KR101974492B1 - Method for determining the presence or absence of different aneuploidies in a sample - Google Patents

Abstract

The present invention provides a method for determining the number of copies of a sequence of interest (CNV) of a sequence of interest in a test sample comprising a mixture of nucleic acids that are known to be different or different in the amount of one or more sequences of interest. The method includes statistical approaches that describe process-related, variable-order stems resulting from inter-chromosomal and inter-sequencing variability. The method can be used to determine CNVs that are known to be associated with, or are likely to be involved in, various fetal insulting CNVs and various medical disorders. The CNV that can be determined according to the method of the present invention can be detected by sequencing the nucleic acid of the test sample only once, including any one or more of chromosomes 1-22, any one of X and Y, and other chromosomes and other chromosomes Deletion and / or duplication of any one or more of the chromosome, chromosome, and chromosome. Any isomericity can be determined from the sequencing information obtained by sequencing the nucleic acid of the test sample only once.

Description

METHOD FOR DETERMINING THE PRESENCE OR ABSENCE OF DIFFERENT ANEUPLOIDIES IN A SAMPLE < RTI ID = 0.0 >

The present invention relates generally to the field of diagnosis and provides a method for determining the variation in the amount of nucleic acid sequence in a mixture of nucleic acids derived from different dielectrics. In particular, the method can be used to perform non-invasive prenatal diagnosis and to diagnose and monitor metastatic progression in cancer patients.

One of the major efforts in human medical research is the discovery of genetic deformities at the heart of adverse health outcomes. In many cases, specific genes and / or deterministic diagnostic markers have been identified in the portion of the genome that is present as an abnormal number of copies. For example, in prenatal diagnosis, extra or missing copies of the entire chromosome are frequently occurring genetic lesions. In cancer, deletion or increase of the entire chromosome or a copy of a chromosome segment, and high level amplification of specific regions of the genome generally occur.

Most of the information on the variation of the number of radiations was provided by cytogenetic analysis allowing structural abnormalities. Conventional procedures for gene screening and biological dosimetry used invasive procedures such as amniocentesis to obtain cells for karyotyping. As a molecular-cytogenetic method for the analysis of the copy number variation, there is a need for cell culture, in situ fluorescence hybridization (FISH), quantitative fluorescent PCT (QF-PCR) and array-comparative genomic hybridization (array-CGH) A need for faster testing methods has been recognized.

The emergence of techniques for sequencing the entire genome in a relatively short period of time and the discovery of circulating cell-free DNA (cfDNA) provide an opportunity to compare genetic material from one chromosome to another without risk associated with invasive sampling methods Respectively. However, limitations of existing methods, including insufficient susceptibility stemming from a limited level of cfDNA, and sequencing bias of technique steaming from the inherent characteristics of genomic information, have led to reliable changes in the number of copies in various clinical settings Is the basis for a continuing need for non-invasive methods to provide any or all of the specificity, sensitivity, and availability for diagnosis.

The present invention fulfills some of the above needs, and in particular provides advantages in providing at least a non-invasive prenatal diagnosis and in providing a reliable method that can be used to diagnose and monitor metastatic progression in cancer patients.

Summary of the Invention

The present invention provides a method for determining the number of copies of a sequence of interest (CNV) of a sequence of interest in a test sample comprising a mixture of nucleic acids that are known to be different or different in the amount of one or more sequences of interest. The method includes a statistical approach that describes variability stemming from process-related, interchromosomal and inter-sequencing variability. The method can be used to determine CNVs that are known to be associated with, or are likely to be involved in, various fetal insulting CNVs and various medical disorders. The CNV that can be determined according to the method of the present invention can be detected by sequencing the nucleic acid of the test sample only once, including any one or more of chromosomes 1-22, any one of X and Y, and other chromosomes and other chromosomes Deletion and / or duplication of any one or more of the chromosome, chromosome, and chromosome. Any isomericity can be determined from the sequencing information obtained by sequencing the nucleic acid of the test sample only once.

In one embodiment, a method is provided for determining the presence or absence of any four or more different complete fetal chromosomal integrity in a maternal test sample comprising fetal and maternal nucleic acid molecules. The method comprising the steps of: (a) obtaining sequence information for a fetal and parent nucleic acid in a maternal test sample; (b) identifying a plurality of sequence tags for each of four or more interest chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and determining a plurality of sequence tags for each of the four or more chromosomes Identifying a plurality of sequence tags for the sequence; (c) calculating a single chromosome dose for each of four or more interest chromosomes using the number of identified sequence tags for each of the four or more interest chromosomes and the number of sequence tags identified for each normalized chromosome; (d) determining the presence or absence of any four or more different complete fetal chromosomal aberrations in the maternal test sample by comparing each single chromosome dose for each of the four or more interest chromosomes with a threshold for each of the four or more chromosomes of interest . Step (a) may comprise sequencing at least a portion of the nucleic acid molecule of the test sample to obtain said sequence information for the fetal and parent nucleic acid molecules of the test sample. In some embodiments, step (c) comprises determining the single chromosome capacity 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 normalized chromosome sequence for each of the chromosomes of interest Lt; / RTI > In some other embodiments, step (c) comprises: (i) associating the number of sequence tags identified for each of the chromosomes of interest in step (b) with the length of each of the chromosomes of interest, Calculate the density ratio; (ii) calculating the sequence tag density ratio for each normalized chromosome sequence in relation to the length of each normalized chromosome, the number of sequence tags identified for the sequence in step (b); (iii) calculating a single chromosomal capacity for each of the chromosomes of interest using the sequence tag density ratio calculated in steps (i) and (ii), wherein the chromosomal capacity comprises a sequence tag density ratio And the sequence tag density ratio for the normalized chromosome sequence for each of the chromosomes of interest.

In another embodiment, a method is provided for determining the presence or absence of any four or more different complete fetal chromosomal integrity in a maternal test sample comprising fetal and maternal nucleic acid molecules. The method comprising the steps of: (a) obtaining sequence information for a fetal and parent nucleic acid in a maternal test sample; (b) identifying a plurality of sequence tags for each of four or more chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and identifying a plurality of sequence tags for each of the four or more chromosomes Identifying the sequence tag of; (c) calculating a single chromosome dose for each of four or more interest chromosomes using the number of identified sequence tags for each of the four or more interest chromosomes and the number of sequence tags identified for each normalized chromosome; (d) determining the presence or absence of any four or more different complete fetal chromosomal aberrations in the maternal test sample by comparing each single chromosome dose for each of the four or more interest chromosomes with a threshold for each of the four or more chromosomes of interest Wherein any four or more chromosomes of interest selected from chromosomes 1-22, X, and Y comprise at least 20 chromosomes selected from chromosomes 1-22, X, and Y, and at least 20 different complete chromosomal chromosomes Is determined. Step (a) may comprise sequencing at least a portion of the nucleic acid molecule of the test sample to obtain said sequence information for the fetal and parent nucleic acid molecules of the test sample. In some embodiments, step (c) comprises determining the single chromosome capacity 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 normalized chromosome sequence for each of the chromosomes of interest Lt; / RTI > In some other embodiments, step (c) comprises: (i) associating the number of sequence tags identified for each of the chromosomes of interest in step (b) with the length of each of the chromosomes of interest, Calculate the density ratio; (ii) calculating the sequence tag density ratio for each normalized chromosome sequence in relation to the length of each normalized chromosome in the number of sequence tags identified for the normalized chromosome sequence in step (b); (iii) calculating a single chromosomal capacity for each of the chromosomes of interest using the sequence tag density ratio calculated in steps (i) and (ii), wherein the chromosomal capacity comprises a sequence tag density ratio And the sequence tag density ratio for the normalized chromosome sequence for each of the chromosomes of interest.

In another embodiment, a method is provided for determining the presence or absence of any four or more different complete fetal chromosomal integrity in a maternal test sample comprising fetal and maternal nucleic acid molecules. The method comprising the steps of: (a) obtaining sequence information for a fetal and parent nucleic acid in a maternal test sample; (b) identifying a plurality of sequence tags for each of four or more chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and identifying a plurality of sequence tags for each of the four or more chromosomes Identifying the sequence tag of; (c) calculating a single chromosome dose for each of four or more interest chromosomes using the number of identified sequence tags for each of the four or more interest chromosomes and the number of sequence tags identified for each normalized chromosome; (d) determining the presence or absence of any four or more different complete fetal chromosomal aberrations in the maternal test sample by comparing each single chromosome dose for each of the four or more interest chromosomes with a threshold for each of the four or more chromosomes of interest Wherein any four or more chromosomes of interest selected from chromosomes 1-22, X, and Y are all chromosomes 1-22, X, and Y, and the complete fetal chromosomal integrity of all chromosomes 1-22, X, Is determined. Step (a) may comprise sequencing at least a portion of the nucleic acid molecule of the test sample to obtain said sequence information for the fetal and parent nucleic acid molecules of the test sample. In some embodiments, step (c) comprises determining the single chromosome capacity 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 normalized chromosome sequence for each of the chromosomes of interest Lt; / RTI > In some other embodiments, step (c) comprises: (i) associating the number of sequence tags identified for each of the chromosomes of interest in step (b) with the length of each of the chromosomes of interest, Calculate the density ratio; (ii) calculating the sequence tag density ratio for each normalized chromosome sequence in relation to the length of each normalized chromosome in the number of sequence tags identified for the normalized chromosome sequence in step (b); (iii) calculating a single chromosomal capacity for each of the chromosomes of interest using the sequence tag density ratio calculated in steps (i) and (ii), wherein the chromosomal capacity comprises a sequence tag density ratio And the sequence tag density ratio for the normalized chromosome sequence for each of the chromosomes of interest.

In any of the above embodiments, the normalized chromosome sequence may be a single chromosome selected from chromosomes 1-22, X, and Y. Alternatively, the normalized chromosome sequence may be a group of chromosomes selected from chromosomes 1-22, X, and Y. [

In another embodiment, a method is provided for determining the presence or absence of any one or more different complete fetal chromosomal integrity in a maternal test sample comprising a fetal and maternal nucleic acid. The method comprising the steps of: (a) obtaining sequence information of a fetal and parent nucleic acid in a sample; (b) identifying a plurality of sequence tags for each of at least one of the interest chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and generating a plurality of sequences for the normalization segment sequence for each of the one or more chromosomes Check tags; (c) calculating a single chromosome dose for each of at least one of the interest chromosomes using the number of sequence tags identified for each of the one or more interest chromosomes and the number of sequence tags identified for the standardization segment sequence; (d) determining the presence or absence of any one or more different complete fetal chromosomal aberrations in the sample by comparing each single chromosome dose for each of the one or more interest chromosomes with a threshold for each of the one or more chromosomes of interest. Step (a) may comprise sequencing at least a portion of the nucleic acid molecule of the test sample to obtain said sequence information for the fetal and parent nucleic acid molecules of the test sample.

In some embodiments, step (c) comprises determining the single chromosome capacity 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 normalization segment sequence for each of the chromosomes of interest Lt; / RTI > In some other embodiments, step (c) comprises: (i) associating the number of sequence tags identified for each of the chromosomes of interest in step (b) with the length of each of the chromosomes of interest, Calculate the density ratio; (ii) calculating the sequence tag density ratio for each normalized segment sequence by relating the number of sequence tags identified for the normalized segment sequence in step (b) to the length of each normalized chromosome; (iii) calculating a single chromosomal capacity for each of the chromosomes of interest using the sequence tag density ratio calculated in steps (i) and (ii), wherein the chromosomal capacity comprises a sequence tag density And the ratio of the sequence tag density ratio to the normalized segment sequence for each of the non-interest and interest chromosomes.

In another embodiment, a method is provided for determining the presence or absence of any one or more different complete fetal chromosomal integrity in a maternal test sample comprising a fetal and maternal nucleic acid. The method comprising the steps of: (a) obtaining sequence information for a fetal and parent nucleic acid in a sample; (b) identifying a plurality of sequence tags for each of at least one of the interest chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and generating a plurality of sequences for the normalization segment sequence for each of the one or more chromosomes Check tags; (c) calculating a single chromosome dose for each of at least one of the interest chromosomes using the number of sequence tags identified for each of the one or more interest chromosomes and the number of sequence tags identified for the standardization segment sequence; (d) determining the presence or absence of one or more different complete fetal chromosomalemias in the sample by comparing each single chromosomal dose for each of the one or more interest chromosomes with a threshold for each of the one or more chromosomes of interest, Any one or more of the interest chromosomes selected from 1-22, X, and Y comprise at least 20 chromosomes selected from chromosomes 1-22, X, and Y, and the presence or absence of at least 20 different complete fetal chromosomal anomalies is determined . Step (a) may comprise sequencing at least a portion of the nucleic acid molecule of the test sample to obtain said sequence information for the fetal and parent nucleic acid molecules of the test sample. In some embodiments, step (c) comprises determining the single chromosome capacity 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 normalization segment sequence for each of the chromosomes of interest Lt; / RTI > In some other embodiments, step (c) comprises: (i) associating the number of sequence tags identified for each of the chromosomes of interest in step (b) with the length of each of the chromosomes of interest, Calculate the density ratio; (ii) calculating the sequence tag density ratio for each normalized segment sequence by relating the number of sequence tags identified for the normalized segment sequence in step (b) to the length of each normalized chromosome; (iii) calculating a single chromosomal capacity for each of the chromosomes of interest using the sequence tag density ratio calculated in steps (i) and (ii), wherein the chromosomal capacity comprises a sequence tag density And the ratio of the sequence tag density ratio to the normalized segment sequence for each of the non-interest and interest chromosomes.

In another embodiment, a method is provided for determining the presence or absence of any one or more different complete fetal chromosomal integrity in a maternal test sample comprising a fetal and maternal nucleic acid. The method comprising the steps of: (a) obtaining sequence information of a fetal and parent nucleic acid in a sample; (b) identifying a plurality of sequence tags for each of at least one of the interest chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and generating a plurality of sequences for the normalization segment sequence for each of the one or more chromosomes Check tags; (c) calculating a single chromosome dose for each of at least one of the interest chromosomes using the number of sequence tags identified for each of the one or more interest chromosomes and the number of sequence tags identified for the standardization segment sequence; (d) determining the presence or absence of one or more different complete fetal chromosomalemias in the sample by comparing each single chromosomal dose for each of the one or more interest chromosomes with a threshold for each of the one or more chromosomes of interest, 1-22, X, and Y are all chromosomes 1-22, X, and Y, and the presence or absence of complete fetal chromosomal integrity of all chromosomes 1-22, X, and Y is determined . Step (a) may comprise sequencing at least a portion of the nucleic acid molecule of the test sample to obtain said sequence information for the fetal and parent nucleic acid molecules of the test sample. In some embodiments, step (c) comprises determining the single chromosome capacity 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 normalization segment sequence for each of the chromosomes of interest Lt; / RTI > In some other embodiments, step (c) comprises: (i) associating the number of sequence tags identified for each of the chromosomes of interest in step (b) with the length of each of the chromosomes of interest, Calculate the density ratio; (ii) calculating the sequence tag density ratio for each normalized segment sequence by relating the number of sequence tags identified for the normalized segment sequence in step (b) to the length of each normalized chromosome; (iii) calculating a single chromosomal capacity for each of the chromosomes of interest using the sequence tag density ratio calculated in steps (i) and (ii), wherein the chromosomal capacity comprises a sequence tag density And the ratio of the sequence tag density ratio to the normalized segment sequence for each of the non-interest and interest chromosomes.

In any one of the above embodiments, different complete chromosomal aberrations are selected from complete chromosomal trisomes, complete chromosomal single chromosomes, and complete chromosomes. The different complete chromosomal aberrations are selected from the complete isomorphism of either chromosome 1-22, X, For example, the different complete fetal chromosomal anomalies may include chromosome 2 trisomy 2, trisomy 8, trisomy 9, trisomy 21, trisomy 13, trisomy 16, Chromosome 22, trisomy 22, 47, XXY, 47, XXX, 47, XYY, and X chromosome.

In any one of the above embodiments, steps (a) - (d) are repeated for a test sample from a different maternal subject, wherein the method comprises the presence of any four or more different complete fetal chromosomal anomalies ≪ / RTI >

In one such embodiment, the method may further comprise calculating a normalized chromosomal value (NCV), wherein the NCV relates the chromosomal dose to an average of the corresponding chromosomal dose as follows in a set of qualified samples :

및

는 적격 샘플의 세트에서 j번째 염색체 용량에 대한 각기 추정 평균 및 표준 편차이고,

는 시험 샘플 i에 대해 관찰된 j번째 염색체 용량이다.In this formula,

And

Is the respective estimated mean and standard deviation for the j < th > chromosome dose in the set of eligible samples,

Is the jth chromosome capacity observed for test sample i.

In another embodiment, a method is provided for determining the presence or absence of different partial fetal chromosomal integrity in a maternal test sample comprising a fetal and maternal nucleic acid. The method comprising the steps of: (a) obtaining sequence information for a fetal and parent nucleic acid in a sample; (b) identifying a plurality of sequence tags for each of any one or more segments of any one or more of the chromosomes of interest selected from chromosomes 1-22, X and Y using said sequence information and identifying any one or more Identifying a plurality of sequence tags for the normalized segment sequence for each of the segments; (c) the number of sequence tags identified for each of the at least one segment of any one or more of the chromosomes of interest and the number of sequence tags identified for the normalized segment sequence, Calculate a single segment capacity for each of the segments; (d) comparing each single segment capacity for each of the one or more segments of any one or more of the interest chromosomes with a threshold for each of the one or more chromosome segments of any one or more of the chromosomes of interest, And determining the presence or absence of chromosomal integrity. Step (a) may comprise sequencing at least a portion of the nucleic acid molecule of the test sample to obtain said sequence information for the fetal and parent nucleic acid molecules of the test sample.

In some embodiments, step (c) comprises comparing the single segment capacity for each of the one or more segments of any one or more of the interest chromosomes to the number of identified sequence tags for each of the one or more segments of any one or more of the chromosomes of interest As the ratio of the number of sequence tags identified for the normalized segment sequence for each of the one or more segments of one or more chromosomes of interest. In some other embodiments, step (c) comprises: (i) associating the number of sequence tags identified for each of the segments of interest in step (b) with the length of each of the segments of interest, Calculate the density ratio; (ii) calculating the sequence tag density ratio for each normalized segment sequence by relating the number of sequence tags identified for the normalized segment sequence to the length of each normalized segment sequence in step (b); (iii) calculating a single segment capacity for each segment of interest using the sequence tag density ratio calculated in steps (i) and (ii), wherein the segment capacity is a sequence tag density ratio And the sequence tag density ratio for the normalized segment sequence for each of the segments of interest. The method may further comprise calculating a normalized segment value (NSV), wherein the NSV relates the segment capacity to an average of the corresponding segment capacity as follows in a set of qualifying samples:

및

는 적격 샘플의 세트에서 j번째 세그먼트 용량에 대한 각기 추정 평균 및 표준 편차이고,

는 시험 샘플 i에 대해 관찰된 j번째 세그먼트 용량이다.In this formula,

And

Is the respective estimated mean and standard deviation for the jth segment capacity in the set of eligible samples,

Is the jth segment capacity observed for test sample i.

In an embodiment of the method described in which the chromosomal capacity or segment capacity is determined using the normalized segment sequence, the normalized segment sequence may be any one or more single segments of chromosomes 1-22, X, and Y. Alternatively, the normalized segment sequence may be a group of any one or more of the segments of chromosome 1-22, X, and Y. [

The steps (a) - (d) of the method for determining the presence or absence of partial fetal chromosomal integrity are repeated for a test sample from a different maternal subject, said method comprising the steps of: And determining the presence or absence of the isomerism. The partial fetal chromosomal aberration that can be determined according to the present method includes the partial imbalance of any segment of any chromosome. Partial completeness can be selected from partial redundancy, partial increment, partial insertion, and partial deletion. Examples of the partial diplomacy that can be determined according to the present method are a partial single chromosome of chromosome 1, a partial single chromosome of chromosome 4, a partial single chromosome of chromosome 5, a partial single chromosome of chromosome 7, Partial chromosomes of chromosome 15, partial single chromosome of chromosome 17, partial single chromosome of chromosome 18, and partial single chromosome of chromosome 22.

