified for each of said chromosomes of interest and the number of sequence tags identified for said normalizing segment sequence for each of said chromosomes of interest.
  • step (c) comprises calculating a sequence tag ratio for a chromosome of interest by relating the number of sequence tags obtained for the chromosome of interest to the length of the chromosome of interest, and relating the number of tags for the corresponding normalizing segment sequence for the chromosome of interest to the length of the normalizing segment sequence, and calculating a chromosome dose for the chromosome of interest as a ratio of the sequence tags density of the chromosome of interest and the sequence tag density for the normalizing segment sequence. The calculation is repeated for each of all chromosomes of interest. Steps (a)-(d) can be repeated for test samples from different patients.
  • NCV normalized chromosome value
  • fij and ⁇ are the estimated mean and standard deviation, respectively, for the -th chromosome dose in a set of qualified samples, and is the observed j ' -th chromosome dose for test sample i.
  • the presence or absence of one complete chromosomal aneuploidy is determined. In other embodiments, the presence or absence of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, or twenty four complete chromosomal aneuploidies are determined in a sample, wherein twenty-two of the complete chromosomal aneuploidies correspond to complete chromosomal aneuploidies of any one or more of the autosomes; the twenty-third and twenty fourth chromosomal aneuploidy correspond to a complete chromosomal aneuploidy of chromosomes X and Y.
  • aneuploidies can comprise trisomies, tetrasomies, pentasomies and other polysomies, and the number of complete chromosomal aneuploidies varies in different diseases and in different stages of the same disease, the number of complete chromosomal aneuploidies that are determined according to the present method are at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 complete, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more chromosomal aneuploidies.
  • the method comprises determining the presence or absence of up to 200 or more chromosomal aneuploidies in a sample form a patient suspected or known to be suffering from cancer e.g. colon cancer.
  • the chromosomal aneuploidies include losses of one or more complete chromosomes
  • hypodiploidies gains of complete chromosomes including trisomies, tetrasomies, pentasomies, and other polysomies. Gains and/or losses of segments of chromosomes can also be determined as described elsewhere herein. The method is applicable to determining the presence or absence of different aneuploidies in samples from patients suspected or known to be suffering from any cancer as described elsewhere herein.
  • any one of chromosomes 1-22, X and Y can be the chromosome of interest in determining the presence or absence of any one or more different complete chromosomal aneuploidies in a patient test sample as described above.
  • two or more chromosomes of interest are selected from any two or more of chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21 , 22, X, or Y.
  • any one or more chromosomes of interest are selected from chromosomes 1-22, X, and Y comprise at least twenty chromosomes selected from chromosomes 1-22, X, and Y, and wherein the presence or absence of at least twenty different complete chromosomal aneuploidies is determined.
  • any one or more chromosomes of interest selected from chromosomes 1-22, X, and Y is all of chromosomes 1-22, X, and Y, and wherein the presence or absence of complete chromosomal aneuploidies of all of chromosomes 1-22, X, and Y is determined.
  • Complete different chromosomal aneuploidies that can be determined include complete chromosomal monosomies of any one or more of chromosomes 1-22, X and Y; complete chromosomal trisomies of any one or more of
  • chromosomes 1-22, X and Y complete chromosomal tetrasomies of any one or more of chromosomes 1-22, X and Y; complete chromosomal pentasomies of any one or more of chromosomes 1-22, X and Y; and other complete chromosomal polysomies of any one or more of chromosomes 1-22, X and Y. Determination of Partial Chromosomal Aneuploidies in Patient Samples
  • the invention provides a method for determining the presence or absence of any one or more different partial chromosomal aneuploidies in a patient test sample comprising nucleic acid molecules.
  • the steps of the method comprise (a) obtaining sequence information for the patient nucleic acids in the sample; and (b) using the sequence information to identify a number of sequence tags for each of any one or more segments of any one or more chromosomes of interest selected from chromosomes 1-22, X, and Y and to identify a number of sequence tags for a normalizing segment sequence for each of any one or more segments of any one or more chromosomes of interest.
  • the normalizing segment sequence can be a single segment of a chromosome or it can be a group of segments form one or more different chromosomes.
  • the method further uses in step (c) the number of sequence tags identified for each of any one or more segments of any one or more chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence to calculate a single segment dose for each of any one or more segments of any one or more chromosome of interest; and (d) comparing each of the single chromosome doses for each of any one or more segments of any one or more chromosomes of interest to a threshold value for each of said any one or more chromosomal segments of any one or more chromosome of interest, and thereby determining the presence or absence of one or more different partial chromosomal aneuploidies in said sample.
  • step (c) comprises calculating a single segment dose for each of any one or more segments of any one or more chromosomes of interest as the ratio of the number of sequence tags identified for each of any one or more segments of any one or more chromosomes of interest and the number of sequence tags identified for the normalizing segment sequence for each of any one or more segments of any one or more chromosomes of interest.
  • step (c) comprises calculating a sequence tag ratio for a segment of interest by relating the number of sequence tags obtained for the segment of interest to the length of the segment of interest, and relating the number of tags for the corresponding normalizing segment sequence for the segment of interest to the length of the normalizing segment sequence, and calculating a segment dose for the segment of interest as a ratio of the sequence tags density of the segment of interest and the sequence tag density for the normalizing segment sequence. The calculation is repeated for each of all chromosomes of interest. Steps (a)-(d) can be repeated for test samples from different patients.
  • a means for comparing segment doses of different sample sets is provided by determining a normalized segment value (NSV), which relates the segment dose in a test sample to the mean of the of the corresponding segment dose in a set of qualified samples.
  • NSV normalized segment value
  • NSVi] - x ⁇ i
  • fij and &j are the estimated mean and standard deviation, respectively, for the -th segment dose in a set of qualified samples, and y is the observed j ' -th segment dose for test sample i.
  • the presence or absence of one partial chromosomal aneuploidy is determined. In other embodiments, the presence or absence of two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, or more partial chromosomal aneuploidies are determined in a sample. In one embodiment, one segment of interest selected from any one of chromosomes 1-22, X, and Y is selected from chromosomes 1-22, X, and Y.
  • two or more segments of interest selected from chromosomes 1-22, X, and Y are selected from any two or more of chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, or Y.