In any of the embodiments described above, the test sample may be a maternal sample selected from blood, plasma, serum, urine, and saliva samples. In either embodiment, the test sample may be a plasma sample. The nucleic acid molecule of the maternal sample is a mixture of DNA molecules free of fetal and maternal cells. Sequencing of nucleic acids can be performed using next generation sequencing (NGS). In some embodiments, sequencing is massively parallel sequencing using sequencing-by-synthesis with a reversible dye terminator. In another embodiment, the sequencing is sequencing-by-ligation. In yet another embodiment, the sequencing is single molecule sequencing. Optionally, the amplification step is performed prior to sequencing.

In another embodiment, a method is provided for determining the presence or absence of any more than twenty different complete fetal chromosomal integra- tions in a maternal plasma test sample comprising a mixture of DNA molecules free of fetal and maternal cells. (A) sequencing at least a portion of a cell-free DNA molecule to obtain sequence information for a DNA molecule free of fetal and maternal cells in a sample; (b) identifying a plurality of sequence tags for each of at least 20 interest chromosomes selected from chromosomes 1-22, X, and Y using the sequence information, and identifying a plurality of sequence tags for each of the 20 or more chromosomes Identifying the sequence tag of; (c) calculating a single chromosome dose for each of the 20 or more chromosomes of interest using the number of sequence tags identified for each of the 20 or more interest chromosomes and the number of sequence tags identified for each normalized chromosome; (d) determining the presence or absence of any of the twenty or more different complete fetal chromosomal aberrations in the sample by comparing each single chromosome dose for each of the twenty or more interest chromosomes with a threshold for each of the twenty or more interest chromosomes .

In another embodiment, the present invention provides a method for identifying a copy of a sequence of interest, such as a clinically relevant sequence, in a test sample, said method comprising the steps of: (a) Wherein said test sample comprises test nucleic acid molecules and said plurality of qualifying samples comprises elutable nucleic acid molecules; (b) obtaining sequence information for said fetal and parent nucleic acid in said sample; (c) calculating, based on said sequencing of said competent nucleic acid molecule, the qualifying sequence capacity for said affinity sequence at each of said plurality of qualifying samples, wherein said calculation of qualifying sequence capacity comprises comparing said qualifying sequence with said affinity sequence and Determining a parameter for the qualifying normalization sequence; (d) identifying one or more qualifying normalization sequences based on the qualifying sequence capacity, wherein the one or more qualifying standardization sequences have the smallest variability and / or greatest variability in sequence capacity of the plurality of qualifying samples ); (e) calculating a test sequence volume for the test sequence of interest, based on the sequencing of the nucleic acid molecule in the test sample, wherein the calculation of test sequence volume is based on the test sequence of interest and one or more standardization test sequences Wherein the one or more normalization test sequences correspond to the one or more qualifying standardization sequences; (f) comparing the test sequence capacity to one or more thresholds; And (g) evaluating the number of copies of the sequence of interest in the test sample based on the result of step (f). In one embodiment, the parameters of interest qualifying sequences and one or more qualifying standardization sequences relate to the number of sequence tags mapped to the qualifying sequence relative to the number of tags mapped to the qualifying standardization sequence, The test sequence and the parameter for one or more normalization test sequences relate to the number of sequence tags mapped to the test sequence of interest relative to the number of tags mapped to the normalization test sequence. In some embodiments, step (b) comprises sequencing at least a portion of the qualifying and test nucleic acid molecules, wherein the sequencing comprises a test and interest sequence and a plurality of mapped sequences for one or more tests and one or more qualifying standardization sequences Providing a tag; At least a portion of the nucleic acid molecule of the test sample is sequenced to obtain sequence information for the fetal and parent nucleic acid molecules of the test sample. In some embodiments, the sequencing step is performed using a next generation sequencing method. In some embodiments, the sequencing method may be a massively parallel sequencing method that utilizes sequencing through synthesis by a reversible dye terminator. In another embodiment, the sequencing method is sequencing through ligation. In some embodiments, sequencing includes amplification. In another embodiment, sequencing is single molecule sequencing. The CNV of the sequence of interest is heterologous, and may be chromosomal or partial. In some embodiments, the chromosomal aneuploidies are selected from the group consisting of: trisomy 2, trisomy 8, trisomy 9, trisomy 16, trisomy 21, trisomy 13, trisomy 18, 22, and 47, XXY, 47, XXX, 47, XYY, and X chromosomes. In other embodiments, partial isomerism is partial chromosome deletion or partial chromosome insertion. In some embodiments, the CNV identified by the present method is a chromosomal or partial isomerism associated with cancer. In some embodiments, the test and qualifying sample is a biological fluid sample, such as a plasma sample, obtained from a pregnant subject, such as a pregnant human subject. In another embodiment, the test and the eligible biological fluid sample, such as a plasma sample, are obtained from a subject known or suspected of having a cancer.

Although the present embodiments are directed to humans and the language is primarily concerned with humans, the concepts of the present invention may be applied to any plant or animal genome.

Include references

All patents, patent applications, and other publications, including all sequences listed in the references cited herein, are expressly incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Are incorporated by reference. All cited documents are incorporated herein by reference in their entirety. However, citation of any document should not be construed as an admission that it is prior art to the present invention.

The novel features of the present invention are disclosed herein alone in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The features and advantages of the invention will be better understood with reference to the following detailed description set forth in the illustrative examples, which utilize the principles of the invention, and the accompanying drawings, in which:
Figure 1 is a flow diagram (100) of a method for determining the presence or absence of a copy number variation in a test sample comprising a mixture of nucleic acids.
Figure 2 shows the distribution of chromosomal capacity for chromosome 21 determined by sequencing cfDNA extracted from a set of 48 blood samples obtained from a human subject, each of which was male or female, respectively. Chromosome 21 capacity for chromosome 21, i.e., normal (O), and 21 trisomy 3 (△) test sample, was measured on chromosome 1-12 and X (FIG. 2A) and on chromosome 1-22 and X ). ≪ / RTI >
Figure 3 shows the distribution of chromosomal capacity for chromosome 18 determined by sequencing cfDNA extracted from a set of 48 blood samples obtained from human subjects each of which is male or female. Chromosome 18 capacity for chromosome 18, ie, normal (O), and 18 trisomy 3 (△) test samples, was measured on chromosome 1-12 and X (FIG. 3a), and on chromosome 1-22 and X ). ≪ / RTI >
Figure 4 shows the distribution of chromosome doses for chromosome 13 determined by sequencing cfDNA extracted from a set of 48 blood samples obtained from human subjects each of which was male or female. Chromosome 13 capacity for chromosome 13, i.e., normal (O), and 13 trisomy stained (?) Test samples, was measured on chromosome 1-12 and X (FIG. 4A) and on chromosome 1-22 and X ). ≪ / RTI >
Figure 5 shows the distribution of chromosomal capacity for chromosome X determined by sequencing cfDNA extracted from a set of 48 test blood samples obtained from human subjects each of which was male or female. Boy (46, XY; (○)), girl (46, XX; (△)); Chromosome X capacity for the X-monochrome (45, X; (+)), and complex karyotype (Cplx ). ≪ / RTI >
Figure 6 shows the distribution of chromosomal capacity for chromosome Y determined by sequencing cfDNA extracted from a set of 48 test blood samples obtained from human subjects each of which was male or female pregnant. Boy (46, XY; (○)), girl (46, XX; (△)); The chromosome Y capacity for the X monochrome (45, X; (+)), and complex karyotype (Cplx (X)) samples is shown for chromosome 1-12 (FIG. 6A) and chromosome 1-22 do.
Figure 7 shows the coefficient of variation CV for chromosomes 21 (1), 18 () and 13 () determined from the capacities shown in Figures 2, 3 and 4, respectively.
Fig. 8 shows the coefficient of variation (CV) for the chromosomes X (1) and Y (2) determined from the capacities shown in Figs. 5 and 6, respectively.
Figure 9 shows the cumulative distribution of GC fractions by the human chromosome. The vertical axis represents the frequency of the chromosome with the CG content below the value shown on the horizontal axis.
Figure 10 shows the sequence capacity of a segment (81000082-103000103bp) of chromosome 11 determined by sequencing cfDNA extracted from a set of seven qualifying samples (?) Obtained and one test sample (?) From a pregnant human subject (Y-axis). A sample ()) from a subject carrying a fetus with partial isomerism of chromosome 11 was identified.
Fig. 11 is a graph comparing the mean standard deviation (Y-axis) of the chromosome 21 (A), chromosome 18 (B), chromosome 13 (C), chromosome X (D) The distribution of normalized chromosomal capacity for Y (E) is illustrated.
Figure 12 shows the normalized chromosomal values for chromosomes 21 (O), 18 (DELTA), and 13 (& squ &) determined in the samples from Training Set 1 using the standardized chromosome described in Example 6.
Figure 13 shows the normalized chromosome values for chromosomes 21 (O), 18 (DELTA), and 13 (& squ &) determined in the samples from test set 1 using the standardized chromosome described in Example 6.
Figure 14 shows the normalized chromosome values for chromosomes 21 (O) and 18 (DELTA) determined in the samples from test set 1 using the standardization method of Chiu et al. (The number of sequence tags identified for the chromosome of interest is shown in the sample Normalized to the number of sequence tags obtained for the remaining chromosomes; see Example 7 elsewhere herein).
Figure 15 shows the normalized chromosomal values for chromosomes 21 (O), 18 (DELTA), and 13 (& squ &) determined in the samples from the training set 1 using the systematic determined standardized chromosomes (as described in Example 7 ).
Figure 16 shows the standardized chromosomal values for chromosomes 21 (O), 18 (DELTA), and 13 (& squ &) determined on samples from test set 1 using a systematic determined standard chromosome (as described in Example 7 ).
Figure 17 shows the normalized chromosome values for chromosome 9 (O) determined in the samples from test set 1 using the systematically determined normalized chromosomes (as described in Example 7).
Figure 18 shows the normalized chromosome values for chromosome X (X-axis) and Y (Y-axis). The arrows show five (Fig. 18A) and three (Fig. 18B) X-ray chromosome samples identified in the training and test sets, respectively, as described in Example 7.
Figure 19 shows the normalized chromosome values for chromosome 1-22 determined in the samples from test set 1 using the systematically determined normalized chromosomes (as described in Example 7).

The present invention provides a method for determining the number of copies of a sequence of interest (CNV) of interest in a test sample comprising a mixture of nucleic acids that are known to be different or different in the amount of one or more sequences of interest. The sequence of interest includes a kilobase (kb) to megabase (Mb) range of genomic sequences for the entire chromosome that is known or thought to be associated with a genetic or disease condition. Examples of sequences of interest include segments of partial trisomy 3 in well known miRNAs, such as chromosomes associated with trisomy 21, and chromosomes increased in diseases such as cancer, such as acute myelogenous leukemia. The CNVs that can be determined according to the present method are usually chromosomes 1-22 and any one or more of the male chromosomes and trisomes of sex chromosomes X and Y, such as 45, X, 47, XXX, 47, XXY and 47, XYY, Deletion and / or duplication of any one or more of the chromosomes of the outer chromosome, including, but not limited to, chromosomes and chromosomes, including XXXX, XXXXX, XXXXY and XYYYY, and chromosomes.

The method includes a statistical approach that describes the variable stamming resulting from process-related, interchromosomal (in-process) and intersequencing (inter-process) variability. The method can be used to determine CNVs that are known to be associated with, or are likely to be involved in, various fetal insulting CNVs and various medical disorders.

Unless otherwise indicated, the practice of the present invention encompasses conventional techniques commonly used in the art of molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and recombinant DNA within the skill of the art. Such techniques are known to those skilled in the art and are described in a number of documents and references (e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual", Third Edition (Cold Spring Harbor, and Ausubel et al., " Current Protocols in Molecular Biology " [1987]).

The numerical range includes a number defining the range. All maximum numerical limitations provided throughout this specification are intended to encompass such lower numerical limitations, as all lower numerical limitations are explicitly stated herein. All minimum numerical limitations provided throughout this specification will include such upper numerical limitations, as all upper numerical limitations are explicitly stated herein. All numerical ranges provided throughout this specification will encompass such narrower numerical ranges, all within a broader numerical range, as all narrower numerical ranges are all expressly recited herein.

The subject matter provided herein is not intended to limit the various aspects or embodiments of the invention that may be collectively referred to herein. Thus, as indicated above, the terms immediately below are collectively defined more fully in connection with the present specification.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Various scientific dictionaries, including the terms contained herein, are well known and available to those skilled in the art. Although any methods and materials similar or equivalent to those described herein are used in the practice or testing of the present invention, some preferred methods and materials are described. Accordingly, the terms defined immediately below are generally more fully described in connection with the present specification. It is to be understood that the invention is not limited to the particular methods, protocols and reagents described, as the methods, protocols and reagents may vary according to the circumstances used by those skilled in the art.

Justice

The singular terms used herein include a plurality of references unless the context clearly dictates otherwise. Unless otherwise indicated, each nucleic acid is written from left to right in 5 'to 3' orientation, and the amino acid sequence is written from left to right in amino to carboxy orientation.

The term " evaluating " herein refers to characterizing a state of chromosomal integration by one of three types of calls: "normal", "affected" and "no-call" . For example, in the presence of a trisomy, a "normal" call is determined by the value of a parameter that is below the threshold of user-defined confidence, such as the test chromosome capacity, and the "altered" call is a threshold of user- Parameters determined by the above parameters, such as the test chromosome capacity, and the "no-call" result being a parameter that lies between the user-defined confidence thresholds that result in a "normal" or "altered" call, .

The term " copy number variation " as used herein refers to a variation in the number of copies of a nucleic acid sequence of 1 kb or more present in a test sample relative to the number of copies of the nucleic acid sequence present in the qualifying sample. &Quot; Radix water variant " refers to a sequence of 1 kb or greater of the nucleic acid found by comparing the sequence of interest in the test sample with the sequence present in the test sample. Copy number variants / mutations include deletions involving microdeletions, insertions including microinjection, duplication, increase, reversal, dislocation and complex multi-site variants. CNV includes chromosomal and partial isomerism.

The term " isomerism " as used herein means an imbalance of a genetic material caused by the loss or acquisition of a whole chromosome, or a portion of a chromosome.

The terms " chromosomal " and " complete chromosomal " refer to an imbalance of genetic material caused by loss or acquisition of the entire chromosome, including germline and mosaic.

As used herein, the terms " partial isomerism " and " partial chromosomal " refer to an imbalance of genetic material, such as partial heterosomes and partial trisomy, caused by loss or gain of chromosomes, And the like.

The term " isomeric sample " herein refers to a sample that represents a subject having a chromosomal content that is not polyploid, i.e., the sample represents a subject having an abnormal chromosomal copy number.

The term " isomeric chromosome " as used herein refers to a chromosome that is known or determined to be present in the sample as an abnormal number of radiations.

The term " plurality " is used herein to refer to a plurality of nucleic acid molecules or sequence tags sufficient to identify significant differences in the number of copies (e.g., chromosome capacity) in a test sample and an eligible sample using the methods of the invention . In some embodiments, at least about 3 x 10 ⁶ sequence tags comprising 20 to 40 bp reads, 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 are obtained for each test sample.

The terms " polynucleotide ", " nucleic acid " and " nucleic acid molecule " are used interchangeably and the 3 'position of the pentose of one nucleotide is linked by a phosphodiester group to the 5' Refers to a covalently linked sequence of nucleotides (i. E., Ribonucleotides in the case of RNA and deoxyribonucleotides in the case of DNA), including but not limited to sequences of any form of nucleic acid including RNA, DNA and cfDNA molecules do. The term " polynucleotide " includes, but is not limited to, single- and double-stranded polynucleotides.

The term " part " is used herein in the context of the amount of sequence information of a fetal and maternal nucleic acid molecule in a biological sample, i.e., less than the sequence information of < 1 human genome.

The term " test sample " as used herein refers to a sample comprising a mixture of nucleic acids comprising at least one nucleic acid sequence having a number of copies that are believed to have undergone a mutation. The nucleic acid present in the test sample is referred to as " test nucleic acid ".

The term " qualifying sample " herein refers to a sample comprising a mixture of nucleic acids present in the number of known radiations in which the nucleic acid in the test sample is compared, which is a normal, i.e. non-isomeric sample for the sequence of interest, Eligible samples used to identify the standardized chromosomes are samples that are not trisomy 21 samples.

The term " training set " as used herein refers to a set of samples that may include altered and undisturbed samples. The unmodified samples in the training set are used as qualifying samples to identify the normalization sequences, such as the standardized chromosomes, and the chromosomal capacity of the unmodified sample is used to set the threshold for the sequence of interest, e.g., chromosome. The altered sample in the training set can be used to demonstrate that the altered test sample can be easily distinguished from the unaltered sample.

The term " competent nucleic acid " is a sequence that is used interchangeably with " competent sequence " and the amount of test sequence or test nucleic acid is compared. An eligibility sequence is preferably a sequence present in the biological sample in a known representation, i.e., the amount of which is known. An " affinity qualifying sequence " is a sequence associated with a difference in sequence representation in an individual having a medical disease, wherein the amount in the qualifying sample is a known qualifying sequence.

The term " sequence of interest " herein refers to a nucleic acid sequence associated with a difference in sequence representation in a healthy individual versus a diseased individual. A sequence of interest may be a sequence for a chromosome that is misrepresented in a disease or genetic condition, i. E., An over-or under-representation. The sequence of interest may also be part of a chromosome, i.e. a chromosome segment or a chromosome. For example, the sequence of interest may be a gene that encodes an over-expressed chromosome in an ischemic condition, or a tumor-inhibitor that is under-expressed in cancer. Sequences of interest include sequences that are over- or under-expressed in a population, or a subset of the cells of the subject. An " affinity sequence " is a sequence of interest in an eligible sample. &Quot; Affinity test sequence " is the sequence of interest in the test sample.

The term " normalized sequence " herein refers to a sequence that most closely resembles that of the sequence of interest used as a normalization parameter and which can best distinguish altered samples from one or more unoriented samples, and a sequence tag Quot; refers to a sequence that exhibits variability in the number of nucleotides. A "normalized chromosome" or "normalized chromosome sequence" is an example of a "normalized sequence". A " normalized chromosomal sequence " may consist of a single chromosome or a group of chromosomes. &Quot; Normalized segment " is another example of " normalized sequence ". A " normalized segment sequence " may be composed of a single segment of a chromosome, or it may consist of two or more segments of the same or different chromosomes.

As used herein, the term " differentiation " means a feature of a normalized chromosome that allows one or more altered, i.e., unmodified, i.e., normal, samples to be distinguished from an isomeric sample.

The term " sequence capacity " as used herein refers to a parameter that associates the sequence tag sequence of the sequence of interest with the tag density of the normalization sequence. &Quot; Test sequence capacity " is a parameter that associates the sequence tag sequence of a sequence of interest, such as chromosome 21, with the sequence tag sequence of a normalization sequence, such as chromosome 9, as determined in the test sample. Similarly, an "affinity sequence dose" is a parameter that associates the sequence tag sequence of a sequence of interest with the sequence tag sequence of a normalization sequence determined in an eligible sample.

The term " sequence tag density " as used herein refers to the number of sequence leads mapped to the reference genome sequence, for example, the sequence tag density for chromosome 21 is the number of sequence leads generated by the sequencing method mapped to chromosome 21 of the reference genome Number. The term " sequence tag density ratio " herein means the ratio of the number of sequence tags mapped to the chromosome of the reference genome, such as chromosome 21, to the length of the reference genome chromosome 21.

The term " next generation sequencing (NGS) " as used herein refers to a sequencing method that enables bulk parallel sequencing of nucleic acid molecules and single nucleic acid molecules amplified by the clones. Non-limiting examples of NGS include sequencing through synthesis using a reversible dye terminator, and sequencing through ligation.

The term " parameter " herein refers to a numerical value characterizing a numerical correlation between a quantitative data set and / or a quantitative data set. For example, the ratio (or function of ratio) between the number of sequence tags mapped for a chromosome and the length of the chromosome to which the tag is mapped is a parameter.