  • any one or more segments of interest are selected from chromosomes 1-22, X, and Y comprise at least one, five, ten, 15, 20, 25, 50, 75, 100 or more segments selected from chromosomes 1-22, X, and Y, and wherein the presence or absence of at least one, five, ten, 15, 20, 25, 50, 75, 100, or more different partial chromosomal aneuploidies is determined.
  • Different partial chromosomal aneuploidies that can be determined include chromosomal aneuploidies include partial duplications, partial multiplications, partial insertions and partial deletions.
  • Samples that can be used for determining the presence or absence of a chromosomal aneuploidy (partial or complete) in a patient can be any of the biological samples described elsewhere herein.
  • the type of sample or samples that can be used for the determination of aneuploidy in a patient will depend on the type of disease from which the patient is known or suspected to be suffering.
  • a stool sample can be chosen as a source of DNA to determine the presence or absence of aneuploidies associated with colorectal cancer.
  • the method is also applicable to tissue samples as described herein.
  • the sample is a biological sample that is obtained by noninvasive means e.g. a plasma sample.
  • sequencing of the nucleic acids in the patient sample can be performed using next generation sequencing (NGS) as described elsewhere herein.
  • NGS next generation sequencing
  • sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators.
  • sequencing is sequencing-by-ligation.
  • sequencing is single molecule sequencing.
  • an amplification step is performed prior to sequencing.
  • the presence or absence of an aneuploidy is determined in a patient suspected to be suffering from a cancer as described elsewhere herein e.g. lung, breast, kidney, head and neck, ovary, cervix, colon, pancreas, esophagus, bladder and other organs, and blood cancers.
  • Blood cancers include cancers of the bone marrow, blood, and lymphatic system, which includes lymph nodes, lymphatic vessels, tonsils, thymus, spleen, and digestive tract lymphoid tissue.
  • Leukemia and myeloma which start in the bone marrow, and lymphoma, which starts in the lymphatic system, are the most common types of blood cancer.
  • the determination of the presence or absence of one or more chromosomal aneuploidies in a patient sample can be made without limitation to determine the predisposition of the patient to a particular cancer, to determine the presence or absence of a cancer as part of routine screen in patients known and not known to be predisposed to the cancer in question, to provide a prognosis for the disease, to assess the need for adjuvant therapy, and to determine the progress or regress of the diseases.
  • the invention provides a computer program product for generating an output indicating the presence or absence of a fetal aneuploidy in a test sample.
  • the computer product comprises a computer readable medium having a computer executable logic recorded thereon for enabling a processor to diagnose a fetal aneuploidy comprising: a receiving procedure for receiving sequencing data from at least a portion of nucleic acid molecules from a maternal biological sample, wherein said sequencing data comprises a calculated chromosome; computer assisted logic for analyzing a fetal aneuploidy from said received data; and an output procedure for generating an output indicating the presence, absence or kind of said fetal aneuploidy.
  • the method of the invention can be performed using a computer-readable medium having stored thereon computer-readable instructions for carrying out a method for identifying any CNV e.g. chromosomal or partial aneuploidies.
  • the invention provides a computer- readable medium having stored thereon computer-readable instructions for carrying out a method for identifying complete and partial chromosomal aneuploidies e.g. fetal aneuploidies.
  • the method of the invention can also be performed using a computer processing system which is adapted or configured to perform a method for identifying any CNV e.g. chromosomal or partial aneuploidies.
  • the invention provides a computer processing system which is adapted or configured to perform a method as described herein.
  • the apparatus comprises a sequencing device adapted or configured for sequencing at least a portion of the nucleic acid molecules in a sample to obtain the type of sequence information described elsewhere herein.
  • Peripheral blood samples were collected from pregnant women in their first or second trimester of pregnancy and who were deemed at risk for fetal aneuploidy. Informed consent was obtained from each participant prior to the blood draw. Blood was collected before amniocentesis or chorionic villus sampling. Karyotype analysis was performed using the chorionic villus or amniocentesis samples to confirm fetal karyotype.
  • Peripheral blood drawn from each subject was collected in ACD tubes.
  • One tube of blood sample (approximately 6-9 mL/tube) was transferred into one 15-mL low speed centrifuge tube.
  • Blood was centrifuged at 2640 rpm, 4°C for 10 min using Beckman Allegra 6 R centrifuge and rotor model GA 3.8.
  • the upper plasma layer was transferred to a 15-ml high speed centrifuge tube and centrifuged at 16000 x g, 4°C for 10 min using Beckman Coulter Avanti J-E centrifuge, and JA-14 rotor. The two centrifugation steps were performed within 72 h after blood collection.
  • Cell-free plasma was stored at -80°C and thawed only once before DNA extraction.
  • Cell-free DNA was extracted from cell-free plasma by using QIAamp DNA Blood Mini kit
  • All sequencing libraries were prepared from approximately 2 ng of purified cfDNA that was extracted from maternal plasma. Library preparation was performed using reagents of the NEBNextTM DNA Sample Prep DNA Reagent Set 1 (Part No. E6000L; New England Biolabs, Ipswich, MA), for Illumina® as follows. Because cell-free plasma DNA is fragmented in nature, no further fragmentation by nebulization or sonication was done on the plasma DNA samples.
  • the overhangs of approximately 2 ng purified cfDNA fragments contained in 40 ⁇ 1 were converted into phosphorylated blunt ends according to the NEBNext® End Repair Module by incubating in a 1.5ml microfuge tube the cfDNA with 5 ⁇ 1 10X phosphorylation buffer, 2 ⁇ 1 deoxynucleotide solution mix (10 mM each dNTP), 1 ⁇ of a 1 :5 dilution of DNA Polymerase I, 1 ⁇ T4 DNA Polymerase and 1 ⁇ T4
  • Polynucleotide Kinase provided in the NEBNextTM DNA Sample Prep DNA Reagent Set 1 for 15 minutes at 20°C. The enzymes were then heat inactivated by incubating the reaction mixture at 75°C for 5 minutes. The mixture was cooled to 4°C, and dA tailing of the blunt-ended DNA was accomplished using ⁇ of the dA-tailing master mix containing the Klenow fragment (3' to 5' exo minus) (NEBNextTM DNA Sample Prep DNA Reagent Set 1), and incubating for 15 minutes at 37°C. Subsequently, the Klenow fragment was heat inactivated by incubating the reaction mixture at 75°C for 5 minutes.