The terms " threshold " and " qualifying threshold ", as used herein, refer to any number that is calculated using a set of qualified data and that functions as a threshold for diagnosis of variation in the number of radiations in an organism, e. If the results obtained by the practice of the present invention exceed a threshold value, the subject can be diagnosed as having a radiopaque variation, for example, trisomy 21. Appropriate threshold values for the methods described herein can be ascertained by analyzing the normalized values calculated for the training set of samples (e.g., chromosome dose, NCV or NSV). Thresholds may be identified using a qualitative (i.e., unaltered) sample in a training set that includes both qualifying (i.e., unaltered) and altered samples. Using a sample of a training set known to have a chromosomality (i.e., altered sample), it can be confirmed that the selected threshold value is useful for distinguishing a sample that has been altered from a sample that has not been altered in the test set (see embodiments herein) do it). The choice of threshold depends on the degree of confidence that the user thinks the classification should be performed. In some embodiments, the training set used to identify the appropriate threshold value includes 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 be desirable to use a larger set of qualified samples to improve diagnostic usability of the threshold.

The term " normalization value " herein refers to the number of sequence tags identified for a sequence of interest (e.g., a chromosome or a chromosome segment) with the number of sequence tags identified for a normalization sequence (e.g., a normalized chromosome or a normalized chromosome segment) Means a numeric value. For example, a " normalization value " may be a chromosomal dose as described elsewhere herein, or may be a NCV (normalized chromosome value) as described elsewhere herein, or may be an NSV (normalized segment value) as described elsewhere herein.

The term " read " refers to DNA of sufficient length (e.g., at least about 30 bp) that can be used to identify a larger sequence or region, such as an aligned and specifically designated chromosomal or genomic region or gene &Lt; / RTI &gt;

The term " sequence tag " is used herein interchangeably with the term " mapped sequence tag " to refer specifically to a larger sequence, such as a reference sequence, by alignment, meaning a mapped sequence leader. The mapped sequence tags are uniquely mapped to the reference dielectric, i. E. They are assigned to a single position relative to the reference dielectric. Tags that can be mapped to more than one location on the reference dielectric, i.e., unmapped tags, are not included in the analysis.

As used herein, the term " aligned, " " aligned, " or " aligned " means that one or more sequences are identified as sequences conforming to the nucleic acid molecule relative to known sequences from the reference genome. Such alignment can be performed manually or by computer algorithms, examples of which include the Efficient Local Alignment of the Nucleotide Data (ELAND) computer program distributed as part of the Illumina Genomics Analysis pipeline. At alignment, the sequence matches may be 100% sequence matches or less than 100% (incomplete matches).

As used herein, the term " reference genome " refers to any particular known genomic sequence of any organism or virus that may be utilized in a sequence that is partially or completely referenced from a subject. For example, reference genomes used in human subjects as well as in many other organisms are found at the National Center for Biotechnology Information at www.ncbi.nlm.nih.gov. &Quot; Dielectric " means complete genetic information of an organism or virus that is expressed in a nucleic acid sequence.

The term " clinically-related sequence " as used herein means a nucleic acid sequence which is known or suspected to be associated with or related to a genetic or disease condition. Determining the absence or presence of a clinically-related sequence may be useful in determining a diagnosis of a medical disease, confirming a diagnosis, or providing a prognosis for the occurrence of the disease.

The term "derived" as used with reference to a nucleic acid or mixture of nucleic acids refers to means by which they are derived from a source which is the source of the nucleic acid (s) herein. For example, in one embodiment, a mixture of nucleic acids derived from two different dielectrics means that the nucleic acid, e.g., cfDNA, is naturally released by the cell through a spontaneous process such as necrosis or apoptosis. In another embodiment, a mixture of nucleic acids derived from two different dielectrics means that the nucleic acid has been extracted from two different types of cells from the subject.

The term " mixed sample " herein means a sample containing a mixture of nucleic acids derived from different dielectrics.

The term " maternal sample " herein refers to a biological sample obtained from a pregnant subject, such as a woman.

The term " biological fluid " as used herein means a liquid obtained from a biological source such as blood, serum, plasma, sputum, washings, cerebrospinal fluid, urine, semen, sweat, tears, saliva and the like. As used herein, the terms " blood, " " plasma, " and " serum " explicitly encompass fractions or processed portions thereof. Similarly, samples are taken from biopsies, swab specimens, smear samples and the like, and the " sample " explicitly includes processed fractions or portions derived from biopsies, swab specimens,

The term " parent nucleic acid " and " fetal nucleic acid " are used herein to refer to the nucleic acid of a pregnant female subject and the fetal nucleic acid possessed by a pregnant woman, respectively.

As used herein, the term " corresponding " is meant to be present in the genome of different subjects and not necessarily necessarily of the same sequence in all genomes, but rather the genetic information of interest sequences, such as genes or chromosomes, ), &Lt; / RTI &gt; such as a gene or a chromosome.

As used herein, the term " substantially cell free " includes preparations of the sample from which naturally associated components have been removed from the desired sample. For example, a plasma sample is essentially free of cells by removing blood cells that are naturally associated with it, such as red blood cells. In some embodiments, a substantial free sample is processed to remove cells that would otherwise contribute to the desired genetic material to be tested for CNV.

As used herein, the term " fetal fraction " refers to a fraction of fetal nucleic acid present in a sample comprising fetal and maternal nucleic acids.

The term " chromosome " as used herein means a genetic-containing gene carrier of living cells derived from chromatin and comprising DNA and protein components (particularly histones). Conventional individual human genome chromosome numbering systems that are internationally recognized are used herein.

The term " polynucleotide length " as used herein means the absolute number of nucleic acid molecules (nucleotides) in the region of the sequence or reference genome. The term " chromosome length " refers to the known length of a chromosome provided in a base pair provided in the NCBI36 / hg18 assembly of the human chromosome, for example, found in the World Wide Web genome.ucsc.edu/cgi-bin/hgTracks?hgsid=167155613&chromInfoPage= it means.

The term " subject " herein refers to a human subject such as mammals, invertebrates, vertebrates, fungi, yeast, bacteria, and virus as well as non-human subjects. Although the embodiments of the present application are related to humans and languages are mainly concerned with humans, the concept of the present invention can be used for a genome from any plant or animal, and is useful in fields such as veterinary science, animal husbandry, and research.

The term " disease " as used herein means a "medical disorder" as a broad term including all diseases and diseases, and includes any disease or disorder that can affect human health, benefit from medical help, It can include normal health conditions such as [damage] and pregnancy.

The term " complete " is used herein to refer to chromosomal integrity to indicate the acquisition or loss of the entire chromosome.

The term " partial " as used herein in reference to chromosomal integrity refers to the acquisition or loss of a portion of a chromosome.

The term " mosaic " herein is referred to to indicate the presence of two groups of cells with different karyotypes in one individual occurring in a single fertilized egg. The mosaic phenomenon can result from mutations during an occurrence that only reproduce as a subset of adult cells.

The term " non-mosaic " as used herein means an organism composed of cells of a karyotype, such as a human fetus.

The term " using a chromosome " as used with respect to determining the chromosomal capacity refers to the use of the sequence information obtained for the chromosome herein, i.e. the number of sequence tags obtained for the chromosome.

As used herein, the term " sensitivity " is equal to the number of actual positives divided by the sum of actual positive and negative negative.

The term " specificity " as used herein is equal to the number of actual voices divided by the sum of the actual voice and the voice.

As used herein, the term " patient sample " refers to a biological sample obtained from a patient, i. E., From a medical attention, nursing or therapeutic receptor. The patient sample may be any of the samples described herein. Preferably, the patient sample is obtained by a non-invasive procedure, such as a peripheral blood sample or a stool sample.

The term " low-diploid " as used herein refers to the number of chromosomes one or more lower than the normal multiple of a chromosome characteristic of a species.

Explanation

The present invention provides a method for determining the number of copies of a different sequence of interest (CNV) in a test sample comprising a mixture of nucleic acids derived from two different dielectrics that are known to be different or different in the amount of one or more sequences of interest to provide. The variation in the number of copies determined by the method of the present invention is dependent on the acquisition or loss of the entire chromosome, a change involving a very large chromosome segment as seen by the microscope, and a change in the DNA segment of the kilobase (kb) to the mega base (Mb) Includes an abundance of hyperspectral radiator number variations. The method includes statistical approaches that describe process-related, variable-order stems resulting from inter-chromosomal and inter-sequencing variability. The method can be used to determine CNVs that are known to be associated with, or are likely to be involved in, various fetal insulting CNVs and various medical disorders. The CNV that can be determined according to the method of the present invention can be detected by sequencing the nucleic acid of the test sample only once, including any one or more of chromosomes 1-22, any one of X and Y, and other chromosomes and other chromosomes Deletion and / or duplication of any one or more of the chromosome, chromosome, and chromosome. Any isomericity can be determined from the sequencing information obtained by sequencing the nucleic acid of the test sample only once.

CNV of the human genome has a significant impact on human diversity and susceptibility to disease (Redon et al., Nature 23: 444-454 [2006], Shaikh et al. Genome Res 19: 1682-1690 [2009]). It is known that CNV contributes to genetic disorders through different mechanisms and causes imbalance in gene amount or gene disruption in most pathologies. In addition to the direct association of CNV with genetic disease, CNV is known to mediate potentially harmful phenotypic changes. Recently, several studies have highlighted the potential pathogenicity of rare or unique CNVs in complex disorders such as autism, ADHD, and schizophrenia, reporting an increased burden of rare or new CNVs compared to normal controls (Sebat et al., 316: 445-449 [2007]; Walsh et al., Science 320: 539-543 [2008]). CNV arises mainly from dielectric rearrangement due to deletion, redundancy, insertion, and unbalanced potential events.

The methods described herein utilize a next generation sequencing technique (NGS) in which DNA templates amplified by clones or single DNA molecules are sequenced in bulk parallel fashion in flow cells (see, e.g., Volkerding et al. Clin Chem 55: 641-658 [2009]; Metzker M Nature Rev 11: 31-46 [2010]). In addition to high-throughput sequence information, NGS provides quantitative information, where each sequence leader is a countable " sequence tag " representing a single clonal DNA template or single DNA molecule. Sequencing techniques of NGS include pyrosequencing, sequencing through synthesis using reversible dye terminators, sequencing by oligonucleotide probing, and ionic semiconductor sequencing. DNA from individual samples can be sequenced individually (i.e., singleplex sequencing). DNA from multiple samples is pooled and sequenced as an exponential genomic molecule on a single sequencing run (i. E., Multiplex sequencing ), And can generate several hundred billion leads of the DNA sequence. Examples of sequencing techniques that can be used to obtain sequence information according to the present methods are described below.

How to Sequence

Some of the sequencing techniques are commercially available, such as those described by Affymetrix Inc. &lt; RTI ID = 0.0 &gt; Sequencing-by-hybridization platform from 454 Life Sciences (Bradford, CT), Illumina / Solexa (Hayward, Calif.) And Helicos Biosciences (Cambridge, There is a sequencing-by-lygation platform from Applied Biosystems (Foster City, Calif.). In addition to single molecule sequencing performed using sequencing-by-synthesis of Helicos Biosciences, other single molecular sequencing techniques have been described by Pacific Biosciences' SMRT ™ technique, the Ion Torrent ™ technique, and the Nanopore Sequencing. While the automated Sanger method is considered as a "first generation" technique, Sanger sequencing, including automated Sanger sequencing, can also be used in the method of the present invention. Additional sequencing methods are nucleic acid imaging techniques, such as atomic force microscopy (AFM) or transmission electron microscopy (TEM). An exemplary sequencing technique is described below.

In one embodiment, the method includes obtaining sequence information for a nucleic acid of a test sample, e.g., cfDNA of a maternal sample, using a single molecular sequencing technique of the Helicos True Single Molecule Sequencing (tSMS) technique [Harris TD et al., Science 320: 106-109 [2008]). In the tSMS technique, a DNA sample is truncated to a strand of about 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 fluorescently labeled adenosine nucleotides. The DNA strand is then hybridized to a flow cell containing millions of Oligo-T capture sites immobilized on the surface of the flow cell. The mold may have a density of about 100 million molds / cm ² . The flow cells are then loaded onto a machine such as a HeliScope ™ sequencer and the laser is irradiated onto the surface of the flow cell to reveal the location of each template. The CCD camera can map the position of the template on the flow cell surface. The template fluorescent label is then cut and washed. Sequencing reactions are initiated by introducing DNA polymerase and fluorescently labeled nucleotides. Oligo-T nucleic acid functions as a primer. The polymerase incorporates the labeled nucleotides into the primers in a template driven manner. Remove the polymerase and the unincorporated nucleotides. The template that induced the incorporation of fluorescently labeled nucleotides was identified by imaging the surface of the flow cell. After imaging, the cleavage step removes the fluorescent label and repeats the process with the other fluorescently labeled nucleotides until the desired lead length is achieved. Sequence information is collected using each nucleotide addition step. Complete dielectric sequencing by a single molecular sequencing technique excludes PCR-based amplification in the manufacture of sequencing libraries, and the immediacy of sample preparation enables direct measurement of the sample, rather than the measurement of the copy of the sample.

In another embodiment, the method comprises obtaining sequence information for a nucleic acid of a test sample, e. G., The cfDNA of a test sample of the mock, using 454 sequencing (Roche) (see, e.g., Margulies, M. et al. Nature 437: 376-380 [2005]). 454 Sequencing involves two steps. In the first step, the DNA shear with approximately 300-800 base pairs of fragment, and the fragment is blunt-ended. The oligonucleotide adapter is then ligated to the end of the fragment. The adapter functions as a primer for amplification and sequencing of the fragments. The fragments may be attached to DNA capture beads, such as streptavidin-coated beads, using, for example, adapter B, containing 5'-biotin tags. The fragments attached to the beads are PCR amplified within the point of the oil-water emulsion. The results are multiple copies of the DNA fragment amplified by the clone in each bead. In the second step, the beads are captured in the wells (pico-liter size). Fatigue sequencing is performed simultaneously for each DAN fragment. The addition of one or more nucleotides generates an optical signal that is recorded by the CCD camera in a sequencing machine. The signal intensity is proportional to the number of nucleotides incorporated. Fatigue sequencing utilizes pyrophosphate (PPi) released upon addition of nucleotides. PPi is converted to ATP by ATP sulfurylase in the presence of adenosine 5 'phosphodiesulfonate. Luciferase uses ATP to convert luciferin to oxyl luciferin, and this reaction generates light that is measured and analyzed.

In another embodiment, the method comprises obtaining sequence information for a nucleic acid of a test sample, e. G., The cfDNA of a test sample, using the SOLiD (TM) technique (Applied Biosystems). In SOLiD ™ sequencing via ligation, the genomic DNA is sheared into fragments and the adapter is attached to the 5 'and 3' ends of the fragment to generate a fragment library. Alternatively, an internal adapter can be used to ligate the adapter to the 5 ' and 3 ' ends of the fragment, to cyclize the fragment, to disassemble the cyclized fragment to create an internal adapter, 3 ' termini to produce a mate-paired library. Next, clonal bead populations are prepared in a microreactor containing beads, primers, templates, and PCR components. After the PCR, the template is denatured and the beads become enriched to separate the beads with the extended template. The template on the selected bead undergoes a 3 ' modification to allow bonding to the glass slide. Sequences can be determined by sequential hydriding and ligation of partially arbitrary oligonucleotides having a central crystal base (or pair of bases) identified by a specific fluorophore. After recording the color, the process is repeated after cleavage and removal of the ligated oligonucleotide.

In another embodiment, the method comprises obtaining sequence information for a nucleic acid of a test sample, e. G., CfDNA of a maternal test sample, using Pacific Biosciences single-molecule, real-time (SMRT) sequencing techniques. In SMRT sequencing, the persistent incorporation of dye-labeled nucleotides is imaged during DNA synthesis. The fused nucleotides are incorporated into a growing primer strand while a single DNA polymerase molecule is attached to the bottom surface of an individual zero-mode wavelength detector (ZMW detector) that obtains sequence information. ZMW is a constraint structure that allows the observation of the incorporation of a single nucleotide by a DNA polymerase into the background of a fluorescent nucleotide that quickly diffuses out of the ZMW (after a microsecond). It takes several milliseconds for the nucleotides to get into the growing strand. During this time, the fluorescent label is excited to generate a fluorescent signal, and the fluorescent tag is cleaved. Measurement of the corresponding fluorescence of the dyes indicates that the base is incorporated. The above process is repeated.

In another embodiment, the method includes obtaining sequence information for a nucleic acid of a test sample, e.g., cfDNA of a maternal test sample, using nanopore sequencing (see, for example, Soni GV and Meller A. Clin Chem 53 : 1996-2001 [2007]). Nanopore sequencing DNA analysis techniques are being developed industrially by many companies, including Oxford Nanopore Technologies (Oxford, United Kingdom). Nanopore sequencing is a single-molecule sequencing technique in which a single molecule of DNA is passed through a nanopore and then sequenced. The nanopore is a small hole with a diameter of about 1 nanometer. The application of nanopore immersion and potential across it in a conductive fluid generates some current due to the conduction of ions through the nanopore. The amount of current flowing is sensitive to the size and shape of the nanopore. As DNA molecules pass through the nanopore, each nucleotide on the DNA molecule interferes with the nanopore to different degrees, varying the magnitude of the current through the nanopore to varying degrees. Thus, this change in current due to the DNA molecule passing through the nanopore represents the reading of the DNA sequence.

In another embodiment, the method includes obtaining a nucleic acid of a test sample, such as a cfDNA of a mock test sample, using a chem-sensitive field effect transistor (chemFET) array (see, for example, 20090026082). In one example of the technique, a DNA molecule can be positioned in a reaction chamber, and the template molecule can be hybridized to a sequencing primer bound to a polymerase. The incorporation of one or more triphosphates from the 3 ' end of the sequencing primer into the new nucleic acid strand can be identified by a change in current by the chemFET. The array can have multiple chemFET sensors. In another example, a single nucleic acid can be attached to the bead, the nucleic acid can be amplified on the bead, and individual beads can be transferred to the individual reaction chambers on the chemFET array using respective chambers with chemFET sensors , The nucleic acid can be sequenced.

In another embodiment, the method comprises obtaining sequence information for a nucleic acid of a test sample, e.g., cfDNA, of a test sample of a host using a Halcyon molecular technique using a transmission electron microscope (TEM). Individual Molecule Placement The method named Rapid Nano Transfer (IMPRNT) utilizes single atom-resolution transmission electron microscopy imaging of selectively labeled high molecular weight (150 kb or more) DNA with a chelator marker and converts these molecules into a coherent base- (3nm-strand-to-strand) parallel arrays with base spacing on the ultra-thin layer. The electron microscope is used to image the molecules on the film to determine the location of the marker and to extract the nucleotide sequence information from the DNA. The method is further described in PCT patent publication WO 2009/046445. The method allows sequencing of a complete human genome within 10 minutes.

In another embodiment, the DNA sequencing technique is a technique of directly translating chemically encoded information (A, C, G, T) into digital information (0, 1) on a semiconductor chip by pairing a semiconductor technique with a simple sequencing chemistry Ion Torrent is single molecule sequencing. In fact, when the nucleotide is incorporated into the strand of DNA by the polymerase, the hydrogen ion is released as a by-product. Ion Torrent uses a high-density array of micro-mechanical wells to perform these biochemical processes in massively parallel fashion. Each well has a different DNA molecule. Below the well is an ion-sensitive layer and below it is an ion sensor. When the nucleotide, for example C, is added to the DNA template and then incorporated into the strand of DNA, the hydrogen ion will be released. The charge from that ion will change the pH of the solution, which can be detected by the Ion Torrent ion sensor. The sequencer - essentially the smallest solid-state pH meter in the world - calls the base, moving directly from chemical information to digital information. The ion-permanent-dielectric-mechanical (PGM ™) sequencer then sequentially immerses the chip into one nucleotide after the other. If the next nucleotide to flood the chip is not matched, no voltage change will be recorded and no base will be called. If two identical bases are present on the DNA strand, the voltage will be doubled and the chip will record the two identical bases called. It is possible to record nucleotide incorporation after a few seconds by direct detection.

In another embodiment, the method comprises obtaining sequence information for a nucleic acid of a test sample, e. G., The cfDNA of a maternal test sample, using sequencing by hybridization. Sequencing through hybridization involves contacting a plurality of polynucleotide sequences with a plurality of polynucleotide probes, wherein each of the plurality of polynucleotide probes may not be tethered or bound to a substrate. The substrate may be a flat surface comprising an array of known nucleotide sequences. The hybridization pattern for the array can be used to determine the polynucleotide sequence present in the sample. In other embodiments, each probe is tied to a bead, such as a magnetic bead. Hybridization to beads can be measured and used to identify multiple polynucleotide sequences in a sample.