  • ⁇ ⁇ of a 1 :5 dilution of Illumina Genomic Adaptor Oligo Mix (Part No. 1000521 ; Illumina Inc., Hayward, CA) was used to ligate the Illumina adaptors (Non-Index Y-Adaptors) to the dA-tailed DNA using 4 ⁇ 1 of the T4 DNA ligase provided in the NEBNextTM DNA Sample Prep DNA Reagent Set 1, by incubating the reaction mixture for 15 minutes at 25 °C.
  • the mixture was cooled to 4°C, and the adaptor-li gated cfDNA was purified from unligated adaptors, adaptor dimers, and other reagents using magnetic beads provided in the Agencourt AMPure XP PCR purification system (Part No. A63881 ; Beckman Coulter Genomics, Danvers, MA). Eighteen cycles of PCR were performed to selectively enrich adaptor-ligated cfDNA using Phusion ® High-Fidelity Master Mix (Finnzymes, Woburn, MA) and Illumina' s PCR primers complementary to the adaptors (Part No. 1000537 and 1000537).
  • the adaptor-ligated DNA was subjected to PCR (98°C for 30 seconds; 18 cycles of 98°C for 10 seconds, 65°C for 30 seconds, and 72°C for 30 seconds; final extension at 72°C for 5 minutes, and hold at 4°C) using Illumina Genomic PCR Primers (Part Nos. 100537 and 1000538) and the Phusion HF PCR Master Mix provided in the NEB NextTM DNA Sample Prep DNA Reagent Set 1, according to the manufacturer's instructions.
  • the amplified product was purified using the Agencourt AMPure XP PCR purification system (Agencourt Bioscience Corporation, Beverly, MA) according to the manufacturer's instructions available at www.beckmangenomics.com/products/AMPureXPProtocol_000387v001.pdf.
  • the purified amplified product was eluted in 40 ⁇ 1 of Qiagen EB Buffer, and the concentration and size distribution of the amplified libraries was analyzed using the Agilent DNA 1000 Kit for the 2100 Bioanalyzer (Agilent technologies Inc., Santa Clara, CA).
  • the amplified DNA was sequenced using Illumina' s Genome Analyzer II to obtain single-end reads of 36bp. 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 36 bp reads were obtained, covering approximately 10% of the genome.
  • the Illumina "Sequencer Control Software” transferred image and base call files to a Unix server running the Illumina "Genome Analyzer Pipeline” software version 1.51.
  • the sequence data generated from the above procedure that uniquely aligned to the genome was read from Gerald output (export.txt files) by a program (c2c.pl) running on a computer running the Linnux operating system. Sequence alignments with base mis-matches were allowed and included in alignment counts only if they aligned uniquely to the genome. Sequence alignments with identical start and end coordinates (duplicates) were excluded.
  • plasma cfDNA obtained from peripheral blood of 48 volunteer pregnant subjects was extracted and sequenced as described in Example 1, and analyzed as follows.
  • sequence tag density The total number of sequence tags that were mapped to each chromosome (sequence tag density) was determined.
  • the number of mapped sequence tags may be normalized to the length of the chromosome to generate a sequence tag density ratio.
  • the normalization to chromosome length is not a required step, and can be performed solely to reduce the number of digits in a number to simplify it for human interpretation. Chromosome lengths that can be used to normalize the sequence tags counts can be the lengths provided on the world wide web at
  • the resulting sequence tag density for each chromosome was related to the sequence tag density of each of the remaining chromosomes to derive a qualified chromosome dose, which was calculated as the ratio of the sequence tag density for the chromosome of interest e.g. chromosome 21 , and the sequence tag density of each of the remaining chromosomes i.e. chromosomes 1-20, 22 and X.
  • Table 1 provides an example of the calculated qualified chromosome dose for chromosomes of interest 13, 18, 21, X, and Y, determined in one of the qualified samples.
  • Chromosomes doses were determined for all chromosomes in all samples, and the average doses for chromosomes of interest 13, 18, 21, X and Y in the qualified samples are provided in Tables 2 and 3, and depicted in Figures 2-6.
  • Figures 2-6 also depict the chromosome doses for the test samples.
  • the chromosome doses for each of the chromosomes of interest in the qualified samples provides a measure of the variation in the total number of mapped sequence tags for each chromosome of interest relative to that of each of the remaining chromosomes.
  • qualified chromosome doses can identify the chromosome or a group of chromosomes i.e.
  • FIGS. 7 and 8 depict the calculated average chromosome doses determined in a population of qualified samples for chromosomes 13, 18, and 21 , and chromosomes X and Y.
  • the best normalizing chromosome may not have the least variation, but may have a distribution of qualified doses that best distinguishes a test sample or samples from the qualified samples i.e. the best normalizing chromosome may not have the lowest variation, but may have the greatest differentiability.
  • differentiability accounts for the variation in chromosome dose and the distribution of the doses in the qualified samples.
  • Tables 2 and 3 provide the coefficient of variation as the measure of variability, and student t- test values as a measure of differentiability for chromosomes 18, 21, X and Y, wherein the smaller the T-test value, the greatest the differentiability.
  • the differentiability for chromosome 13 was determined as the ratio of difference between the mean chromosome dose in the qualified samples and the dose for chromosome 13 in the only T13 test sample, and the standard deviation of mean of the qualified dose.
  • the qualified chromosome doses also serve as the basis for determining threshold values when identifying aneuploidies in test samples as described in the following.
  • Example 3 Examples of diagnoses of T21, T13, T18 and a case of Turner syndrome obtained using the normalizing chromosomes, chromosome doses and differentiability for each of the chromosomes of interest are described in Example 3.
  • chromosome doses for assessing aneuploidy in a biological test sample
  • maternal blood test samples were obtained from pregnant volunteers and cfDNA was prepared, sequenced and analyzed as described in Examples 1 and 2.
  • Table 4 provides the calculated dose for chromosome 21 in an exemplary test sample (#11403).
  • the calculated threshold for the positive diagnosis of T21 aneuploidy was set at > 2 standard deviations from the mean of the qualified (normal) samples. A diagnosis for T21 was given based on the chromosome dose in the test sample being greater than the set threshold.
  • Chromosomes 14 and 15 were used as normalizing chromosomes in separate calculations to show that either a chromosome having the lowest variability e.g. chromosome 14, or a chromosome having the greatest differentiability e.g. chromosome 15, can be used to identify the aneuploidy. Thirteen T21 samples were identified using the calculated chromosome doses, and the aneuploidy samples were confirmed to be T21 by karyotype.
  • Table 5 provides the calculated dose for chromosome 18 in a test sample (#11390).
  • the calculated threshold for the positive diagnosis of T18 aneuploidy was set at 2 standard deviations from the mean of the qualified (normal) samples. A diagnosis for T18 was given based on the chromosome dose in the test sample being greater than the set threshold.
  • Chromosome 8 was used as the normalizing chromosome. In this instance chromosome 8 had the lowest variability and the greatest differentiability. Eight T18 samples were identified using chromosome doses, and were confirmed to be Tl 8 by karyotype.
  • Chromosome Dose for a T18 aneuploidy (sample #11390, 47 XY +18)
  • Table 6 provides the calculated dose for chromosome 13 in a test sample (#51236).
  • the calculated threshold for the positive diagnosis of T13 aneuploidy was set at 2 standard deviations from the mean of the qualified samples.
  • a diagnosis for T13 was given based on the chromosome dose in the test sample being greater than the set threshold.
  • the chromosome dose for chromosome 13 was calculated using either chromosome 5 or the group of chromosomes 3, 4, 5, and 6 as the normalizing chromosome. One T13 sample was identified.
  • sequence tag density for chromosomes 3-6 is the average tag counts for chromosomes 3-
  • a group of chromosomes can be used as the normalizing chromosome to determine chromosome doses and identify aneuploidies.
  • Table 7 provides the calculated dose for chromosomes X and Y in a test sample (#51238).
  • the calculated threshold for the positive diagnosis of Turner Syndrome was set for the X chromosome at ⁇ -2 standard deviations from the mean, and for the absence of the Y chromosome at ⁇ -2 standard deviations from the mean for qualified (normal) samples.
  • Chromosome Dose for a Turners (XO) aneuploidy (sample #51238, 45 X)
  • a sample having an X chromosome dose less than that of the set threshold was identified as having less than one X chromosome.
  • the same sample was determined to have a Y chromosome dose that was less than the set threshold, indicating that the sample did not have a Y chromosome.
  • the combination of chromosome doses for X and Y were used to identify the Turner Syndrome
  • the method provided enables for the determination of CNV of chromosomes.
  • the method enables for the determination of over- and under-representation chromosomal aneuploidies by massively parallel sequencing of maternal plasma cfDNA and identification of normalizing chromosomes for the statistical analysis of the sequencing data.
  • the sensitivity and reliability of the method allow for accurate first and second trimester aneuploidy testing.
  • sequence doses was applied for assessing partial aneuploidy in a biological test sample of cfDNA that was prepared from blood plasma, and sequenced as described in Example 1. The sample was confirmed by karyotyping to have been derived from a subject with a partial deletion of chromosome 11.
  • the average sequence dose, standard deviation, and coefficient of variation were calculated for all 20 megabase segments in the entire genome, and the 20-megabase sequence having the least variability was the identified normalizing sequence on chromosome 5 (13000014-33000033bp) (See Table 8), which was used to calculate the dose for the sequence of interest in the test sample (see Table 9).
  • Table 8 provides the sequence dose for the sequence of interest on chromosome 11 (810000082- 103000103bp) in the test sample that was calculated as the ratio of sequence tags mapped to the sequence of interest and the sequence tags mapped to the identified normalizing sequence.
  • Figure 10 shows the sequence doses for the sequence of interest in the 7 qualified samples (O) and the sequence dose for the corresponding sequence in the test sample (0).
  • the mean is shown by the solid line, and the calculated threshold for the positive diagnosis of partial aneuploidy that was set 5 standard deviations from the mean is shown by the dashed line.
  • a diagnosis for partial aneuploidy was based on the sequence dose in the test sample being less than the set threshold.
  • the test sample was verified by karyotyping to have deletion q21-q23 on chromosome 11.
  • the method of the invention can be used to identify partial aneuploidies.
  • Sequencing data obtained for the samples described in Examples 2 and 3, and shown in figures 2-6 were further analyzed to illustrate the sensitivity of the method in successfully identifying aneuploidies in maternal samples.
  • Normalized chromosome doses for chromosomes 21, 18, 13 X and Y were analyzed as a distribution relative to the standard deviation of the mean (Y-axis) and shown in Figure 11.
  • the normalizing chromosome used is shown as the denominator (X-axis).
  • Figure 11 (A) shows the distribution of chromosome doses relative to the standard deviation from the mean for chromosome 21 dose in the unaffected samples (o) and the trisomy 21 samples (T21; ⁇ ) when using chromosome 14 as the normalizing chromosome for chromosome 21.
  • Figure 11 (B) shows the distribution of chromosome doses relative to the standard deviation from the mean for chromosome 18 dose in the unaffected samples (o) and the trisomy 18 samples (T18; ⁇ ) when using chromosome 8 as the normalizing chromosome for chromosome 18.
  • Figure 11 (C) shows the distribution of chromosome doses relative to the standard deviation from the mean for chromosome 13 dose in the unaffected samples (o) and the trisomy 13 samples (T13; ⁇ ), using the average sequence tag density of the group of chromosomes 3, 4, 5, and 6 as the normalizing chromosome to determine the chromosome dose for chromosome 13.
  • Figure 11 (D) shows the distribution of chromosome doses relative to the standard deviation from the mean for chromosome X dose in the unaffected female samples (o), the unaffected male samples ( ⁇ ), and the monosomy X samples (XO; +) when using chromosome 4 as the normalizing chromosome for chromosome X.
  • Figure 11 (E) shows the distribution of chromosome doses relative to the standard deviation from the mean for chromosome Y dose in the unaffected male samples (o the unaffected female sample s ( ⁇ ), and the monosomy X samples (+), when using the average sequence tag density of the group of chromosomes 1-22 and X as the normalizing chromosome to determine the chromosome dose for chromosome Y.
  • the method provided is sensitive and specific for determining the presence or absence of chromosomal aneuploidies in a maternal blood sample.
  • the protocol was designed to provide blood samples and clinical data to support development of noninvasive prenatal genetic diagnostic methods. Pregnant women, age 18 years or older were eligible for inclusion. For patients undergoing clinically indicated CVS or amniocentesis blood was collected prior to performance of the procedure, and results of fetal karyotype was also collected.