In yet another embodiment, the method comprises sequencing nucleic acid of a test sample, such as cfDNA of a maternal test sample, by mass parallel sequencing of millions of DNA fragments using Illumina sequencing through synthesis and reversible terminator-based sequencing chemistry (As described, for example, in Bentley et al., Nature 6: 53-59 [2009]). The template DNA may be a genomic DNA, such as cfDNA. In some embodiments, genomic DNA from isolated cells is used as a template, which is fragmented to a length of several hundred base pairs. In other embodiments, cfDNA is used as a template, and since cfDNA exists as a short fragment, fragmentation is unnecessary. For example, fetal cfDNA circulates as a fragment of about 170 base pairs in length (Fan et al., Clin Chem 56: 1279-1286 [2010]) and no fragmentation of DNA is required before sequencing . The Illumina sequencing technique relies on attaching fragmented genomic DNA to a planar optically transparent surface to which an oligonucleotide anchor is attached. Template DNA is end-repaired to produce a 5'-phosphorylated smooth end, and a single A base is added to the 3 'end of the smooth-phosphorylated DNA fragment using the polymerase activity of the Klenow fragment. This addition produces a DNA fragment for ligation to an oligonucleotide adapter, which has an overhang of a single T base at its 3 'end to increase ligation efficiency. Adapter oligonucleotides are complementary to flow cell anchors. Under limited dilution conditions, the adapter-modified single-stranded DNA template is added to the flow cells and fixed by hybridization with an anchor. The attached DNA fragments are extended and the bridges are amplified to produce ultra-dense sequential flow cells with several hundred million clusters each containing ~ 1,000 copies of the same template. In one embodiment, arbitrarily fragmented genomic DNA, e. G., CfDNA, is amplified using PCR and cluster amplified. Alternatively, an amplification-free genomic library method is used, and arbitrarily fragmented genomic DNA, such as cfDNA, is enriched using only cluster amplification (Kozarewa et al., Nature Methods 6: 291-295 [2009 ]). Sequencing the template using a robust 4-color DNA sequencing-by-synthesis technique using a reversible terminator with removable fluorescent dye. Laser excitation and total internal reflection optics are used to achieve sensitive fluorescence detection. A short sequence lead of about 20-40 bp, e.g., 36 bp, is aligned for the repeat-masked reference dielectric and the unique mapping of short sequence leads to the reference dielectric is verified using specially developed data analysis pipeline software . Repeated-unmasked reference dielectrics are also available. Only the leads that are uniquely mapped to the reference dielectric, whether repetitive-masked or repetitive-unmasked reference dielectric, are used. After completion of the first reading, the template may be regenerated in situ so that a second reading is possible from the opposite end of the fragment. Thus, single-ended or paired end sequencing of the DNA fragment can be used. Partial sequencing of the DNA fragments present in the sample is performed and sequence tags containing a predetermined length, e.g., 36 bp leads, are mapped and counted against a known reference dielectric. In one embodiment, the reference genomic sequence is the NCBI36 / hg18 sequence, which is available on the World Wide Web at genome.ucsc.edu/cgi-bin/hgGateway?org=Human&db=hg18&hgsid=166260105). Alternatively, the reference genomic sequence is GRCh37 / hg19, 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 DDBJ (the DNA Databank of Japan). A number of computer algorithms for aligning sequences are available including but not limited to BLAST (Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993), FASTA (Person & Lipman, 1988), BOWTIE Genome Biology 10: R25.1-R25.10 [2009]), or ELAND (Illumina, Inc., San Diego, Calif., USA). In one embodiment, one end of a copy propagated by a clone of plasma cfDNA molecules is sequenced and processed by bioinformatic sorting analysis on an Illumina dielectric analyzer using Efficient Large-Scale Alignment of Nucleotide Databases (ELAND) software do.

In some embodiments of the methods described herein, the mapped sequence tag is about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, , About 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, Lt; RTI ID = 0.0 &gt; 500 bp. &Lt; / RTI &gt; It is anticipated that advances in technology will enable single-ended leads of greater than 500 bp, allowing a lead of greater than about 1000 bp when a paired terminal lead is generated. In one embodiment, the mapped sequence tag comprises a sequence leader of 36 bp. The mapping of the sequence tag is accomplished by determining the chromosomal origin of the sequenced nucleic acid (e.g., cfDNA) molecule by comparing the sequence of the tag with the sequence of the reference, and no specific genetic sequence information is required. A small degree of mismatch (0-2 mismatches per sequence tag) due to a small number of polymorphisms that may exist between the reference dielectric and the dielectric of the mixed sample can be tolerated.

A plurality of sequence tags are obtained for the sample. In some embodiments, at least about 3 x 10 ⁶ sequence tags comprising 20 to 40 bp leads, such as 36 bp, 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 are obtained by mapping the leads to the reference dielectric per sample. In one embodiment, all sequence leads are mapped for all regions of the reference dielectric. In one embodiment, all regions of the reference genome, such as mapped tags for all chromosomes, are counted and the CNV of the sequence of interest, such as its chromosome or portion, is determined in the mixed DNA sample, i.e., over- or under-representation. The method does not require the distinction of the two dielectrics.

The accuracy required to accurately determine whether CNV, e.g., isomerism, is present or absent in a sample is a function of the number of sequence tags (interchromosomal variability) mapped to the reference genome among the samples in the sequencing progression, Variations in the number of sequence tags mapped to the dielectric (intersequence variability) are predicted. For example, the mutation may be specifically indicated for a tag that is mapped to a GC-rich or GC-deficient reference sequence. Other variations may result from different protocols for extraction and purification of nucleic acids, the manufacture of sequencing libraries, and the use of different sequencing platforms. The method is based on the knowledge of a normalization sequence (normalized chromosomal sequence or normalized segment sequence) due to inherent chromosomal (in progress), and variable sequencing and platform-dependent variability resulting from sequencing (progression) (Chromosome capacity, or segment capacity). The chromosomal capacity is based on knowledge of a single chromosome, or of a normalized chromosome sequence that can consist of two or more chromosomes selected from chromosomes 1-22, X, and Y. Alternatively, the normalized chromosome sequence may consist of a single chromosome segment, or one chromosome, or two or more segments of two or more chromosomes. Segment capacity is based on the knowledge of a single segment of any single chromosome, or of a standardized segment sequence, which can consist of two or more segments of any two or more of chromosomes 1-22, X, and Y.

Determination of the Normalized Sequence in the Eligible Sample: Normalized Chromosomal Sequence and Standardization Segment  order

The normalization sequence is identified using sequence information from a set of qualifying samples obtained from a subject known to contain cells of any sequence of interest, for example, a cell having a normal number of copies for its chromosome or segment. The determination of the normalization sequence is summarized in steps 100, 120, 130, 140, and 145 of the embodiment of the method depicted in FIG. The sequence information obtained from the qualifying sample is also used to determine a statistically significant confirmation of chromosomal integration in the test sample (step 155, Figure 1, and Example).

Figure 1 provides a flow diagram (100) of an embodiment of the method of the present invention for determining the CNV of a sequence of interest, e.g., its chromosome or segment, in a biological sample. In some embodiments, the biological sample comprises a mixture of nucleic acids obtained from a subject and provided by different dielectrics. Different dielectrics can be provided to a sample by two individuals, for example, different dielectrics are provided by the fetus and the mother bearing the fetus. Alternatively, the genome is provided to a sample by a canine cancerous cell and a normal squamous cell from the same subject, such as a plasma sample from a cancer patient.

A set of qualifying samples was obtained to identify the qualifying normalization sequence and to provide a variance value used to determine a statistically significant confirmation of CNV in the test sample. In step 110, a plurality of biologically competent samples are obtained from a plurality of subjects known to contain cells with a normal number of copies for any sequence of interest. In one embodiment, a qualifying sample is obtained from a pregnant host with a fetus identified as having a normal copy number of chromosomes using cytogenetic means. The biologically competent sample may be a biological fluid, such as plasma, or any suitable sample described below. In some embodiments, a qualifying sample contains a mixture of nucleic acid molecules, such as cf DNA molecules. In some embodiments, the qualifying sample is a maternal plasma sample containing a mixture of fetal and maternal cfDNA molecules. Sequence information for normalized chromosomes and / or segments thereof is obtained by sequencing at least a portion of nucleic acids, such as fetal and parent nucleic acids, using any known sequencing method. Preferably, the fetal and maternal nucleic acids are sequenced as single or cloned amplified molecules using any of the next generation sequencing (NGS) methods described elsewhere herein.

At step 120, at least a portion of each of each eligible nucleic acid contained in the eligible sample is sequenced to generate millions of sequence leads, such as 36bp leads, aligned against a reference genome such as hg18. In some embodiments, the sequence leader is selected from the group consisting of about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, About 90 bp, about 95 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, It is anticipated that advances in technology will enable single-ended leads of greater than 500 bp, allowing a lead of greater than about 1000 bp when a paired terminal lead is generated. In one embodiment, the mapped sequence leader comprises 36 bp. The sequence leads are aligned with respect to the reference dielectric, and the uniquely mapped leads to the reference dielectric are known as sequence tags. In one embodiment, at least about 3 x 10 ⁶ qualifying sequence tags comprising 20 to 40 bp leads, at least about 5 x 10 ⁶ qualifying sequence tags, at least about 8 x 10 ⁶ qualifying 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 &lt; ⁶ &gt; qualifying sequence tags are obtained from the leads that are uniquely mapped to the reference dielectric.

At step 130, the nucleic acid of the qualifying sample is sequenced and all tags obtained are counted to determine the qualitative sequence tag density. In one embodiment, the sequence tag density is determined as the number of qualifying sequence tags mapped to the sequence of interest on the reference genome. In another embodiment, the qualitative sequence tag densities are determined as the number of qualifying sequence tags mapped to the sequence of interest normalized to the length of the interest qualifying sequence to which they are mapped. The sequence tag density determined as the ratio of the tag density to the length of the sequence of interest is referred to herein as the tag density ratio. Standardization to the length of the sequence of interest is unnecessary and can be included as a step to simplify this in human interpretation by reducing the number of numbers in a number. Since all qualifying sequence tags are mapped and counted in each eligible sample, the sequence tag sequence for a sequence of interest, such as a clinically-related sequence, is determined in the qualifying sample, which is then compared to the sequence tag It is the same density.

In some embodiments, the sequence of interest is a chromosome associated with complete chromosomal accessibility such as chromosome 21, the qualifying normalization sequence is not associated with chromosomal accessibility, and the variation in sequence tag density is a variation of the sequence of interest (i. E., Chromosome) Is the most complete chromosome. Any one or more of chromosomes 1-22, X, and Y may be of interest, and one or more chromosomes may be identified as the normalization sequence for any one of chromosomes 1-22, X, and Y in each of the eligible samples . Standardized chromosomes may be individual chromosomes or may be groups of chromosomes as described elsewhere herein.

In another embodiment, the sequence of interest is a segment of a chromosome associated with partial isomerism such as a chromosomal deletion or insertion, or an unbalanced chromosomal translocation, wherein the normalization sequence is not related to the partial isomerism and the variation in the sequence tag density is a partial isomerism Is the closest chromosome segment to the chromosome segment variation associated with. Any one or more segments of any one or more of the chromosomes 1-22, X, and Y may be of interest.

In all embodiments, whether a single sequence or a group of sequences is identified in an eligible sample as a standardization sequence for any one or more sequences of interest, the qualifying standardization sequence is most similar to the variation in sequence tag sequence of the sequence of interest determined in the qualifying sample . For example, an eligibility normalization sequence is the sequence with the smallest variability, i.e., the variability of the normalization sequence is closest to the variability of the sequence of interest.

In some embodiments, the normalization sequence is the sequence that best distinguishes one or more qualifying samples from one or more altered samples, which is the sequence with the greatest differentiation, i.e., the differentiation of the normalization sequence, To provide an optimal identification of the sequence of interest in the altered test sample in order to distinguish it from the intact samples from the outside. In other embodiments, the normalized sequence is the sequence with the greatest differentiability from the smallest variant. The level of differentiation can be determined as a statistical difference between the chromosomal capacity or the capacity of a segment in the population of eligible samples as shown in the following examples and in the sequential capacity such as the segment capacity and the chromosomal dose (s) in one or more test samples. For example, the discrimination can be expressed numerically as a T-test value, which represents the chromosomal capacity in the population of eligible samples and the statistical difference between the chromosomal dose (s) in one or more test samples. Alternatively, the discrimination can be expressed numerically as a normalized chromosome value (NCV), which is the z-score for chromosomal capacity as long as the distribution to NCV is normal. Similarly, the differentiability can be expressed numerically as a T-test value, which represents the segment capacity in a population of eligible samples and the statistical difference between segment capacity (s) in one or more test samples. Alternatively, the differentiation of segment capacity can be expressed numerically as a normalized segment value (NSV), which is the z-score for chromosomal capacity as long as the distribution to NSV is normal. In determining the z-score, the mean and standard deviation of the chromosome or segment capacity in a set of qualifying samples may be used. Alternatively, the mean and standard deviation of the chromosome or segment capacity can be used in a training set comprising an eligible sample and a corrupted sample. In other embodiments, the normalized sequence is the sequence with the greatest differentiability from the smallest variant.

The method has inherently similar characteristics and tends to have similar variations between the sample and sequencing progress and identifies sequences useful for determining sequence capacity in test samples.

In qualifying samples, the sequence capacity (i. E., Chromosome capacity or Segment  Capacity)

In step 140, based on the calculated qualification tag density, the qualifying sequence capacity for the sequence of interest, i. E., The chromosome capacity or segment capacity, is compared to the sequence tag density for the sequence of interest and subsequently the additional sequence Is determined as the ratio of the qualitative sequence tag density to the &lt; RTI ID = 0.0 &gt; Sequence capacity is then determined in the test sample using the identified normalization sequence.

 In one embodiment, the sequence capacity of an eligible sample is a calculated chromosome dose as a ratio of the number of sequence tags to the chromosome of interest and the number of sequence tags to the normalized chromosome sequence in the eligible sample. The normalized chromosome sequence may be a single chromosome, a group of chromosomes, a segment of one chromosome, or a group of segments from different chromosomes. Thus, the chromosomal capacity for the chromosomes of interest can be determined from (i) the number of tags for a chromosome of interest and the number of tags for a standard chromosome sequence composed of a single chromosome, (ii) the number of tags for a chromosome of interest, and The ratio of the number of tags to the standardized chromosome sequence composed of the above chromosomes, or (iii) the ratio of the number of tags to the standardized segment sequence composed of the number of tags for the chromosome of interest and the single segment of the chromosome, (iv) The number of tags for a chromosome of interest and the ratio of the number of tags to a standardized segment sequence consisting of two or more segments from one chromosome, or (v) the number of tags for a chromosome of interest and a normalized segment sequence consisting of two or more segments of two or more chromosomes As a ratio of the number of tags to the number of tags. An example of determining the chromosome capacity for the chromosome 21 of interest according to (i) - (v) is as follows: The chromosomal capacity for the chromosome of interest, for example chromosome 21, is determined by the sequence tag density of chromosome 21 and all remaining chromosomes, 1-20, chromosome 22, chromosome X, and chromosome Y, respectively (i); (Ii) measuring the chromosomal capacity for a chromosome of interest, such as chromosome 21, as the ratio of the sequence tag density for chromosome 21 to the sequence tag density for all possible combinations of two or more remaining chromosomes; Measuring the chromosome capacity for the chromosome of interest, such as chromosome 21, as the ratio of the sequence tag density for chromosome 21 to the sequence tag density for another chromosome segment, such as chromosome 9; Measuring the chromosomal capacity for a chromosome of interest, such as chromosome 21, as the ratio of the sequence tag density of chromosome 21 to the two tag segments of another chromosome, e.g., two segments of chromosome 9 (iv); The chromosome capacity for a chromosome of interest, such as chromosome 21, is measured as the ratio of the sequence tag density of chromosome 21 and the two tagged segments of two different chromosomes, such as the segment of chromosome 9 and the sequence tag of the segment of chromosome 14.

In another embodiment, the sequence capacity of an eligible sample is the calculated segment capacity as a ratio of the number of sequence tags for the segment of interest and the number of sequence tags for the normalized segment sequence in the eligible sample. The normalized segment sequence may be a segment of one chromosome, or a group of segments from different chromosomes. Thus, the segment capacity for a segment of interest can be determined from (i) the number of tags for the segment of interest and the number of tags for a normalized segment sequence consisting of a single segment of the chromosome, (ii) the number of tags for the segment of interest And (iii) the number of tags for a segment of interest and the number of tags for a normalized segment sequence consisting of two or more segments of two or more different chromosomes .

The chromosomal capacity for one or more chromosomes of interest is determined in all eligible samples, and the normalized chromosomal sequence is identified in step 145. Similarly, the segment capacity for one or more segments of interest is determined in all eligible samples, and the normalized segment sequence is identified in step 145.

Identification of the normalized sequence from the qualifying sequence capacity

In step 145, the normalization sequence is identified for the sequence of interest as a sequence based on the calculated sequence capacity, i. E., The sequence resulting in the smallest variability in sequence capacity for the sequence of interest over all eligible samples. The method has inherently similar characteristics and tends to have similar variations between the sample and sequencing progress and identifies sequences useful for determining sequence capacity in test samples.

The normalization sequence for one or more sequences of interest can be identified in a set of qualifying samples and the sequences identified in the qualifying sample are subsequently identified as one or more of the interest in each test sample in order to determine the presence or absence of the & Is used to calculate the sequence capacity for the sequence (step 150). The identified normalization sequences for the chromosomes or segments of interest may be different when different sequencing platforms are utilized and / or when there is a difference in the preparation of the sequencing library and / or the purification of the nucleic acids to be sequenced. The use of standardization sequences in accordance with the methods of the present invention provides a specific and sensitive measure of variation in the number of copies of a chromosome or segment thereof, regardless of the sample preparation and / or sequencing platform used.

In some embodiments, more than one normalization sequence is identified, i.e., various standardization sequences can be determined for one sequence of interest, and a plurality of sequence doses can be determined for one sequence of interest. For example, a variation such as the coefficient of variation in chromosome capacity for the chromosome 21 of interest is minimal when the sequence tag density of chromosome 14 is utilized. However, 2, 3, 4, 5, 6, 7, 8 or more normalization sequences can be identified for use in determining the sequence capacity for a sequence of interest in a test sample. As an example, the second dose for chromosome 21 in any one test sample can be determined using chromosome 7, chromosome 9, chromosome 11, or chromosome 12 as a normalized chromosome sequence, since all of these chromosomes have a CV for chromosome 14 (See Example 2, Table 2). Preferably, when a single chromosome is selected as the normalized chromosome sequence for a chromosome of interest, the normalized chromosome sequence will be a chromosome that produces a chromosomal capacity for a chromosome of interest with the least variability across all tested samples, such as an eligible sample .

Standardized chromosomal sequences as normalization sequences for chromosome (s)

In other embodiments, the normalized chromosomal sequence may be a single sequence, or it may be a group of sequences. For example, in some embodiments, the normalization sequence is a group of sequences identified as a normalization sequence for one or more of chromosome 1-22, X and Y, such as a group of chromosomes. The group of chromosomes containing the normalization sequence for the chromosomes of interest, i.e. the normalized chromosome sequence, is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, or 22 chromosomes, and includes or excludes one or both of chromosomes X and Y. [ A group of chromosomes identified as normalized chromosome sequences is a group of chromosomes that generate chromosomal capacity for the chromosomes of interest with the least variability across all tested samples, such as qualifying samples. Preferably, the individual chromosomes and groups of chromosomes are tested together for their ability to best mimic the behavior of the sequence of interest selected as the normalized chromosome sequence.

 In one embodiment, the normalization sequence for chromosome 21 includes 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. In another embodiment, the normalization sequence for chromosome 21 is selected from chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14. Alternatively, the normalization sequence for chromosome 21 includes 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, A chromosome 14, a chromosome 15, a chromosome 16, and a chromosome 17. In another embodiment, the group of chromosomes is a group selected from chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14.