  • Peripheral blood samples (two tubes or -20 mL total) were drawn from all subjects in acid citrate dextrose (ACD) tubes (Becton Dickinson). All samples were de-identified and assigned an anonymous patient ID number. Blood samples were shipped overnight to the laboratory in temperature controlled shipping containers provided for the study. Time elapsed between blood draw and sample receipt was recorded as part of the sample accessioning.
  • cfDNA was sent to Prognosys Biosciences, Inc. (La Jolla, CA) for sequencing library preparation (cfDNA blunt ended and ligated to universal adapters) and sequencing using standard manufacturer protocols with the Illumina Genome Analyzer IIx instrumentation (http://www.illumina.com/). Single-end reads of 36 base pairs were obtained. Upon completion of the sequencing, all base call files were collected and analyzed. For the test set samples, sequencing libraries were prepared and sequencing carried out on Illumina Genome Analyzer IIx instrument. Sequencing library preparation was performed as follows.
  • the full-length protocol described is essentially the standard protocol provided by Illumina, and only differs from the Illumina protocol in the purification of the amplified library: the Illumina protocol instructs that the amplified library be purified using gel electrophoresis, while the protocol described herein uses magnetic beads for the same purification step.
  • the Illumina protocol instructs that the amplified library be purified using gel electrophoresis, while the protocol described herein uses magnetic beads for the same purification step.
  • Approximately 2 ng of purified cfDNA that had been extracted from maternal plasma was used to prepare a primary sequencing library using NEBNextTM DNA Sample Prep DNA Reagent Set 1 (Part No. E6000L; New England Biolabs, Ipswich, MA) for Illumina® essentially according to the manufacturer's instructions.
  • the overhangs of approximately 2 ng purified cfDNA fragments contained in 40 ⁇ 1 were converted into phosphorylated blunt ends according to the NEBNext® End Repair Module by incubating the 40 ⁇ 1 cfDNA with 5 ⁇ 1 10X phosphorylation buffer, 2 ⁇ 1 deoxynucleotide solution mix (10 mM each dNTP), ⁇ of a 1:5 dilution of DNA Polymerase I, ⁇ T4 DNA Polymerase and ⁇ T4 Polynucleotide Kinase provided in the NEBNextTM DNA Sample Prep DNA Reagent Set 1 in a 200 ⁇ microfuge tube in a thermal cycler for 30 minutes at 20°C.
  • the sample was cooled to 4°C, and purified using a QIAQuick column provided in the QIAQuick PCR Purification Kit (QIAGEN Inc., Valencia, CA) as follows.
  • the 50 ⁇ 1 reaction was transferred to 1.5 ml microfuge tube, and 250 ⁇ 1 of Qiagen Buffer PB were added.
  • the resulting 300 ⁇ 1 were transferred to a QIAquick column, which was centrifuged at 13,000RPM for 1 minute in a microfuge.
  • the column was washed with 750 ⁇ 1 Qiagen Buffer PE, and re-centrifuged. Residual ethanol was removed by an additional centrifugation for 5 minutes at 13,000 RPM.
  • the DNA was eluted in 39 ⁇ 1 Qiagen Buffer EB by centrifugation.
  • dA tailing of 34 ⁇ 1 of the blunt-ended DNA was accomplished using 16 ⁇ 1 of the dA-tailing master mix containing the Klenow fragment (3' to 5' exo minus) (NEBNextTM DNA Sample Prep DNA Reagent Set 1), and incubating for 30 minutes at 37°C according to the manufacturer's NEBNext® dA-Tailing Module.
  • the sample was cooled to 4°C, and purified using a column provided in the MinElute PCR Purification Kit (QIAGEN Inc., Valencia, CA) as follows.
  • the 50 ⁇ 1 reaction was transferred to 1.5 ml microfuge tube, and 250 ⁇ 1 of Qiagen Buffer PB were added.
  • the 300 ⁇ 1 were transferred to the MinElute column, which was centrifuged at 13,000RPM for 1 minute in a microfuge.
  • the column was washed with 750 ⁇ 1 Qiagen Buffer PE, and re-centrifuged. Residual ethanol was removed by an additional centrifugation for 5 minutes at 13,000 RPM.
  • the DNA was eluted in 15 ⁇ 1 Qiagen Buffer EB by centrifugation. Ten microliters of the DNA eluate were incubated with ⁇ ⁇ of a 1:5 dilution of the Illumina Genomic Adapter Oligo Mix (Part No.
  • the Agencourt AMPure XP PCR purification system removes unincorporated dNTPs, primers, primer dimers, salts and other contaminates, and recovers amplicons greater than lOObp.
  • the purified amplified product was eluted from the Agencourt beads in 40 ⁇ 1 of Qiagen EB Buffer and the size distribution of the libraries was analyzed using the Agilent DNA 1000 Kit for the 2100 Bioanalyzer (Agilent technologies Inc., Santa Clara, CA). For both the training and test sample sets, single-end reads of 36 base pairs were sequenced.
  • Intra-run and inter-run sequencing variation in the chromosomal distribution of sequence reads can obscure the effects of fetal aneuploidy on the distribution of mapped sequence sites.
  • a chromosome dose was calculated as the count of mapped sites for a given chromosome of interest is normalized to counts observed on a predetermined normalizing chromosome sequence.
  • a normalized chromosome sequence can be composed of a single chromosome or a group of chromosomes. The normalizing chromosome sequence was first identified in a subset of samples in the training set of samples that were unaffected i.e.
  • chromosomes of interest 21 , 18, 13 and X were qualified samples having diploid karyotypes for chromosomes of interest 21 , 18, 13 and X, considering each autosome as a potential denominator in a ratio of counts with our chromosomes of interest.
  • Denominator chromosomes i.e. normalizing chromosome sequences were selected that minimized the variation of the chromosome doses within and between sequencing runs.
  • Each chromosome of interest was determined to have a distinct normalizing chromosome sequence (denominator) (Table 10). No single chromosome could be identified as a normalizing chromosome sequence for chromosome 13 as no one chromosome was determined to reduce the variability in the dose of chromosome 13 across samples i.e.
  • Chromosomes 2-6 were chosen randomly and tested for their ability as a group to mimic the behavior of chromosome 13. The group of chromosomes 2-6 was found to diminish substantially the variability in the dose for chromosome 13 in the training samples, and was thus chosen as the normalizing chromosome sequence for chromosome 13. As described above, the variability in chromosome dose for chromosome Y is greater than 30 independently of which single chromosome is used as the normalizing chromosome sequence in determining the chromosome Y dose. The group of chromosomes 2-6 was found to diminish substantially the variability in the dose for chromosome Y in the training samples, and was thus chosen as the normalizing chromosome sequence for chromosome Y.
  • the chromosome doses for each of the chromosomes of interest in the qualified samples provides a measure of the variation in the total number of mapped sequence tags for each chromosome of interest relative to that of each of the remaining chromosomes.
  • qualified chromosome doses can identify the chromosome or a group of chromosomes i.e. normalizing chromosome sequence that has a variation among samples that is closest to the variation of the chromosome of interest, and that would serve as ideal sequences for normalizing values for further statistical evaluation.
  • Chromosome doses for all samples in the training set i.e. qualified and affected also serve as the basis for determining threshold values when identifying aneuploidies in test samples as described in the following.
  • a normalizing value was determined and used to determine the presence or absence of an aneuploidy.
  • the normalizing value was calculated as a chromosome dose that can be further computed to provide a normalized chromosome value (NCV).
  • a chromosome dose was calculated for each chromosome of interest, 21, 18, 13, X and Y for every sample.
  • the chromosome dose for chromosome 21 was calculated as a ratio of the number of tags in the test sample that mapped to chromosome 21 in the test sample, and the number of tags in the test sample that mapped to chromosome 9;
  • the chromosome dose for chromosome 18 was calculated as a ratio of the number of tags in the test sample that mapped to chromosome 18 in the test sample, and the number of tags in the test sample that mapped to chromosome 8;
  • the chromosome dose for chromosome 13 was calculated as a ratio of the number of tags in the test sample that mapped to chromosome 13 in the test sample, and the number of tags in the test sample that mapped to chromosomes 2-6;
  • the chromosome dose for chromosome X was calculated as a ratio of the number of tags in the test sample that mapped
  • NCV normalized chromosome value
  • NC Vij where fijANO Oj are the estimated training set mean and standard deviation respectively for the j-th chromosome dose, and xij is the observed j-th chromosome dose for sample i.
  • the NCV is equivalent to a statistical z-score for the doses. No significant departure from linearity is observed in a quantile-quantile plot of the NCVs from unaffected samples. In addition, standard tests of normality for the NCVs fail to reject the null hypothesis of normality.
  • an NCV was calculated for each chromosome of interest, 21, 18, 13, X and Y for every sample. To insure a safe and effective classification scheme, conservative boundaries were chosen for aneuploidy classification.
  • a NCV > 4.0 was required to classify the chromosome as affected (i.e. aneuploid for that chromosome) and a NCV ⁇ 2.5 to classify a chromosome as unaffected. Samples with autosomes that have an NCV between 2.5 and 4.0 were classified as "no call".
  • Sex chromosome classification in the test was performed by sequential application of NCVs for both X and Y as follows:
  • Cardiac Defect 78 (9.4) 0 (0.0) 3 (6.4)
  • AMA Advanced Maternal Age
  • NT nuchal translucency
  • the training set study selected 71 samples from the initial sequential accumulation of 435 samples that were collected between April 2009 and December 2009. All subjects with affected fetus' (abnormal karyotypes) in this first series of subjects were included for sequencing and a random selection and number of non-affected subjects with adequate sample and data. Clinical characteristics of the training set patients were consistent with the overall study demographics as shown in Table 11. The gestational age range of the samples in the training set ranged from 10 weeks, 0 days to 23 weeks 1 day. Thirty-eight underwent CVS, 32 underwent amniocentesis and 1 patient did not have the invasive procedure type specified (an unaffected karyotype 46, XY).
  • the number of unique sequence sites i.e. tags identified with unique sites in the genome
  • the number of unique sequence sites varied from 2.2M in the early phases of the training set study to 13.7M in the latter phases due to improvements in sequencing technology over time.
  • different unaffected samples were run at the beginning and end of the study.
  • the average number of unique sites was 3.8M and the average chromosome doses for chromosome 21 and chromosome 18 were 0.314 and 0.528, respectively.
  • the average number of unique sites was 10.7M and the average chromosome doses for chromosome 21 and chromosome 18 were 0.316 and 0.529, respectively. There was no statistical difference between the chromosome doses for chromosome 21 and chromosome 18 over the time of the training set study.
  • the training set NCVs for chromosomes 21, 18 and 13 are shown on Figure 12.
  • the results shown in Figure 12 are consistent with an assumption of normality in that roughly 99% of the diploid NCVs would fall within + 2.5 standard deviations of the mean.
  • 8 samples with clinical karyotypes indicating T21 had NCVs ranging from 6 to 20.
  • Four samples having clinical karyotypes indicative of fetal T18 had NCVs ranging from 3.3 to 12
  • the two samples having karyotypes indicative of fetal trisomy 13 (T13) had NCVs of 2.6 and 4.
  • the spread of the NCVs in affected samples is due to their dependence on the percentage of fetal cfDNA in the individual samples.
  • the means and standard deviations for the sex chromosomes were established in the training set.
  • the sex chromosome thresholds allowed 100% identification of male and female fetuses in the training set.
  • chromosome doses means and standard deviations from the training set
  • a test set of 48 samples was selected from samples collected between January 2010 and June 2010 from 575 total samples.
  • One of the samples from a twin gestation was removed from the final analysis leaving 47 samples in the test set.
  • Personnel preparing samples for sequencing and operating the equipment were blinded to the clinical karyotype information.
  • the gestational age range was similar to that seen in the training set (Table 11). 58% of the invasive procedures were CVS, higher than that of the overall procedural demographics, but also similar to the training set. 50% of subjects were Caucasian, 27% Hispanic, 10.4% Asian and 6.3% African American.
  • the number of unique sequence tags varied from approximately 13M to 26M.
  • the chromosome doses for chromosome 21 and chromosome 18 were 0.313 and 0.527, respectively.
  • the test set NCVs for chromosome 21, chromosome 18 and chromosome 13 are shown in Figure 13 and the classifications are given in Table 12.
  • test set 13/13 subjects having clinical karyotypes that indicated fetal T21 were correctly identified having NCVs ranging from 5 to 14. Eight/eight subjects having karyotypes that indicated fetal T18 were correctly identified having NCVs ranging from 8.5 to 22.