In some embodiments, the method is further improved using standardization sequences determined by systematic computations of all chromosome doses, individually using each chromosome and in all possible combinations using all remaining chromosomes (see Example 7 ). For example, a systematically determined normalization chromosome can be used to determine whether a single chromosome or group of chromosomes is a standardized chromosome that produces a variability in the minimum chromosomal capacity for the chromosome of interest over the set of qualified samples, Y, and any combination of two or more of chromosomes 1-22, X, and Y (see Example 7) by systematically calculating all possible chromosome doses (see Example 7) . Thus, in one embodiment, the standardized chromosome sequence systematically calculated for chromosome 21 is a group of chromosomes consisting of chromosome 4, chromosome 14, chromosome 16, chromosome 20, and chromosome 22. A single chromosome or group of chromosomes can be determined for all chromosomes in the genome.

In one embodiment, the normalization sequence for chromosome 18 includes chromosome 8, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 9, chromosome 10, chromosome 11, chromosome 12, chromosome 13, 14 &lt; / RTI &gt; Preferably, the normalization sequence for chromosome 18 is selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14. Alternatively, the normalization sequence for chromosome 18 includes 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 &Lt; / RTI &gt; Preferably, the group of chromosomes is a group selected from chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14.

In another embodiment, the normalization sequence for chromosome 18 is determined by a systematic calculation of all possible chromosome doses using each possible standardization chromosome individually and using all possible combinations of standardization chromosomes (as described elsewhere herein) ). Thus, in one embodiment, the normalization sequence for chromosome 18 is a standardized chromosome composed of a group of chromosomes consisting of chromosome 2, chromosome 3, chromosome 5, and chromosome 7.

In one embodiment, the normalization sequence for chromosome X includes 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. Preferably, the normalization sequence for chromosome X is selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8. Alternatively, the normalization sequence for chromosome X includes 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, A chromosome 14, a chromosome 15, and a chromosome 16. Preferably, the group of chromosomes is a group selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.

In another embodiment, the normalization sequence for chromosome X is determined by a systematic calculation of all possible chromosome doses using each possible standardization chromosome individually and using all possible combinations of normalization chromosomes (as described elsewhere herein) ). Thus, in one embodiment, the normalization sequence for chromosome X is a standardized chromosome composed of chromosome 4 and a group of chromosome 8.

In one embodiment, the normalization sequence for chromosome 13 includes 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. Preferably, the normalization sequence for chromosome 13 is a chromosome selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8. In another embodiment, the normalization sequence for chromosome 13 includes 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 14 18, and chromosome 21, respectively. Preferably, the group of chromosomes is a group selected from chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8.

In another embodiment, the normalization sequence for chromosome 13 is determined by a systematic calculation of all possible chromosome doses using each possible standardization chromosome individually and using all possible combinations of normalization chromosomes (as described elsewhere herein) ). Thus, in one embodiment, the normalization sequence for chromosome 13 is a standardized chromosome comprising chromosome 4 and a group of chromosome 5. In another embodiment, the normalization sequence for chromosome 13 is a standardized chromosome composed of chromosome 4 and a group of chromosome 5.

The variation in chromosome dose for chromosome Y exceeds 30, apart from that used for determining chromosome Y capacity, by the normalized chromosome. Therefore, any one chromosome, or a group of two or more chromosomes selected from chromosome 1-22 and chromosome X, can be used as a normalization sequence for chromosome Y. [ In one embodiment, the at least one normalized chromosome is a group of chromosomes consisting of chromosome 1-22, and chromosome X. In another embodiment, the group of chromosomes is comprised of chromosome 2, chromosome 3, chromosome 4, chromosome 5, and chromosome 6.

In another embodiment, the normalization sequence for chromosome Y is determined by a systematic calculation of all possible chromosome doses using each possible standardization chromosome individually and using all possible combinations of normalization chromosomes (as described elsewhere herein) ). Thus, in one embodiment, the normalization sequence for chromosome Y is a normalized chromosome comprising a group of chromosomes consisting of chromosome 4 and chromosome 6. In another embodiment, the normalization sequence for chromosome Y is a standardized chromosome composed of a group of chromosomes consisting of chromosome 4 and chromosome 6.

The normalization sequences used to calculate the different interest chromosomes, or the volumes of different interest segments, may be the same, or they may be different normalization sequences for different interest chromosomes or segments. For example, a normalization sequence for a chromosome of interest A, such as a normalized chromosome (one or a group) may be the same, or it may be different from a normalization sequence for the chromosome B of interest, such as a normalized chromosome (one or a group).

The normalization sequence for a complete chromosome may be a complete chromosome or a complete chromosome group, or it may be a segment of a chromosome or a group of segments of one or more chromosomes.

Standardized sequences for chromosome (s) Segment  order

In another embodiment, the normalization sequence for a chromosome may be a normalization segment sequence. The normalized segment sequence may be a single segment, or it may be a group of segments of one chromosome, or they may be segments from two or more different chromosomes. The normalized segment sequence can be determined by a systematic calculation of all combinations of segment sequences in the genome. For example, the normalized segment sequence for chromosome 21 may be a single segment larger or smaller than the size of chromosome 2, which is approximately 47 Mbp (million base pairs) from chromosome 9, which is approximately 140 Mbp. Alternatively, the normalization sequence for chromosome 21 may be a combination of a sequence from chromosome 1 and a sequence from chromosome 12.

In one embodiment, the normalization sequence for chromosome 21 is a standardized segment sequence of one segment of chromosomes 1-20, 22, X, and Y, or a group of two or more segments. In another embodiment, the normalization sequence for chromosome 18 is a segment or a group of segments of chromosome 1-17, 19-22, X, and Y. In another embodiment, the normalization sequence for chromosome 13 is a segment or a group of segments of chromosome 1-12, 14-22, X, and Y. In another embodiment, the normalization sequence for chromosome X is chromosome 1-22, and a segment or segment group of Y. In another embodiment, the normalization sequence for chromosome Y is chromosome 1-22, and a segment or segment group of X. The standardized segment sequence of a single or segmented group can be determined for all chromosomes in the genome. Two or more segments of a normalized segment sequence may be segments from one chromosome, or two or more segments may be segments of two or more different chromosomes. As described for the normalized chromosome sequences, the normalized segment sequence may be identical for two or more different chromosomes.

chromosome To the segment (s)  Standardization as a standardized sequence Segment  order

The presence or absence of CNV of the sequence of interest can be determined when the sequence of interest is a segment of the chromosome. Variation in the number of copies of a chromosome segment makes it possible to determine the presence or absence of partial chromosomal integration. Examples of partial chromosomal aberrations associated with various fetal anomalies and disease conditions are described below. Segments of a chromosome can have any length. For example, it may range from kilo base to hundreds of megabases. The human genome occupies more than 3 billion DNA bases, which can be divided into tens, thousands, hundreds of thousands, and millions of different sized segments, where the number of copies can be determined according to the method. The normalization sequence for a segment of a chromosome may be a normalized segment sequence that may be a single segment from either chromosome 1-22, X or Y, or it may be a group of segments from any one or more of chromosomes 1-22, X, Lt; / RTI &gt;

The normalized sequence for the segment of interest is a sequence with the closest variability to the variability of the segment of interest across the chromosome and sample. The determination of the normalization sequence may be performed as described to determine the normalization sequence for the chromosome of interest when the normalization sequence is a group of any one or more of the segments of chromosome 1-22, X and Y. [ The normalized segment sequence of a segment or group of segments may be a standardized sequence for a segment of interest in each sample of one segment and a set of samples that are known to be diploid for an eligible sample, , And the normalization sequence is determined to provide the segment capacity with the smallest variability for the segment of interest over all eligible samples as described above for the normalized chromosome sequence.

For example, for a segment of interest that is 1 Mb (megabase), the remaining 3 million segments of the about 3 g human genome (minus 1 mg of interest segment) may be used individually or in combination with each other to identify interest By calculating the segment capacity for a segment, one can determine whether a segment or group of segments is eligible and functions as a normalized segment sequence for the test sample. The segment of interest may range from about 1000 bases to several tens of megabases. The normalized segment sequence may consist of one or more segments of the same size as the sequence of interest. In other embodiments, the normalized segment sequence may consist of segments that are different from and / or different from those of the sequence of interest. For example, a normalized segment sequence for a sequence of 10,0000 base-long may be 20,000 base-long and may comprise a combination of sequences of different lengths, such as 7,000 + 8,000 + 5,000 bases. As described elsewhere herein with respect to normalized chromosome sequences, the normalized segment sequence is determined by the systematic calculation of all possible chromosomal and / or segmental capacities using each possible standardized chromosomal segment individually and using all possible combinations of normalized segments (As described elsewhere herein). A single segment or group of segments may be determined for all segments and / or chromosomes of the genome.

The normalization sequence used to calculate the capacity of different interest chromosomal segments may be the same, or it may be a different normalization sequence for different interest chromosomal segments. For example, a normalization sequence for a chromosome segment of interest A, such as a normalization segment (one or a group), may be the same or may be different from a normalization sequence for a chromosome segment of interest B, such as a normalization segment (one or a group).

Determination of the water-solubility in the test sample

Based on the identification of the normalization sequence (s) in the qualifying sample, the sequence capacity is determined for the sequence of interest in a test sample comprising a mixture of nucleic acids derived from different dielectrics in one or more sequences of interest.

At step 115, the test sample is obtained from a subject known or suspected to have a clinically-related CNV of the sequence of interest. The test sample may be a biological fluid, such as plasma, or any suitable sample described below. In some embodiments, the test sample contains a mixture of nucleic acid molecules, such as cfDNA molecules. In some embodiments, the test sample is a maternal plasma sample containing a mixture of fetal and maternal cfDNA molecules.

At step 125, at least a portion of the test nucleic acid in the test sample is sequenced as described for the qualifying sample to generate millions of sequence leads, such as 36bp leads. As in step 120, the leads generated by sequencing the nucleic acid in the test sample are uniquely mapped to the reference genome. At least about 3 x 10 ⁶ qualifying sequence tags comprising 20 to 40 bp leads, at least about 5 x 10 ⁶ qualifying sequence tags, at least about 8 x 10 ⁶ qualifying 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 tag , Or at least about 50 x 10 &lt; ⁶ &gt; elongation sequence tags are obtained from the leads that are uniquely mapped to the reference genome.

In step 135, all the tags obtained by sequencing the nucleic acid in the test sample are counted to determine the test sequence tag density. In one embodiment, the number of test sequence tags mapped to the sequence of interest is normalized to the known length of the mapped sequence of interest to provide a test sequence tag density ratio. As described for the qualifying sample, normalization to a known length of the sequence of interest is not necessary, and may be included as a step to simplify this in a human interpretation by reducing the number of numbers in the number. Since all mapped test sequence tags are counted in the test sample, the sequence tag sequence for a sequence of interest, such as a clinically-related sequence, of the test sample is determined, which is an additional sequence corresponding to one or more of the normalization sequences identified in the qualifying sample &Lt; / RTI &gt;

At step 150, the test sequence capacity is determined for the sequence of interest of the test sample, based on the identity of one or more normalization sequences in the qualifying sample. As described elsewhere herein, one or more of the normalization sequences may be a single sequence or a group of sequences. The sequence volume for the sequence of interest of the test sample is the ratio of the sequence tag density determined for the sequence of interest to the sequence of interest in the test sample and the sequence tag density of the at least one normalization sequence determined in the test sample wherein the normalization sequence of the test sample is for a particular sequence of interest This corresponds to the standardized sequence identified in the eligible sample. For example, if it is determined that the identified normalization sequence for chromosome 21 in a qualifying sample is a chromosome, such as chromosome 14, then the test sequence volume for chromosome 21 (the sequence of interest) is the sequence tag for chromosome 21 Density and the sequence tag density for chromosome 14. [ Similarly, the chromosomal capacity for chromosome 13, 18, X, Y, and other chromosomes associated with chromosomal aberration is determined. The normalization sequence for a chromosome of interest may be a chromosome or a group of chromosomes, or a group of chromosome segments or chromosome segments. As noted above, the sequence of interest may be part of a chromosome, such as a chromosome segment. Thus, the capacity for a chromosome segment can be determined as the ratio of the sequence tag density determined for the segment in the test sample to the sequence tag density for the normalized chromosome segment in the test sample, where the normalized segment of the test sample is eligible for a particular segment of interest Correspond to the normalized segments identified in the sample (a single segment or group of segments). The chromosome segment may range in size from kilobase (kb) to megabases (Mb).

At step 155, the threshold value is derived from the established sequence deviations determined for a plurality of eligibility samples and the sequence capacity determined for a sample known to be biologically compatible with the sequence of interest. The precise classification is dependent on the difference between the probabilistic distributions for different classes, that is, for the imperative type. Preferably, the threshold value is selected from an empirical distribution for each type of imperative, e.g., trisomy 21. Possible thresholds include the use of a method of sequencing cfDNA extracted from a maternal sample comprising a mixture of fetal and maternal nucleic acids to determine chromosomal integrity, as described in the Examples, trisomy 13, trisomy 18 , Trisomy 21, and X-chain chromosome aberration. The threshold value determined to distinguish the altered sample against the chromosomal integrity may be the same or different from the threshold value determined to distinguish the altered sample for different isomerizations. As set forth in the examples, the threshold for each interesting chromosome is determined from the variability in the capacity of the chromosome of interest over the course of the sample and sequencing. The less variable the chromosomal capacity for any given chromosome of interest, the narrower the diversity in dose for the chromosome of interest over all of the unaltered samples, which is used to establish thresholds for determining different isomericity.

In step 160, the variation in the number of copies of the sequence of interest is determined in the test sample by comparing the test sequence capacity for the sequence of interest to one or more thresholds established from the qualifying sequence capacity.

At step 165, the calculated capacity for the test sequence of interest is compared to that set as the selected threshold value according to the threshold of user-defined confidence to classify the sample as " normal ", " altered " The "no call" sample is a sample that can not be reliably diagnosed.

Another embodiment of the present invention provides a method for providing a prenatal diagnosis of fetal chromosomal integrity in a biological sample comprising fetal and maternal nucleic acid molecules. Wherein the diagnosis is based on sequencing at least a portion of a mixture of fetal and maternal nucleic acid molecules from a biological test sample, such as a maternal plasma sample, to obtain sequence information, and / or one or more standard chromosome capacities for one or more chromosomes of interest from the sequencing data and / The standardized segment capacity for the segment of interest is calculated and the difference between the segment capacity for the chromosome capacity and / or segment of interest for each chromosome of interest and the statistically significant difference between the thresholds established in the plurality of qualifying (normal) , And to provide prenatal diagnosis based on statistical differences. A diagnosis of normal or altered is made, as described in step 165 of the method. A " no call " is provided for an event in which a diagnosis of normal or altered can not be reliably performed.

Sample

The sample used to determine the isomerism and partial isomerism of CNV, e.g., chromosomes, includes nucleic acids that are present in cells or are " cell free ". In some embodiments of the invention, it is advantageous to obtain cell-free nucleic acids, such as cell-free DNA (cfDNA). Cell-free nucleic acids containing cell-free DNA can be obtained by a variety of methods known in the art, including, but not limited to, plasma and serum-containing biological samples (Chen et al., Nature Med. 2: 1033- 1035 [1996]; Lo et al., Lancet 350: 485-487 [1997]). To separate acellular DNA from cells, fractionation, centrifugation (e.g., density gradient centrifugation), DNA-specific precipitation, or high throughput cell sorting and / or isolation methods may be used.

A sample comprising a mixture of nucleic acids using the methods described herein is a biological sample, such as a tissue sample, a biological fluid sample, or a cell sample. In some embodiments, the mixture of nucleic acids is purified or separated from the biological sample by any of the known methods. The sample may be composed of purified or isolated polynucleotides or may comprise a biological sample such as a tissue sample, a biological fluid sample, or a cell sample. Biological fluids include, but are not limited to, blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, lymph, saliva, cerebrospinal fluid, rabages, bone marrow suspension, vaginal flow, transcervical lavage, brain fluid, ascites, breast milk, respiratory tract, intestinal, and urogenital gland secretions, amniotic fluid, and leukophoresis samples. In some embodiments, the sample is a sample that can be readily obtained by non-invasive procedures such as blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear effusions, saliva or faeces. Preferably, the biological sample is a peripheral blood sample, or plasma and serum fraction. In other embodiments, the biological sample is a swab specimen or a smear specimen, a biopsy specimen, or a cell culture fluid. In another embodiment, the sample is a mixture of two or more biological samples, e.g., the biological sample may comprise two or more of a biological fluid sample, a tissue sample, and a cell culture sample. As used herein, the terms " blood ", " plasma ", and " serum " explicitly encompass fractions or processed parts thereof. Similarly, when a sample is taken from a biopsy, swab specimen, smear, etc., the " sample " includes a portion derived from an apparently processed fraction or biopsy, swab specimen, smear specimen,

In some embodiments, the sample includes, but is not limited to, different individuals, different developmental stages of the same or different individuals, individuals suffering from different diseases (e.g., individuals who have cancer or are believed to have a genetic condition) A sample, a sample obtained at different stages of the disease in a subject, a sample obtained from a subject receiving different treatments for the disease, a subject exposed to different environmental factors, or a subject suffering from pathology, or an infectious disease agent (e.g., RTI ID = 0.0 &gt; HIV). &Lt; / RTI &gt;

In one embodiment, the sample is a maternal sample obtained from a pregnant female, such as a pregnant woman. In this example, the sample may be analyzed using the methods described herein to provide prenatal diagnosis over a potential chromosome in the fetus. The maternal sample can be a tissue sample, a biological fluid sample, or a cell sample. Biological fluids include, but are not limited to, blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear effusion, lymph, saliva, cerebrospinal fluid, rabbis, bone marrow suspension, vaginal effusions, , Secretions from breast milk, respiratory tract, intestines, and genitourinary tract, and leukophoresis samples. In another embodiment, the maternal sample is a mixture of two or more biological samples, e.g., the biological sample may comprise two or more of a biological fluid sample, a tissue sample, and a cell culture sample. In some embodiments, the sample is a sample that can be readily obtained by non-invasive procedures such as blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear effusions, saliva or faeces. In some embodiments, the biological sample is a peripheral blood sample, or plasma and serum fraction. In another embodiment, the biological sample is a swab specimen or a smear specimen, a biopsy specimen, or a cell culture fluid. As discussed above, the terms " blood ", " plasma ", and " serum " explicitly encompass fractions or processed portions thereof. Similarly, when a sample is taken from a biopsy, swab specimen, smear, etc., the " sample " includes a portion derived from an apparently processed fraction or biopsy, swab specimen, smear specimen,

The sample may be obtained from an in vitro cultured tissue, cell, or other polynucleotide-containing source. The cultured sample may be cultured (e.g., tissue or cells) maintained in different media and conditions (e.g., pH, pressure, or temperature), culture media (e.g., tissue or cells) maintained for different periods, (E.g., tissue or cells) treated with a factor or reagent (e.g., a drug candidate, or a modulator), or a source comprising a culture of a different type of tissue or cell.

Methods for separating nucleic acids from biological sources are well known and will vary depending on the nature of the source. One skilled in the art can readily separate nucleic acids from a source as required by the methods described herein. In some instances, it may be advantageous to fragment the nucleic acid molecule of the nucleic acid sample. Fragmentation can be random or specific, for example, as achieved using restriction endonuclease digestion. Random fragmentation methods are well known in the art and include, for example, limited DNAse digestion, alkaline treatment, and physical shearing. In one embodiment, the sample nucleic acid is obtained as unfragmented cfDNA. In other embodiments, the sample nucleic acid is obtained as a genomic DNA fragmented into fragments of about 500 base pairs or more and the NGS method can be readily utilized.

Before birth  For diagnosis CNV Determination of

Cell-free fetal DNA and RNA circulation can be used in maternal blood for early noninvasive prenatal diagnosis (NIPD) of an increased number of genetic diseases for pregnancy management and to assist in reproductive physician-decision making. The presence of circulating cell-free DNA in the bloodstream has been known for over 50 years. More recently, the presence of circulating small amounts of fetal DNA has been found in maternal bloodstream during pregnancy (Lo et al., Lancet 350: 485-487 [1997]). Considering that the cell-free fetal DNA (cfDNA) originated from a perinuclear placental cell, the DNA was typically found to consist of short fragments of less than 200 bp in length (Chan et al., Clin Chem 50: 88-92 [ 2004), which is early known to be distinguishable at 4 weeks of gestation (Illanes et al., Early Human Dev 83: 563-566 [2007]) and is removed from maternal circulation during delivery time (Lo et al. Am J Hum Genet 64: 218-224 [1999]). In addition to cfDNA, fragments of cell-free fetal RNA (cfRNA) also originate from genes transcribed in the fetus or placenta and can be distinguished from maternal bloodstream. Extraction and subsequent analysis of these fetal genetic elements from maternal blood samples presents new opportunities for NIPD.