  • the single sample having a karyotype classified as T13 in this test set was classified as a no call with an NCV of approximately 3.
  • test data set all male samples were correctly identified including a sample with complex karyotype, 46,XY + marker chromosome (unidentifiable by cytogenetics) (Table 3).
  • twin gestation sample in the test set was classified correctly as non-affected for T21 (all twins showed diploid karyotype for chromosome 21).
  • twin gestation sample in the test set karyotype was only established for Twin B (46,XX) and the algorithm correctly classified as non-affected for T21.
  • the data show that massively parallel sequencing can be used to determine a plurality abnormal fetal karyotypes from the blood of pregnant women. These data demonstrate that 100% correct classification of samples with trisomy 21 and trisomy 18 can be identified using independent test set data. Even in the case of fetuses with abnormal sex chromosome karyotypes, none of the samples were incorrectly classified with the algorithm of the method. Importantly, the algorithm also performed well in determining the presence of T21 in two sets of twin pregnancies having at least one affected fetus, which has never been shown previously.
  • this study examined a variety of sequential samples from multiple centers representing not only the range of abnormal karyotypes that one is likely to witness in a commercial clinical setting, but showing the significance of accurately classifying pregnancies non-affected by common trisomies to address the unacceptably high false positive rates that remain in prenatal screening today.
  • the data provide valuable insight into the vast capabilities of employing this method in the future. Analysis of subsets of the unique genomic sites showed increases in the variance consistent Poisson counting statistics.
  • the method utilized in the Chiu, et al paper simply uses the number of sequence tags on the chromosome of interest, in their case chromosome 21, normalized by the total number of tags in the sequencing run.
  • the challenge for this approach is that the distribution of tags on each chromosome can vary from sequencing run to sequencing run, and thus increases the overall variation of the aneuploidy determination metric.
  • the test data for chromosomes 21 and 18 was reanalyzed using the method recommended by Chiu, et al. as shown in Figure 14.
  • Ehrich, et al also focused only on T21 and used the same algorithm as Chiu, et al., (Ehrich et al, Am J Obstet Gynecol 204:205 el-el 1 [2011]).
  • they after observing a shift in their test set z-score metric from the external reference data i.e. training set, they retrained on the test set to establish the classification boundaries.
  • this approach is feasible, in practice it would be challenging to decide how many samples are required to train and how often one would need to retrain to ensure that the classification boundaries are correct.
  • One method of mitigating this issue is to include controls in every sequencing run that measure the baseline and calibrate for quantitative behavior.
  • the data obtained using the present method show that massively parallel sequencing is capable of determining multiple fetal chromosomal abnormalities from the plasma of pregnant women when the algorithm for normalizing the chromosome counting data is optimized.
  • the present method for quantification not only minimizes random and systematic variations between sequencing runs, but also allows for effective classification of aneuploidies across the entire genome, most notably T21 and T18. Larger sample collections are required to test the algorithm for T13 determination. To this end, a prospective, blinded, multi-site clinical study to further demonstrate the diagnostic accuracy of the present method is being performed.
  • test set 1 To demonstrate the capability of the method to determine the presence or absence of any chromosomal aneuploidy in each of a set of maternal test samples (test set 1; Example 6), systematically determined normalizing chromosome sequences were identified in unaffected samples of the training set (training set 1 ; Example 6), and used to calculate chromosome doses for all chromosomes in each of the test samples. Determination of the presence or absence of any one or more different complete fetal chromosomal aneuploidies in each of the test and training set samples was accomplished from sequencing information obtained from a single sequencing run on each individual sample.
  • chromosome densities i.e. the number of sequence tags identified for each chromosome in each of the samples of the training set described in Example 6, a systematically determined normalizing chromosome sequence consisting of a single chromosome or a group of chromosomes was determined by calculating a single chromosome dose for each of chromosomes 1- 22, X and Y.
  • the systematically determined normalizing chromosome sequence for each of chromosomes 1-22, X, and Y was determined by systematically calculating chromosome doses for each chromosome using every possible combination of chromosomes as the numerator.
  • chromosome doses were calculated as a ratio of (i) the number of sequence tags obtained for chromosome 21 (chromosome of interest) and (ii) the number of sequence tags obtained for each of the remaining chromosomes, and the sum of the number of tags obtained for all possible combinations of the remaining chromosomes (excluding chromosome 21) i.e. 1, 2, 3, 4, 5, etc. up to 20, 21, 22, X, and Y; 1 + 2, 1+3, 1+4, 1+5, etc. up to 1+20, 1+22, 1+X, and 1+Y;, 1+2+3, 1+2+4, 1+2 +5 etc.
  • Chromosome doses were determined in the same manner for chromosome 21 in all training samples, and the systematically determined normalizing chromosome sequence for chromosome 21 was determined as the single or group of chromosomes resulting in a dose for chromosome 21 having the smallest variability across all training samples.
  • Table 13 provides the single or the group of chromosomes that were identified as the systematically determined normalizing sequence for each of chromosomes of interest 1-22, X, and Y.
  • the systematically determined normalizing chromosome sequence was determined to be a single chromosome ⁇ e.g. when chromosome 4 is the chromosome of interest), and for other chromosomes of interest, the systematically determined normalizing chromosome sequence was determined to be a group of chromosomes (e.g. when chromosome 21 is the chromosome of interest).
  • chromosome doses for all chromosomes of interest 1-22, X and Y were determined in each of the samples in the training set, and in each of all samples in the test set described in Example 5 using the corresponding
  • the number of sequence tags obtained in each of the training samples for the systematically determined normalizing chromosome sequences was used to determine the chromosome doses for each chromosome in each of the training samples, and the number of sequence tags obtained in each of the test samples for the systematically determined normalizing chromosome sequence was used to determine the chromosome dose for each chromosome for each of the test samples.
  • the same conservative boundaries were chosen as described in Example 6.
  • a plot of the chromosome doses for chromosomes 21, 18 and 13 in the training set of samples using the systematically determined normalizing chromosome sequence is given in Figure 15.
  • the systematically determined normalizing chromosome sequence i.e. the group of chromosomes 4+14+16+20+22
  • 8 samples with clinical karyotypes indicating T21 had NCVs between 5.4 and 21.5.
  • the systematically determined normalizing chromosome sequence i.e. the group of chromosomes 2+3+5+7
  • 4 samples with clinical karyotypes indicating T18 had NCVs between 3.3 and 15.3.
  • the systematically determined normalizing chromosome sequence i.e.
  • the T21 samples of the training set are shown as the last 8 samples of the chromosome 21 data (O); theT18 samples of the training set are shown as the last 4 samples of the chromosome 18 data ( ⁇ ); and the T13 samples of the training set are shown as the last 2 samples of the chromosome 13 data ( ⁇ ).
  • FIG. 18A shows a plot of NCVs determined for the X chromosome (X-axis) and NCVs determined for the Y chromosome (Y axis) for each of the samples in the training set.
  • a plot of the chromosome doses for chromosomes 21, 18 and 13 in the test samples using the relevant systematically determined normalizing chromosome sequences is given in Figure 16.
  • the systematically determined normalizing chromosome sequence i.e. the group of chromosomes 4+14+16+20+22
  • 13 of 13 samples with clinical karyotypes indicating T21 were correctly identified with NCVs between 7.2 and 16.3.
  • the systematically determined normalizing chromosome sequence i.e. the group of chromosomes 2+3+5+7
  • all 8 samples with clinical karyotypes indicating T18 were identified with NCVs between 12.7 and 30.7.
  • the systematically determined normalizing chromosome sequence i.e.
  • the T21 samples of the test set are shown as the last 13 samples of the chromosome 21 data (O); theT18 samples of the test set are shown as the last 8 samples of the chromosome 18 data ( ⁇ ); and the T13 sample of the test set is shown as the last sample of the chromosome 13 data ( ⁇ ).
  • O the last 13 samples of the chromosome 21 data
  • ⁇ the last 8 samples of the chromosome 18 data
  • ⁇ the T13 sample of the test set is shown as the last sample of the chromosome 13 data ( ⁇ ).
  • FIG. 18B shows a plot of NCVs determined for the X chromosome (X-axis) and NCVs determined for the Y chromosome (Y axis) for each of the samples in the test set
  • the present method allows for determining the presence or absence of a complete, or partial, chromosomal aneuploidy of each of chromosomes 1-22, X, and Y in each sample.
  • the method determined the presence of a trisomy of chromosome 9 in one of the test samples.
  • the systematically determined normalizing chromosome sequence i.e. the group of chromosomes 3+4+8+10+17+19+20+22
  • chromosome of interest 9 a sample having an NCV of 14.4 was identified ( Figure 17).
  • This sample corresponded to the test sample in Example 6 that was suspected of being aneuploid for chromosome 9 following the calculation of an aberrantly low dose for chromosome 21 (for which chromosome 9 was used as the normalizing chromosome sequence in Example 6).
  • Figure 19 shows a plot of the NCVs for each of chromosomes 1-22 in each of the 47 test samples. Medians of NCVs were normalized to zero.
  • the data show that the method of the invention (including the use of systematically determined normalizing chromosome sequences) determined the presence of all 5 types of chromosomal aneuploidies that were present in this test set with 100% sensitivity and 100% specificity, and clearly indicate that the method can identify any complete chromosomal aneuploidy for any one of chromosomes 1-22, X, and Y, in any sample.
  • DiGeorge syndrome (22ql 1.2 deletion syndrome), a disorder caused by a defect in chromosome 22, results in the poor development of several body systems. Medical problems commonly associated with DiGeorge syndrome include heart defects, poor immune system function, a cleft palate, poor function of the parathyroid glands and behavioral disorders. The number and severity of problems associated with DiGeorge syndrome vary greatly. Almost everyone with DiGeorge syndrome needs treatment from specialists in a variety of fields.
  • a blood sample is obtained by venipuncture for the mother, and cfDNA is prepared as described in the Examples above.
  • the purified cfDNA is ligated to adaptors and subjected to cluster amplification using the Illumina cBot cluster station.
  • Massively parallel sequencing is performed using reversible dye terminators to generate millions of 36bp reads.
  • the sequence reads are aligned to the human hgl9 reference genome, and the reads that are uniquely mapped to the reference genome are counted as tags.
  • the number of sequence tags mapped to every single lOOObp segment is used individually to compute segment doses for the 3Mb region of 22ql l.2.
  • all possible combinations of two or more segments are used to determine segment doses for the segment of interest in all qualified samples.
  • the single 3Mb segment or the combination of two or more 3Mb segments that result in the segment dose having the lowest variability across samples is chosen as the normalizing segment sequence.
  • the number of sequence tags mapped to the segment of interest in each of the qualified samples is used to determine a segment dose in each of the qualified samples.
  • the mean and standard deviation of the segment doses in all qualified samples is calculated, and used to set threshold s to which segment doses determined in test samples can be compared.
  • Preferably, normalized segment values (NSV) are calculated for all segments of interest in all qualified samples, and used to set the threshold values.
  • a normalized segment value is calculated for the segment in the test sample as described previously and the NCV of the segment of interest in the test sample is compared to the threshold determined using the qualified samples to determine the presence or absence of a deletion of 22ql 1.2 in the test sample.
  • a test NCV ⁇ -3 indicates that a loss in the segment of interest i.e. partial deletion of chromosome 22 (22ql l.2) is present in the test sample.
  • stage II colon cancers of patients who had relapse of disease showed significantly more losses on
  • stage II colon cancer patients losses on 4q22.1- 4q35.2 have been shown to be associated with worse outcome. Determination of the presence or absence of these genomic alterations may aid in selecting patients for adjuvant therapy (Brosens et al, Analytical Cellular Pathology / Cellular Oncology 33: 95-104 [2010]).
  • stool and/or plasma samples are obtained from the patient(s).
  • Stool DNA is prepared according to the method described by Chen et al, J Natl Cancer Inst 97:1124-1132 [2005]); and plasma DNA is prepared according to the method described in the Examples above.
  • DNA is sequenced according to an NGS method described herein, and the sequence information for the patient(s) sample(s) is used to calculate segment doses for one or more segments spanning the 4q22.1 to 4q35.2 region.
  • Segment doses are determined using normalizing segment sequences that are determined a priori by in a set of qualified stool and/or plasma samples, respectively. Segment doses in the test samples (patient samples) are calculated, and the presence or absence of one or more partial chromosomal deletions within the 4q22.1 to 4q35.2 region is determined by comparing the NSV for each of the segments of interest to the threshold set from the NSV in the set of qualified samples.

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