The method is a polymorphism-independent method for use in NIPD, and fetal cfDNA does not need to be distinguished from maternal cfDNA in order to be able to determine fetal insemination. In some embodiments, the isomerism is a complete chromosome trichome or a single chromosome, or a partial trichome or a single chromosome. Partial isomerism is caused by loss or gain of a portion of the chromosome and includes chromosomal imbalance resulting from unbalanced dislocation, unbalance reversal, deletion and insertion. In the long run, the most common known complication of survival is the Down Syndrome (DS) caused by the presence of some or all of chromosome 21, trisomy 21. Rarely, DS can be caused by inherited defects or sporadic defects due to the extra copies of all or a portion of chromosome 21 attached to another chromosome (usually chromosome 14) to form a single abnormal chromosome. DS is associated with excess mortality caused by long-term health problems such as intellectual disabilities, severe learning disabilities and heart disease. Other biologic features with known clinical significance include Edwards syndrome (trisomy 18) and Patau syndrome (trisomy 13), which are often fatal within the first few months of survival. Abnormalities associated with the number of sex chromosomes are also known, and X-linked chromosomes such as Turner's syndrome (XO) and Xyla syndrome (XXX) and Kleinefelter syndrome (XXY) and XYY syndrome , All of which are associated with a variety of phenotypes including infertility and reduced intellectual function. These and other chromosomal abnormalities can be diagnosed prior to birth using the methods of the present invention.

According to some embodiments of the present invention, the trisomy of the present invention is not limited to trisomy 21 (T21; Down syndrome), trisomy 18 (T18; Edwards syndrome), trisomy 16 (T16), trisomy 22 (T22), trisomy 15 (T15), trisomy 13 (T13), trisomy 13 ; Warkany Syndrome) and XXY (Kleinfelder &apos; s Syndrome), XYY, or XXX trichomes. It will be appreciated that a variety of other complete and partial trisomes can be determined in fetal cfDNA according to the teachings of the present invention. Examples of partial trisomy include, but are not limited to, partial trisomy 1q32-44, 9p trichomatosis with trisomy 3, 4th trisomy mosaic phenomenon, 17p trichomatosis, partial trisomy 4q26-qter , 9 trisomy, partial 2p trisomy, partial 1q trisomy, and / or partial 6p trisomy / 6q trisomy.

The method of the present invention also determines the X chromosome of a chromosome, and a partial heterozygote, such as a single chromosome 13, a single chromosome 15, a single chromosome 16, a single chromosome 21, and a single chromosome 22, which are known to be involved in pregnancy abortion Can be used. Partial single chromosomes of chromosomes typically associated with complete isomerism can also be determined by the methods of the present invention. Chromosome 18p is a rare chromosomal disorder in which all or part of the short arm (p) of chromosome 18 is deleted (single chromosome). The disease is typically characterized by a small height, varying degrees of mental retardation, language delay, malformations of the head and face (craniofacial) region, and / or additional bodily anomalies. Associated craniofacial defects can vary widely in extent and severity from disease to disease. Diseases caused by changes in the structure or number of copies of chromosome 15 include the same part of chromosome 15, the Angelman syndrome and the Frederick's syndrome with loss of gene activity in the 15q11-q13 region. It will be understood that multiple dislocation and microdeletion may be asymptomatic in the recipient parent, but may cause major genetic disorders in the offspring. For example, a healthy parent with a 15q11-q13 microdeletion can give birth to a child with severe neurodegenerative disease, Angelman syndrome. Thus, the present invention can be used to identify such partial deletions and other deletions in the fetus. The partial heterozygosity 13q is a rare chromosomal disease caused by the absence of the fragment of the long arm (q) of chromosome 13 (heteroskeleton). A baby born with a partial heteroskeleton 13q may exhibit low birth weight, malformations of the head and face (craniofacial region), skeletal abnormalities (especially hands and feet), and other physical anomalies. Mental retardation is a hallmark of these diseases. Of the individuals born with these diseases, the mortality rate during childhood is high. Nearly all the pathologies of the partial heteroskeleton 13q occur randomly (sporadically) without apparent reason. The 22q11.2 deletion syndrome, also known as Dijozzi syndrome, is a syndrome caused by deletion of a small fragment of chromosome 22. The deletion (22 q11.2) occurs near the middle of the chromosome on one of the long arm of the chromosome pair. The features of these syndromes vary widely among members of the same family and affect much of the body. Characteristic signs and symptoms include congenital heart disease, defects in the palate (velo-pharyngeal insufficiency) most commonly associated with obstruction of the occlusion, learning disabilities, mild differences in facial characteristics, and recurrent infections And congenital defects such as &lt; RTI ID = 0.0 &gt; The microdeletion of the chromosome region 22q11.2 is associated with a 20 to 30-fold increased risk of schizophrenia. In one embodiment, the method of the present invention may be used to determine a partial chromosome, including, but not limited to, a chromosome 18p, a partial chromosome 15q11-q13 of chromosome 15, and a partial chromosome 13q, The partial heterozygosity of chromosome 22 can also be determined using the above method.

Using the method of the present invention, any one of the parents can determine any degree of completeness if it is a known unknown holder. This is a small over-marker chromosome (SMC); t (11; 14) (p15; p13) dislocation; Unbalanced potential t (8; 11) (p23.2; p15.5); 11q23 microdeletion; Smith-Majenis Syndrome 17p11.2 Fruiting; 22q13.3 deletion; Xp22.3 microdeletion; 10p14 deletion; 20p microdeletion, Dijoroz syndrome [del (22) (q11.2q11.23)], Williams syndrome (7q11.23 and 7q36 deletion); 1p36 deletion; 2p microdeletion; Neurofibromatosis type 1 (17q11.2 microdeletion), Yq deletion; Wolf-Hirschhorn syndrome (WHS, 4p16.3 microdeletion); 1p36.2 Microdeletion; 11q14 deletion; 19q13.2 microdeletion; Rubinstein-Tebee (16 p13.3 microdeletion); 7p21 microdeletion; Miller-Dicker syndrome (17p13.3), 17p11.2 deletion; And a mosaic for 2q37 microdeletion.

Determination of complete fetal chromosomal integrity

In one embodiment, the invention provides a method for determining the presence or absence of any one or more different complete fetal chromosomal integrity in a maternal test sample comprising fetal and maternal nucleic acid molecules. Preferably, the method determines the presence or absence of any four or more different complete chromosomal integrations. The method comprising the steps of: (a) obtaining sequence information for a fetal and parent nucleic acid in a maternal test sample; (b) identifying a plurality of sequence tags for each of at least one of interest chromosomes selected from chromosome 1-22, X and Y using the sequence information, and identifying a plurality of sequences for a standardized chromosome sequence for each of the one or more chromosomes of interest &Lt; / RTI &gt; tag. The normalized chromosome sequence may be a single chromosome, or it may be a group of chromosomes selected from chromosomes 1-22, X, and Y. The method includes determining the number of sequence tags identified for each of one or more of the interest chromosomes for each of the one or more chromosomes of interest in step (c) and the number of identified sequence tags for each of the normalized segment sequences Additional numbers are used; (d) comparing the single chromosome dose for each of the one or more interest chromosomes with a threshold for each of the one or more chromosomes of interest to determine the presence or absence of any one or more different complete fetal chromosomal integrations in the maternal test sample.

 In some embodiments, step (c) calculates 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 standard chromosome for each of the chromosomes of interest .

In another embodiment, step (c) comprises comparing the single chromosome capacity for each of the chromosomes of interest to the number of sequence tags identified for each of the chromosomes of interest and the ratio of the number of sequence tags identified for the chromosome of interest to each of the chromosomes of interest Lt; / RTI &gt; In another embodiment, step (c) relates the number of sequence tags obtained for the chromosome of interest to the length of the chromosome of interest, and determines the number of tags for the corresponding normalized chromosome sequence for the chromosome of interest to the length of the normalized chromosome sequence And associating the chromosome capacity for the chromosome of interest with the sequence tag density of the interest chromosome and the ratio of the sequence tag density to the normalization sequence. This calculation is repeated for each of the interest chromosomes. Steps (a) - (d) may be repeated for test samples from different parent bodies.

Examples of embodiments that determine in a maternal test sample comprising a mixture of four or more complete fetal chromosomal aberrations with DNA molecules without fetal and maternal cells include: (a) sequencing at least a portion of a cell-free DNA molecule, Obtaining sequence information for a cell-free DNA molecule; (b) identifying a plurality of sequence tags for each of at least 20 interest chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and identifying a plurality of sequences for a standardized chromosome for each of 20 or more chromosomes of interest Check tags; (c) calculating a single chromosome dose for each of the 20 or more chromosomes of interest using the number of sequence tags identified for each of the 20 or more interest chromosomes and the number of sequence tags identified for each normalized chromosome; (d) determining the presence or absence of any of the twenty or more different complete fetal chromosomal aberrations in the test sample by comparing each single chromosome dose for each of the twenty or more interest chromosomes with a threshold for each of the twenty or more interest chromosomes do.

In another embodiment, a method of determining the presence or absence of any one or more different complete fetal chromosomal integra- tions in a maternal test sample as described above utilizes a normalized segment sequence to determine the capacity of the chromosome of interest. In this example, the method comprises: (a) obtaining sequence information for the fetal and parent nucleic acid in the sample; (b) identifying a plurality of sequence tags for each of at least one of the interest chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and identifying a plurality of sequence tags for each of the one or more interest chromosomes And identifying the sequence tag. The normalized segment sequence may be a single segment of a chromosome or may be a group of segments from one or more different chromosomes. The method further comprises comparing the number of sequence tags identified for each of the one or more chromosomes of interest with the number of sequences identified for the normalized segment sequence to calculate a single chromosome capacity for each of the one or more interest chromosomes in step (c) Further use the number of tags; (d) comparing each of said single chromosomal capacities for each of said one or more interest chromosomes with a threshold for each of said one or more chromosomes of interest to determine the presence or absence of one or more different complete fetal chromosomalemias of said sample.

In some embodiments, step (c) comprises comparing the single chromosome capacity for each of the interest chromosomes with the number of sequence tags identified for each of the chromosomes of interest and the sequence tag identified for the normalization segment sequence for each of the chromosomes of interest As a ratio of the number of pixels.

In other embodiments, step (c) involves associating the number of sequence tags obtained for the chromosome of interest with the length of the interest chromosome, and determining the number of tags for the corresponding normalized segment sequence for the chromosome of interest to the length of the normalized segment sequence And associating the chromosome capacity for the chromosome of interest with the sequence tag density of the chromosome of interest and the ratio of the sequence tag sequence to the normalized segment sequence. The calculation is repeated for each of the interest chromosomes. Steps (a) - (d) may be repeated for test samples from different parent bodies.

The means for comparing the chromosome capacities of the different sample sets is provided by determining a normalized chromosome value (NCV) that relates the chromosomal capacity of the test sample to an average of the corresponding chromosomal dose in the set of qualified samples. The NCV is calculated as follows:

및

는 적격 샘플의 세트에서 j번째 염색체 용량에 대한 각기 추정 평균 및 표준 편차이고,

는 시험 샘플 i에 대해 관찰된 j번째 염색체 용량이다.In this formula,

And

Is the respective estimated mean and standard deviation for the j &lt; th &gt; chromosome dose in the set of eligible samples,

Is the jth chromosome capacity observed for test sample i.

In some embodiments, the presence or absence of one or more complete fetal chromosomal integrity is determined. In other embodiments, at least two, at least three, at least four, at least five. At least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 The presence or absence of complete fetal chromosomal anomalies is determined in the sample wherein at least 19, at least 20, at least 21, at least 22, at least 23, or 24 complete chromosomal aberrations are present in the sample, Corresponds to the complete chromosomal integrity of the chromosome; The 23rd and 24th chromosomal aberrations correspond to the complete fetal chromosomal integrity of chromosomes X and Y. [ Because the sex chromosomal aberration may include trisomy, contamination staining, and other staining, the number of different complete chromosomal aberrations that can be determined according to the method is at least 24, at least 25, at least 26 , At least 27, at least 28, at least 29, or at least 30 complete chromosomal integrations. Thus, the number of different complete fetal chromosomal aberrations determined is related to the number of chromosomes of interest selected for analysis.

In one embodiment, determining the presence or absence of any one or more of the different complete fetal chromosomalemias in the maternal test sample as described above comprises determining a normalized segment sequence for one chromosome of interest selected from chromosomes 1-22, X, and Y . In other embodiments, two or more chromosomes of interest are 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. In one embodiment, any one or more of the interest chromosomes comprises at least 20 chromosomes selected from chromosomes 1-22, X, and Y and selected from chromosomes 1-22, X, and Y, wherein at least 20 different complete embryos The presence or absence of chromosomal integrity is determined. In other embodiments, any one or more of the chromosomes of interest selected from chromosomes 1-22, X, and Y are all chromosomes 1-22, X, and Y, and all chromosomes 1-22, X, The presence or absence of the water-solubility is determined. Completely different fetal chromosomal aberrations that can be determined include complete chromosomal trichomes, complete chromosomal single chromosomes, and complete chromosomes. Examples of complete fetal chromosomal integrity include, but are not limited to, trisomy of any one or more of the common chromosomes, such as 2 trisomy, 8 trisomy, 9 trisomy, 21 trisomy, 13 trisomy, Trisomy 16, trisomy 18, trisomy 22; Trichromosomes of sex chromosomes, such as 47, XXY, 47 XXX, and 47 XYY; Sex chromosomes of sex chromosomes, such as 48, XXYY, 48, XXXY, 48XXXX, and 48, XYYY; Sex chromosomes, such as 49, XXXYY 49, XXXXY, 49, XXXXX, 49, XYYYY; And an X-linked chromosome. Other complete fetal chromosomal integrity that can be determined according to the method is described below.

Determination of partial fetal chromosomal integration

In yet another embodiment, the invention provides a method for determining the presence or absence of any one or more different partial fetal chromosomalemias in a maternal test sample comprising fetal and maternal nucleic acid molecules. The method comprising: obtaining sequence information for the fetal and parent nucleic acid in the sample; (b) identifying a plurality of sequence tags for each of any one or more segments of any one or more of the chromosomes of interest selected from chromosomes 1-22, X, and Y using the sequence information, And identifying a plurality of sequence tags for the normalized segment sequence for each of the one or more segments. The normalized segment sequence may be a single segment of a chromosome or may be a group of segments from one or more different chromosomes. The method includes determining the number of sequence tags identified for each of the one or more segments of any one or more of the interest chromosomes to calculate a single segment capacity for each of the one or more segments of any one or more of the interest chromosomes in step (c) Further utilizing the number of sequence tags identified for the normalized segment sequence; (d) comparing each of the single chromosome capacities for each of the one or more segments of any one or more of the interest chromosomes with a threshold for each of the one or more chromosome segments of any one or more of the interest chromosomes, To determine the presence or absence of fetal chromosomal integrity.

In some embodiments, step (c) comprises comparing the single chromosomal capacity for each of the one or more segments of any one or more of the interest chromosomes to the number of identified sequence tags for each of the one or more segments of any one or more of the chromosomes of interest As the ratio of the number of sequence tags identified for the normalized segment sequence for each of the one or more segments of one or more chromosomes of interest.

In another embodiment, step (c) relates the number of sequence tags obtained for the segment of interest to the length of the segment of interest and the number of tags for the normalized segment sequence corresponding to the segment of interest to the length of the normalized segment sequence Calculating the sequence tag ratio for the segment of interest by the association and calculating the segment capacity for the segment of interest as the ratio of the sequence tag density of the segment of interest to the sequence tag sequence for the normalized segment sequence. The calculation is repeated for each of the interest chromosomes. Steps (a) - (d) may be repeated for test samples from different parent bodies.

The means for comparing the segment capacity of the different sample sets is provided by determining a normalized segment value (NSV) that relates the segment capacity of the test sample to the average of the corresponding segment capacity in the set of qualified samples. The NSV is calculated as follows:

및

는 적격 샘플의 세트에서 j번째 세그먼트 용량에 대한 각기 추정 평균 및 표준 편차이고,

는 시험 샘플 i에 대해 관찰된 j번째 세그먼트 용량이다.In this formula,

And

Is the respective estimated mean and standard deviation for the jth segment capacity in the set of eligible samples,

Is the jth segment capacity observed for test sample i.

In some embodiments, the presence or absence of a partial fetal chromosomal integrity is determined. In other embodiments, the presence or absence of partial fetal chromosome 2, 3, 4, 5, 6, 7, 8, 9, 10, 15. 20. 25 or more partial fetal chromosomal integrity is determined in the sample. In one embodiment, a segment of interest selected from any one of chromosomes 1-22, X, and Y is selected from chromosomes 1-22, X, and Y. In another embodiment, two or more segments of interest selected from chromosomes 1-22, X, and Y are 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. In one embodiment, any one or more segments of interest are selected from chromosomes 1-22, X, and Y and comprise at least 1, 5, 10, 15, 20, , 25 or more segments, wherein the presence or absence of at least 1, 5, 10, 15, 20, 25 different partial fetal chromosomal aberrations is determined. Different partial fetal chromosomalities that can be determined include partial redundancy, partial increase, partial insertion and partial deletion. Examples of partial fetal chromosomal anomalies include partial heterochromosomes and partial trisomes of normal chromosomes. Usually, the partial chromosomes of chromosome 1 are partially chromosomes of chromosome 1, partial chromosomes of chromosome 4, partial single chromosome of chromosome 5, partial single chromosome of chromosome 7, partial single chromosome of chromosome 11, chromosome 15 A partial single chromosome of chromosome 18, a partial single chromosome of chromosome 18, and a partial single chromosome of chromosome 22. Other partial fetal chromosomal aberrations that may be determined according to the method are described below.

In any of the embodiments described above, the test sample is a maternal sample selected from blood, plasma, serum, urine, and saliva samples. In some embodiments, the matrix test sample is a plasma sample. The nucleic acid molecule of the maternal sample is a mixture of DNA molecules free of fetal and maternal cells. Sequencing of nucleic acids can be performed using the next generation sequencing (NGS) described elsewhere herein. In some embodiments, sequencing is massively parallel sequencing using sequencing through synthesis by a reversible dye terminator. In another embodiment, sequencing is sequencing through ligation. In yet another embodiment, the sequencing is single molecule sequencing. Optionally, the amplification step is performed prior to sequencing.

Clinical disease CNV Determination of

The methods described herein can be used to determine any abnormality in the representation of the gene sequence in the genome, in addition to the early determination of congenital defects.

Blood plasma and serum DNA from a cancer patient can be recovered and contain a measurable amount of tumor DNA that can be used as a surrogate for tumor DNA and the tumor can be characterized by an isomeric or an inadequate number of gene sequences or even a full chromosome Respectively. Thus, determination of the difference in the amount of a given sequence, i.e. the sequence of interest, in a sample from an individual can be used for the diagnosis of a medical disorder. In some embodiments, the method can be used to determine the presence or absence of chromosomal integrity in patients suspected or known to have cancer. The method also includes determining the presence or absence of a disease state; Determining the presence or absence of a nucleic acid of a pathogen such as a virus; Can be used to determine chromosomal abnormalities associated with graft versus host disease (GVHD) and to determine the contribution of an individual in forensic analysis.

Embodiments of the present invention encompass a nucleic acid sequence encoding a sequence of interest, such as a copy number of a clinically-related sequence, in a test sample that includes a mixture of nucleic acids derived from two different dielectrics and is known or considered to be different in the amount of one or more sequences of interest Provides a way to evaluate mutations. A mixture of nucleic acids is derived from two or more types of cells. In one embodiment, the mixture of nucleic acids is derived from normal cells and cancerous cells derived from a subject afflicted with a medical disorder such as cancer.

The occurrence of cancer is often established by changes in the number of entire chromosomes, i.e., by changes in the number of segments of the chromosome, that is, by partial miRNA, caused by a process known as complete chromosomal aberration and / or chromosomal instability (CIN) (Thoma et al., Swiss Med Weekly 2011: 141: w13170). Many solid tumors, such as breast cancer, are believed to progress from onset to metastasis through accumulation of multiple genetic abnormalities [Sato et al., Cancer Res., 50: 7184-7189 [1990]; Jongsma et al., J Clin Pathol: Mol Path 55: 305-309 [2002]). Such genetic abnormalities can confer proliferation benefits, genetic instability and accompanying abilities cumulatively to develop rapid tolerability and enhanced angiogenesis, proteolysis and metastasis. Genetic abnormalities can affect a recessive "tumor suppressor gene" or a predominantly oncogenic gene. Deletion and recombination leading to the loss of heterozygosity (LOH) are believed to play a major role in tumor progression by exposing mutated tumor suppressor alleles.

cfDNA may be used to treat a wide range of conditions including but not limited to lung cancer (Pathak et al. Clin Chem 52: 1833-1842 [2006]), prostate cancer (Schwartzenbach et al. Clin Cancer Res 15: 1032-8 [2009] al. available online at breast-cancer-research.com/content/11/5/R71 [2009]), which was diagnosed as having a malignant tumor. Identification of cancer-associated genetic instability that can be determined in circulating cfDNA in cancer patients is a potential diagnostic and prognostic tool. In one embodiment, the method of the present invention is used to determine the presence or absence of a sequence of interest in a sample comprising a mixture of nucleic acids derived from a subject known or suspected of having a cancer, such as carcinoma, sarcoma, lymphoma, leukemia, Evaluate CNV. In one embodiment, the sample is a plasma sample comprising a mixture of cfDNA derived from (treated) normal and cancer cells from peripheral blood. In another embodiment, the biological sample for which it is desired to determine whether CNV is present may be, but is not limited to, serum, sweat, tears, sputum, urine, sputum, ear effusion, lymph, saliva, cerebrospinal fluid, From other biologic fluids or tissue biopsies, swab specimens, or smears, including vaginal effusions, cervical intraocular fluid, brain fluid, ascites, breast milk, respiratory tract, secretions of intestinal and genitourinary tracts, and leukophoresis samples Lt; RTI ID = 0.0 &gt; cancerous &lt; / RTI &gt; In other embodiments, the biological sample is a feces sample.

A sequence of interest is a nucleic acid sequence that is known or believed to play a role in the development and / or progression of cancer. Examples of sequences of interest include nucleic acid sequences that are amplified or deleted in cancerous cells, i. E., Complete chromosomes and / or segments of chromosomes, as described below.

In one embodiment, the method can be used to determine the presence or absence of chromosome amplification. In some embodiments, chromosome amplification is the acquisition of one or more whole chromosomes. In other embodiments, chromosome amplification is the acquisition of one or more segments of a chromosome. In yet another embodiment, the chromosomal amplification is the acquisition of two or more segments of two or more chromosomes. Chromosomal amplification can involve the acquisition of one or more oncogenes.

The dominantly functioning genes associated with human solid tumors typically exert their effects by over-expressed or altered expression. Gene amplification is a common mechanism that results in upregulation of gene expression. Evidence from cytogenetic studies indicates that significant amplification occurs in over 50% of human breast cancers. Most notably, amplification of the proto-oncogene human epidermal growth factor receptor 2 (HER2) present on chromosome 17 (17 (17q21-q22)) has been shown to result in excessive and aberrant signal transduction in breast cancer and other malignant tumors (Park et al., Clinical Breast Cancer 8: 392-401 [2008]). Various oncogenes have been found to be amplified in other human malignant tumors. Examples of amplification of cell tumor genes in human tumors include c-myc, primary neuroblastoma (III and IV), neuroblastoma cell line, retinoblastoma cell line, and primary tumor in the promyelocytic leukemia cell line HL60 and small cell lung cancer cell line C-myb, epidermal carcinoma cells, and primary glial glioma in L-myc, acute myelogenous leukemia, and colorectal carcinoma cell lines in N-myc and small cell lung carcinomas and tumors, small cell lung carcinoma cell lines and tumors, , CK-ras-2 in primary carcinoma of the colon, bladder and rectum and amplification of N-ras in mammary carcinoma cell lines (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)].

In one embodiment, the method can be used to determine the presence or absence of a chromosomal deletion. In some embodiments, a chromosomal deletion is a loss of one or more whole chromosomes. In other embodiments, the chromosomal deletion is a loss of one or more segments of the chromosome. In yet another embodiment, the chromosomal deletion is a loss of two or more segments of two or more chromosomes. Chromosomal deletions can involve the loss of one or more tumor suppressor genes.

Chromosomal deletions involving tumor suppressor genes can play an important role in the development and progression of solid tumors. The retinoblastoma tumor suppressor gene (Rb-1) located on chromosome 13q14 is the most widely characterized tumor suppressor gene. Protein, the nucleus of the 105 kDa Rb-1 gene product, plays a clear role in cell cycle regulation (Howe et al., Proc Natl Acad Sci (USA) 87: 5883-5887 [1990]). The altered or lost expression of Rb protein is caused by inactivation of both gene alleles through point mutation or chromosome deletion. Rb-i gene alterations have been found not only in retinoblastoma but also in other malignant tumors such as osteosarcoma, small cell lung cancer (Rygaard et al., Cancer Res 50: 5312-5317 [1990]) and breast cancer. The restriction fragment length polymorphism (RFLP) study suggested that such tumor types often lost heterozygosity at 13q suggesting that one of the Rb-1 gene alleles was lost due to chromosome deletion in the gross (Bowcock et al. Am J Hum Genet, 46: 12 [1990]). Overlapping, deletion, and chromosome 1 involving unbalanced dislocation involving chromosome 6 and other partner chromosomes have been implicated in the chronic and progressive progression of myeloproliferative neoplasms in the area of chromosome 1, particularly 1q21-1q32 and 1p11-13, (Caramazza et al., Eur J Hematol 84: 191-200 [2010]). These results suggest that the tumor gene or tumor suppressor gene may be associated with both of the two tumors. Myeloproliferative neoplasms are also associated with deletion of chromosome 5. Complete loss of or loss of chromosome 5 is the most common karyotypic abnormality in myelodysplastic syndrome (MDS). Patients with isolated del (5q) / 5q-MDS have a more favorable prognosis than patients with additional karyotypes, which tend to develop myeloproliferative nephropathy (MPN) and acute myelogenous leukemia. The incidence of asymmetric chromosome 5 deletion has led to the view that 5q has one or more tumor-suppressing genes that play a fundamental role in regulating the growth of hematopoietic stem / progenitor cells (HSC / HPC). Confirmation of candidate tumor-suppressor genes including ribosomal subunit RPS14, transcription factor Egr1 / Krox20 and cytoskeletal remodeling protein, alpha-catenin by cytogenetic mapping of generally deleted regions (CDRs) centered on 5q31 and 5q32 (Eisenmann et al., Oncogene 28: 3429-3441 [2009]). Cytogenetics and allelic genotype studies of new tumor and tumor cell lines showed that allele loss from multiple distinct regions on chromosome 3p, including 3p25, 3p21-22, 3p21.3, 3p12-13 and 3p14, , Is the fastest and most frequent genetic abnormality following a broad spectrum of primary epithelial cancers of the head and neck, ovaries, cervix, colon, pancreas, esophagus, bladder, and other organs. Several tumor suppressor genes have been mapped for the chromosome 3p region and it is believed that the absence or promoter and methylation are preceded by loss of 3p or whole chromosome 3 in the development of carcinomas (Angeloni D., Briefings Functional Genomics 6: 19-39 [2007]).

Neonates and children with Down syndrome (DS) often exhibit congenital acute leukemia and have an increased risk of acute myelogenous leukemia and acute lymphoblastic leukemia. Chromosome 21, which has about 300 genes, can carry many structural abnormalities such as dislocation, deletion, and amplification in leukemia, lymphoma, and solid tumors. Furthermore, it has been confirmed that the gene located on chromosome 21 plays an important role in tumorigenesis. In addition to body numbers, structural chromosomes 21 and more are associated with leukemia and specific genes, including RUNX1, TMPRSS2, and TFF, located at 21q, play a role in tumorigenesis (Fonatsch C Gene Chromosomes Cancer 49: 497-508 [2010 ]).

In one embodiment, the method provides a means for assessing the degree of association between gene amplification and tumor evolution. The correlation between amplification and / or deletion and grade or grade may be important in prognosis, since this information may be used to predict the progression of a disease with a more advanced tumor with the worst prognosis Because it can contribute to the definition of an underlying tumor grade. In addition, information about early amplification and / or deletion events may be useful for correlating such events as predictors of subsequent disease progression. Gene amplification and deletion identified by this method can be used to assess tumor grade, history, Brd / Urd index, hormone status, nodal involvement, tumor size, survival duration and other tumor characteristics available from epidemiological and biostatistical studies May be associated with other known parameters. For example, the tumor DNA to be tested by the method may include atypical hyperplasia, ductal carcinoma in situ, I-III cancer, and metastatic lymph nodes to confirm amplification and deletion and inter-stage association I could. Established relevance may enable effective therapeutic intervention. For example, a region that is consistently amplified may contain an overexpressed gene whose product may be subject to therapeutic attack (e. G., Growth factor receptor tyrosine kinase, p185 ^HER2 ).

Using this method, amplification and / or deletion events associated with tolerance can be identified by determining the number of copies of the nucleic acid sequence from a primary cancer relative to cells transfected to other sites. If gene amplification and / or deletion is an indication of karyotypic instability that allows rapid development of tolerance, more amplification and / or deletion will be expected in primary tumors from chemically resistant patients than in chemo-sensitive patient tumors. For example, if amplification of a particular gene is responsible for the development of tolerance, the region surrounding this gene will be expected to be consistently amplified in tumor cells from pleural effusion in chemically resistant patients, rather than in primary tumors. The discovery of gene amplification and / or association between deleterious and tolerable outbreaks can identify patients who will benefit from adjuvant therapy or who will not benefit.

In a manner similar to that described to determine the presence or absence of complete and / or partial fetal chromosomal integrity in a maternal sample, any patient sample comprising a nucleic acid, such as DNA or cfDNA, &Lt; / RTI &gt; the presence or absence of complete and / or partial chromosomal integrity in a patient sample). The patient sample may be any of the biological sample types described elsewhere herein. Preferably, the sample is obtained by a non-invasive procedure. For example, the sample may be a blood sample, or its serum and plasma fraction. Alternatively, the sample may be a urine sample or a stool sample. In yet another embodiment, the sample is a tissue biopsy sample. In all cases, the sample is purified and comprises a nucleic acid, such as cfDNA or genomic DNA, sequenced using any of the NGS sequencing methods described above.

Both complete and partial chromosomal aberrations associated with cancer formation and progression can be determined according to the method.

Determination of complete chromosomal integrity in patient samples

In one embodiment, the invention provides a method for determining the presence or absence of any one or more different complete chromosomal integrity in a patient test sample comprising a nucleic acid molecule. In some embodiments, the method determines the presence or absence of any one or more different complete chromosomal integra- tions. The method comprising the steps of: (a) obtaining sequence information for a patient nucleic acid in a patient test sample; (b) identifying a plurality of sequence tags for each of at least one of interest chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and identifying a plurality of sequences for a standardized chromosome sequence &Lt; / RTI &gt; tag. The normalized chromosome sequence may be a single chromosome, or it may be a group of chromosomes selected from chromosomes 1-22, X, and Y. The method comprising the step of determining the number of sequence tags identified for each of one or more of the interest chromosomes and the number of sequence tags identified for each of the normalized chromosome sequences to calculate a single chromosome capacity for each of one or more of the interest chromosomes in step (c) Lt; / RTI &gt; (d) comparing the single chromosome dose for each of the one or more interest chromosomes with a threshold for each of the one or more chromosomes of interest, thereby determining the presence or absence of any one or more different complete patient chromosomalemias in the patient test sample.

 In some embodiments, step (c) comprises determining the single chromosome capacity 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 normalized chromosome sequence for each of the chromosomes of interest Lt; / RTI &gt;

In another embodiment, step (c) comprises comparing the single chromosome capacity for each of the chromosomes of interest to the number of sequence tags identified for each of the chromosomes of interest and the ratio of the number of sequence tags identified for the chromosome of interest to each of the chromosomes of interest Lt; / RTI &gt; In another embodiment, step (c) relates the number of sequence tags obtained for the chromosome of interest to the length of the chromosome of interest, and determines the number of tags for the corresponding normalized chromosome sequence for the chromosome of interest to the length of the normalized chromosome sequence And associating the chromosome capacity for the chromosome of interest with the sequence tag density of the interest chromosome and the ratio of the sequence tag density to the normalization sequence. This calculation is repeated for each of the interest chromosomes. Steps (a) - (d) may be repeated for test samples from different patients.

Examples of embodiments in which one or more complete chromosomal aberrations are determined in a cancer patient test sample comprising a cell-free DNA molecule include: (a) sequencing at least a portion of a cell-free DNA molecule to obtain sequence information about the patient- &Lt; / RTI &gt; (b) identifying a plurality of sequence tags for each of at least 20 interest chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and identifying a plurality of sequence tags for each of the 20 or more chromosomes Identify sequence tags; (c) calculating a single chromosome dose for each of the 20 or more chromosomes of interest using the number of sequence tags identified for each of the 20 or more interest chromosomes and the number of sequence tags identified for each normalized chromosome; (d) determining the presence or absence of any more than 20 different complete chromosomalemias in a patient test sample by comparing each single chromosome dose for each of the 20 or more chromosomes of interest with a threshold for each of the 20 or more chromosomes of interest do.

In another embodiment, a method of determining the presence or absence of any one or more different complete chromosomalemias in a patient test sample as described above utilizes a standardized segment sequence to determine the capacity of the chromosome of interest. In this example, the method comprises: (a) obtaining sequence information for a nucleic acid in a sample; (b) identifying a plurality of sequence tags for each of at least one of the interest chromosomes selected from chromosomes 1-22, X and Y using the sequence information, and generating a plurality of sequences for the normalization segment sequence for each of the one or more chromosomes &Lt; / RTI &gt; tag. The normalized segment sequence may be a single segment of a chromosome or may be a group of segments from one or more different chromosomes. The method further comprises comparing the number of sequence tags identified for each of the one or more chromosomes of interest with the number of sequences identified for the normalized segment sequence to calculate a single chromosome capacity for each of the one or more interest chromosomes in step (c) Further use the number of tags; (d) comparing each of the single chromosome capacities for each of the one or more interest chromosomes with a threshold for each of the one or more interest chromosomes to determine the presence or absence of one or more different complete chromosomalemias in the patient sample.

In some embodiments, step (c) comprises comparing the single chromosome capacity for each of the interest chromosomes with the number of sequence tags identified for each of the chromosomes of interest and the sequence tag identified for the normalization segment sequence for each of the chromosomes of interest As a ratio of the number of pixels.

In another embodiment, step (c) involves associating the number of sequence tags obtained for the chromosome of interest with the length of the interest chromosome and associating the number of tags for the corresponding normalized segment sequence with the length of the normalized segment sequence To calculate the sequence tag ratios for the chromosomes of interest and to calculate the chromosomal capacity for the chromosomes of interest as the ratio of the sequence tag density of the interest chromosome to the sequence tag sequence for the normalized segment sequence. The calculation is repeated for each of the interest chromosomes. Steps (a) - (d) may be repeated for test samples from different patients.

The means for comparing the chromosome capacities of the different sample sets is provided by determining a normalized chromosome value (NCV) that relates the chromosomal capacity of the test sample to an average of the corresponding chromosomal dose in the set of qualified samples. The NCV is calculated as follows:

및

는 적격 샘플의 세트에서 j번째 염색체 용량에 대한 각기 추정 평균 및 표준 편차이고,

는 시험 샘플 i에 대해 관찰된 j번째 염색체 용량이다.In this formula,

And

Is the respective estimated mean and standard deviation for the j &lt; th &gt; chromosome dose in the set of eligible samples,

Is the jth chromosome capacity observed for test sample i.

In some embodiments, the presence or absence of one complete chromosomal complement is determined. In other embodiments, 2, 3, 4, 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, or 24 complete chromosomal integrations are determined in the sample, with 22 complete chromosomal aberrations corresponding to complete chromosomal integrity of any one or more of the common chromosomes; The 23rd and 24th chromosomal integrity corresponds to the complete chromosomal integrity of chromosomes X and Y. [ Since the number of complete chromosomal aberrations can be varied in various diseases and at different stages of the same disease, it is possible that the number of chromosomal aberrations that can be determined according to the present method At least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, At least 80, at least 90, at least 100 or more chromosomal integers. The systemic karyotype of the tumor has shown that the number of chromosomes in cancer cells is highly variable from low dichotomies (significantly less than 46 chromosomes) to quadruples and gonadal polyps (less than 200 chromosomes) (Storchova and Kuffer J Cell Sci 121: 3859-3866 [2008]). In some embodiments, the method comprises determining the presence or absence of chromosomal insufficiency of up to 200 or fewer in a sample from a patient known or known to be afflicted with cancer, such as colorectal cancer. Chromosomal aberrations include the acquisition of complete chromosomes, including loss of one or more complete chromosomes (low diploid), trichomes, tetraschromosomes, autosomes, and other chromosomes. Acquisition and / or loss of segments of the chromosome may also be determined as described elsewhere herein. The method can be used to determine the presence or absence of different isomerism in a sample from a patient known or suspected to have had any cancer as described elsewhere herein.

In some embodiments, any one of chromosomes 1-22, X and Y may be a chromosome of interest in determining the presence or absence of any one or more different complete chromosomalemias in a patient test sample as described above. In other embodiments, two or more chromosomes of interest are 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. In one embodiment, any one or more of the interest chromosomes comprises at least 20 chromosomes selected from chromosomes 1-22, X, and Y and selected from chromosomes 1-22, X, and Y, wherein at least 20 different complete chromosomes The presence or absence of the water-solubility is determined. In other embodiments, any one or more of the chromosomes of interest selected from chromosomes 1-22, X, and Y are all chromosomes 1-22, X, and Y, and all chromosomes 1-22, X, The presence or absence of the water-solubility is determined. Completely different chromosomal aberrations that can be determined include chromosomes 1-22, complete chromosomal chromosomes of any one of X and Y; Chromosomes 1-22, complete chromosome trichromosomes of any one of X and Y; Chromosomes 1-22, complete chromosomal chromosomes of any one of X and Y; Chromosomes 1-22, complete chromosomal autosomes of either X or Y; And other complete chromosome chromosomes of any one or more of chromosomes 1-22, X and Y.

Determination of partial chromosomal integrity in patient samples

In yet another embodiment, the invention provides a method for determining the presence or absence of any one or more different partial chromosomal integrity in a patient test sample comprising a nucleic acid molecule. The method comprising: (a) obtaining sequence information for a patient nucleic acid in a sample; (b) identifying a plurality of sequence tags for each of any one or more segments of any one or more of the chromosomes of interest selected from chromosomes 1-22, X, and Y using the sequence information and identifying any one Identifying a plurality of sequence tags for the normalized segment sequence for each of the above segments. The normalized segment sequence may be a single segment of a chromosome or may be a group of segments from one or more different chromosomes. The method includes determining the number of sequence tags identified for each of the one or more segments of any one or more of the interest chromosomes to calculate a single segment capacity for each of the one or more segments of any one or more of the interest chromosomes in step (c) Further utilizing the number of sequence tags identified for the normalized segment sequence; (d) comparing each of the single chromosome capacities for each of the one or more segments of any one or more of the interest chromosomes with a threshold for each of the one or more chromosome segments of any one or more of the interest chromosomes, To determine the presence or absence of chromosomal integration.

In some embodiments, step (c) comprises comparing the single chromosomal capacity for each of the one or more segments of any one or more of the interest chromosomes to the number of identified sequence tags for each of the one or more segments of any one or more of the chromosomes of interest As the ratio of the number of sequence tags identified for the normalized segment sequence for each of the one or more segments of one or more chromosomes of interest.

In another embodiment, step (c) relates the number of sequence tags obtained for the segment of interest to the length of the segment of interest and the number of tags for the normalized segment sequence corresponding to the segment of interest to the length of the normalized segment sequence Calculating the sequence tag ratio for the segment of interest by the association and calculating the segment capacity for the segment of interest as the ratio of the sequence tag density of the segment of interest to the sequence tag sequence for the normalized segment sequence. The calculation is repeated for each of the interest chromosomes. Steps (a) - (d) may be repeated for test samples from different patients.

The means for comparing the segment capacity of the different sample sets is provided by determining a normalized segment value (NSV) that relates the segment capacity of the test sample to the average of the corresponding segment capacity in the set of qualified samples. The NSV is calculated as follows:

및

는 적격 샘플의 세트에서 j번째 세그먼트 용량에 대한 각기 추정 평균 및 표준 편차이고,

는 시험 샘플 i에 대해 관찰된 j번째 세그먼트 용량이다.In this formula,

And

Is the respective estimated mean and standard deviation for the jth segment capacity in the set of eligible samples,

Is the jth segment capacity observed for test sample i.

In some embodiments, the presence or absence of a partial chromosomal complement is determined. In other embodiments, the presence or absence of partial chromosome 2, 3, 4, 5, 6, 7, 8, 9, 10, 15. 20, In one embodiment, a segment of interest selected from any one of chromosomes 1-22, X, and Y is selected from chromosomes 1-22, X, and Y. In another embodiment, two or more segments of interest selected from chromosomes 1-22, X, and Y are 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. In one embodiment, any one or more segments of interest are selected from chromosomes 1-22, X, and Y and comprise at least 1, 5, 10, 15, 20, At least 1, 5, 10, 15, 20, 25, 50, 75, 100, or more segments, The presence or absence of a different partial chromosomal complement thereof is determined. Different partial chromosomal aberrations that may be determined include chromosomal integration including partial overlap, partial increase, partial insertion, and partial deletion.

A sample that may be used to determine the presence or absence of chromosomal integration (partial or complete) in a patient may be any biological sample described elsewhere herein. The type of sample or sample that can be used in determining the biocompatibility in a patient will depend on the type of disease known or suspected to be afflicted by the patient. For example, faecal samples can be selected as the DNA source to determine the presence or absence of the insulin resistance associated with colorectal cancer. The method may also be used in the tissue samples described herein. Preferably, the sample is a biological sample obtained by non-invasive means such as a plasma sample. As described elsewhere herein, sequencing of nucleic acids in a patient sample can be performed using the next generation sequencing (NGS) described elsewhere herein. In some embodiments, sequencing is massively parallel sequencing using sequencing through synthesis by a reversible dye terminator. In other embodiments, sequencing is sequencing through ligation. In yet another embodiment, the sequencing is single molecule sequencing. Optionally, the amplification step is performed prior to sequencing.

In some embodiments, the presence or absence of the biosynthetic agent is selected from the group consisting of those described herein, such as lung cancer, breast cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, colon cancer, pancreatic cancer, esophageal cancer, bladder cancer and other organs, It is determined in patients who are thought to have cancer. Blood cancers include cancer of the lymphatic system, including bone marrow, blood, and lymph nodes, lymphatic vessels, tonsils, thymus, spleen, and digestive tract lymphoid tissue. Leukemia and myeloma originating from the bone marrow, and lymphoma originating from the lymphoid lineage are the most common types of blood cancer.

Determination of the presence or absence of one or more chromosomal aberrations in a patient sample can be performed as part of a conventional screen in patients and unknown patients known to be subject to a subject cancer, To determine the presence or absence of cancer, to provide a prognosis for the disease, to assess the need for adjuvant therapy, and to determine the progression or regression of the disease.

CNV Apparatus and system for determining

Analysis of the sequencing data and diagnostics derived therefrom are typically performed using various computer algorithms and programs. In one embodiment, the invention provides a computer program product for generating an output indicative of the presence or absence of fetal insemination in a test sample. A computer product is a receiving procedure for receiving sequencing data from at least a portion of nucleic acid molecules from a maternal biological sample, the receiving procedure comprising the sequencing data including a computed chromosome; Computer support logic for analyzing fetal anomalies from the received data; And an output procedure for generating an output indicative of the presence, absence or type of fetal anomaly, wherein the computer executable logic enabling the processor to diagnose fetal integrity is recorded thereon .

The method of the present invention may be carried out using a computer readable medium having computer readable instructions stored thereon for performing a method of identifying the completeness or partial completeness of any CNV, e.g., a chromosome. Thus, in one embodiment, the invention provides a computer-readable medium having computer-readable instructions stored thereon for performing a method of identifying complete and partial chromosomal integrity, such as fetal anemia.

The method of the present invention may also be performed using a computer processing system configured or configured to perform a method of identifying the completeness or partial completeness of any CNV, e.g., a chromosome. Thus, in one embodiment, the invention provides a computer processing system configured or configured to perform the methods described herein. In one embodiment, the apparatus includes a sequencing device configured or configured to sequence at least a portion of a nucleic acid molecule in a sample to obtain a type of sequence information as otherwise described herein.

The present invention is described in further detail in the following examples, which are not intended to limit the scope of the invention claimed in any way. The accompanying drawings are intended to be considered an integral part of the specification and description of the present invention. The following examples are provided to illustrate but not limit the claimed invention.

Experiment

Example  One

Sample processing and DNA  extraction

Peripheral blood samples were collected from pregnant women who were in the early or mid-trimester of pregnancy and considered to be at risk for fetal death. Prior consent was obtained from each participant before sampling. Blood was collected before amniocentesis or chorionic villus sampling. Karyotype analysis was performed using chorionic villus or amniotic fluid samples to identify fetal karyotypes.

Peripheral blood collected from each subject was collected in an ACD tube. Blood samples from one tube (approximately 6-9 mL / tube) were transferred to one 15-mL low speed centrifuge tube. Blood was centrifuged at 440C for 10 minutes at 2640 rpm using a Beckman Allegra 6R centrifuge and rotor model GA 3.8.

For the acellular plasma extraction, the upper plasma layer was transferred to a 15-ml high speed centrifuge tube and centrifuged at 16000 x g for 10 minutes at 16000 x g using a Beckman Coulter Avanti J-E centrifuge and a JA-14 rotor. Two centrifugation steps were performed within 72 hours after blood extraction. Cell free plasma was stored at -80 ° C and thawed once before DNA extraction.

Cell-free DNA was extracted from plasma free of cells by using a QIAamp DNA blood mini kit (Qiagen) according to the manufacturer's instructions. Five milliliters of buffer AL and 500 mu l of Qiagen protease were added to 4.5 ml to 5 ml of acellular plasma. The volume was adjusted to 10 ml with phosphate buffered saline (PBS), and the mixture was incubated at 56 [deg.] C for 12 minutes. The precipitated cfDNA was separated from the solution by centrifugation at 8,000 RPM in a Beckman microcentrifuge using multiple columns. The column was washed with AW1 and AW2 buffer and cfDNA eluted with 55 ul of nuclease-free water. About 3.5-7 ng of cfDNA was extracted from plasma samples.

All sequencing libraries were prepared from approximately 2 ng of purified cfDNA extracted from maternal plasma. Library preparation was carried out using the reagents of NEBNext ™ DNA Sample Prep DNA Reagent Set 1 (Part No. E6000L; New England Biolabs, Ipswich, Mass.) Against Illumina® as follows. As the acellular plasma DNA is virtually fragmented, no additional fragmentation by plasma or DNA sonication has been performed on the plasma DNA sample. The excess packing of about 2 ng of purified cfDNA fragment contained in 40 [mu] l was incubated with 5 [mu] l of 10X phosphorylation buffer, 2 [mu] l of deoxynucleotide solution mix (10 mM each dNTP) provided in NEBNext ™ DNA Sample Prep DNA Reagent Set 1, , 1 [mu] l of a 1: 5 dilution of DNA polymerase I, 1 [mu] l of T4 DNA polymerase and 1 [mu] l of T4 polynucleotide kinase were incubated in a 1.5 ml microfused tube for 15 min at 20 [deg.] C, ® End Repair Module into a phosphorylated smooth end. The enzyme was then thermally inactivated by incubating the reaction mixture at 75 ° C for 5 minutes. The mixture was cooled to 4 ° C and DA tailing of the smooth-ended DNA was performed with 10 μl of the da-tailing master mix containing the Klenow fragment (3 'to 5' exon minus) (NEBNext ™ DNA Sample Prep DNA Reagent Set 1) RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; for 15 minutes. Subsequently, the Klenow fragment was heat-inactivated by incubating the reaction mixture at 75 DEG C for 5 minutes. After inactivation of the Klenow fragment, an Illumina adapter (Non-Index Y-adapter) was applied using a 1: 5 dilution of Illumina Dielectric Adapter Oligomix (Part No. 1000521; Illumina Inc., Hayward, CA) The mixture was incubated for 15 minutes at 25 占 폚, and then ligation was performed on dA-tailed DNA using 4 占 of T4 DNA ligase provided in NEBNext 占 DNA Sample Prep DNA Reagent Set 1. The mixture was cooled to 4 ° C and adapter-ligated cfDNA was ligated with magnetic beads provided in the Agencourt AMPure XP PCR purification system (Part No. A63881; Beckman Coulter Genomics, Danvers, MA) Sieves, and other reagents. 18 cycles of PCR were performed to selectively enrich for adapter-lyzed cfDNA using Illumina's PCR primers (Part Nos. 1000537 and 1000537) complementary to the Phusion® High-Fidelity Master Mix (Finnzymes, Woburn, MA) Respectively. NEBNext ™ DNA Sample The adapter-lyzed DNA was PCR amplified using PCR (30 sec for 30 sec) using the Illumina genomic PCR primers (Part Nos. 100537 and 1000538) and the Phusion HF PCR master mix provided in Prep DNA Reagent Set 1, 18 C for 10 seconds, 65 C for 30 seconds, and 72 C for 30 seconds; final extension for 5 minutes at 72 C and holding at 4 C). The amplified product was purified using the Agencourt AMPure XP PCR purification system (Agencourt Bioscience Corporation, Beverly, Mass.) According to the manufacturer's instructions available at www.beckmangenomics.com/products/AMPureXPProtocol_000387v001.pdf. The purified and amplified product was eluted in 40 [mu] l of Qiagen EB buffer and the concentration and size distribution of the amplified library was analyzed using an Agilent DNA 1000 kit (Agilent Technologies Inc., Santa Clara, Calif.) For the 2100 bioanalyzer.

The amplified DNA was sequenced using Illumina's Dielectric Analyzer II to obtain a 36 bp single-ended lead. Only about 30 bp of random sequence information is needed to identify sequences belonging to a particular human chromosome. Longer sequences can uniquely identify more specific targets. In this case, a number of 36 bp leads containing approximately 10% of the dielectric were obtained. Upon completion of the sample sequencing, Illumina "Sequencer Control Software" moved the image and base call files to a Unix server running Illumina "Dielectric Analyzer Pipeline" software version 1.51. By running the Illumina "Gerald" program, by the National Center for Biotechnology Information (NCBI36 / hg18, available on the World Wide Web http://genome.ucsc.edu/cgi-bin/hgGateway?org=Human&db=hg18&hgsid=166260105) Sequences were aligned for the reference human genome derived from the provided hg18 genome. Sequence data generated from the above procedure, which is uniquely aligned to the genome, was read from the Gerald output (export.txt files) by running the program (c2c.pl) on the computer-implemented Linnux operating system. Sequence alignment with base mismatch was allowed and included in the alignment factor only if they were uniquely aligned to the dielectric. (Redundancy) with the same start and end coordinates.

Approximately 5 million to 15 million 36 bp tags with 2 or less mismatches were uniquely mapped to the human genome. All mapped tags were counted and included in the calculation of chromosome capacity in both the test and qualifying samples. A tag derived from a male or female can map regions extending from the base 0 to base 2 x 10 ⁶ , base 10 x 10 ⁶ to base 13 x 10 ⁶ , and base 23 x 10 ⁶ of the Y-chromosome to the ends , These areas were specifically excluded from the analysis.

It has been found that some mutations in the total number of sequence tags mapped to individual chromosomes across the sample have been sequenced in the same sequence (interchromosomal variation), but substantially larger mutations have occurred during the different sequencing proceedings Sequencing progression).

Example  2

The capacity and variability of chromosomes 13, 18, 21, X, and Y

To investigate the degree of chromosomal and sequencing variation in the number of sequence tags mapped for all chromosomes, plasma cfDNA obtained from the peripheral blood of 48 volunteer pregnant subjects was extracted and sequenced as described in Example 1 , And analyzed as follows.

The total number of sequence tags (sequence tag densities) mapped for each chromosome was measured. Alternatively, the number of mapped sequence tags can be normalized to the length of the chromosome to generate a sequence tag density ratio. Normalization of chromosome length is not a necessary step, and may be performed alone to reduce the number of numbers in the number and simplify it for human interpretation. The chromosome length that can be used to normalize the sequence tag coefficient may be the length provided in genome.ucsc.edu/goldenPath/stats.html#hg18 on the World Wide Web.

The sequence tag densities generated for each chromosome are related to the sequence tag densities of each of the remaining chromosomes, and the sequence tag density for the target chromosome, such as chromosome 21, and the remaining chromosomes, i.e., chromosomes 1-20, 22, We derived the qualitative chromosomal capacity calculated as the ratio of the sequence tag density for. Table 1 provides examples of qualified chromosome doses calculated for the chromosomes of interest 13,18, 21, X, and Y determined in one of the eligible samples. The chromosomal capacity is determined for all chromosomes in all samples and the average capacity for the interest chromosomes 13, 18, 21, X and Y in the eligible sample is provided in Tables 2 and 3 and is depicted in FIGS. 2-6. Figures 2-6 also show the chromosomal capacity for the test sample. The chromosomal capacity for each of the chromosomes of interest in the eligible sample provides a measure of variation in the total number of sequence tags mapped for each of the chromosomes of interest relative to the sequence tags mapped for each of the remaining chromosomes. Thus, an eligible chromosome dose can identify a group of chromosomes or chromosomes, that is, a normalized chromosome, that has the closest mutation to the mutation of the interest chromosome in the sample and that will be provided as an ideal sequence for normalization values for further statistical evaluation. Figures 7 and 8 show the calculated average chromosomal capacity as determined in a population of eligible samples for chromosomes 13,18, and 21 and for chromosomes X and Y. [

In some instances, a best-normalized chromosome may not have the smallest variance, but it may have a distribution of the qualifying dose that best distinguishes the sample from the test sample or the qualifying sample, i.e., the best- , But it can have the greatest differentiation. Thus, differentiation accounts for the variation in chromosome capacity and the distribution of capacity in qualifying samples.

Tables 2 and 3 provide the Student's t-test as a measure of variability and a measure of the differentiation for chromosomes 18, 21, X, and Y, the smaller the T-test value, the greater the differentiation. The differentiation for chromosome 13 was determined as the average chromosomal dose in eligible samples and only as a ratio of the difference in dose to chromosome 13 in the T13 test sample, and the standard deviation of the mean of the eligible dose.

Eligible chromosomal capacity also serves as a criterion for determining thresholds when determining the integrity of the test sample as described below.

Table 1

(N = 1; samples # 11342, 46 &amp; cir &amp;) for chromosomes 13,18, XY )

Table 2

Eligible chromosomal capacity, variability and differentiation for chromosomes 21, 18 and 13

Table 3

Qualitative chromosome capacity, variability and differentiation for chromosome 13, X, and Y

A diagnostic example of T21, T13, T18 and Turner's syndrome obtained using standardized chromosomes, chromosome dose and differentiation for each of the chromosomes of interest is described in Example 3.

Example  3

Diagnosis of Fetal Status Using Standardized Chromosomes

Maternal blood test samples were obtained from pregnant volunteers and cfDNA was prepared, sequenced, and analyzed as described in Examples 1 and 2, in order to apply the use of chromosomal capacity to assess the biologic response in biological test samples.

# 21 Trichrome

Table 4 provides the calculated capacity for chromosome 21 in the exemplary test sample (# 11403). Threshold values generated for positive diagnoses of T21 adherence were set at> 2 standard deviations from the mean of eligible (normal) samples. Diagnosis for T21 was provided based on chromosome capacity in test samples larger than the set threshold. Chromosomes 14 and 15 were used as standardized chromosomes in separate calculations to show that chromosomes with the smallest variability, such as chromosome 14, or chromosomes with the greatest differentiation, such as chromosome 15, can be used to confirm the integrity . Thirteen T21 samples were identified using the calculated chromosome capacity, and the aqueous samples were confirmed to be T21 by the karyotype.

Table 4

T21  Chromosome capacity for isomerism (samples # 11403, 47 XY  +21)

18 times Trichrome

Table 5 provides the calculated capacity for chromosome 18 in the test sample (# 11390). The threshold values produced for the positive diagnosis of T18 adherence were set at 2 standard deviations from the mean of the eligible (normal) samples. Diagnosis for T18 was provided based on chromosome capacity in test samples larger than the set threshold. Chromosome 8 was used as a standard chromosome. In this example, chromosome 8 has the greatest differentiability from the smallest variability. Eight T18 samples were identified using chromosomal capacity and T18 was confirmed by karyotype.

These data indicate that standardized chromosomes can have both the smallest variability and the greatest differentiation.

Table 5

T18  The chromosomal capacity for isomerism (samples # 11390, 47 XY  +18)

No. 13 Trichrome

Table 6 provides the calculated capacity for chromosome 13 in the test sample (# 51236). The threshold values produced for the positive diagnosis of T13 adherence were set at 2 standard deviations from the mean of the eligible (normal) samples. Diagnosis for T13 was provided based on chromosomal capacity in test samples larger than the set threshold. Chromosome capacity for chromosome 13 was calculated using chromosome 5 or a group of chromosomes 3, 4, 5, and 6 as a standardized chromosome. One T13 sample was identified.

Table 6

T13  The chromosome capacity for isomerism (samples # 51236, 47 XY  +13)

Sequence tag density for chromosome 3-6 is the average tag count for chromosome 3-6.

The data show that the combination of chromosome 3, 4, 5 and 6 gives the greatest differentiation than the variability of chromosome 5 and the differentiability of any other chromosome.

Therefore, a group of chromosomes can be used as a standardized chromosome for determining the chromosome capacity and confirming the integrity.

Turner syndrome (X Heterochromosome )

Table 7 provides the calculated doses for chromosomes X and Y in the test sample (# 51238). Thresholds produced for the positive diagnosis of Turner syndrome (X chromosomes) were set to <-2 standard deviation from the mean for the X chromosome and <-2 standard deviation from the mean for the qualitative (normal) sample in the absence of the Y chromosome Respectively.

Table 7

Turner  ( XO ) Chromosome capacity for isomerism (Sample # 51238, 45 X)

Samples with an X chromosome capacity lower than the set threshold were found to have less than one X chromosome. The same sample was determined to have a lower Y chromosome capacity than the set threshold, indicating that the sample did not carry the Y chromosome. Thus, a combination of chromosomal capacities for X and Y was used to identify Turner syndrome (X-only chromosome) samples.

Thus, the method provided enables the determination of chromosomal CNV. In particular, the method enables determination of over- or under-representation chromosomal integrity by massively parallel sequencing of maternal plasma cfDNA and identification of standardized chromosomes for statistical analysis of sequencing data. The sensitivity and certainty of the method permits precise early and mid-tricolor trials.

Example  4

Measure partial impermeability

The utilization of the sequential doses was made from blood plasma and was applied to assess partial isomerism in the biological test sample of sequenced cfDNA as described in Example 1. [ It was confirmed that the sample was derived from a specimen in which chromosome 11 was partially deleted by karyotyping.

Analysis of the sequencing data for partial isomerism (partial deletion of chromosome 11, i.e. q21-q23) was performed as described for chromosomal integration in the previous examples. Sequence tags in the test samples were mapped to chromosome 11 and compared to the tag counts obtained for the corresponding sequences for chromosome 11 in the eligible samples (data not shown), the tags between base pairs 81000082-103000103 on the q arm of the chromosome And a significant loss of the coefficient. Sequence tags mapped to the sequence of interest (810000082-103000103bp) for chromosome 11 in each eligible sample, and sequence tags mapped for all 20 megabase segments in the entire genome of the eligible sample, i. E., Qualitative sequence tag densities Qualitative sequence capacity was determined as a ratio of the tag density in all eligible samples. The mean sequence volume, standard deviation, and coefficient of variation were calculated for all 20 megabase segments in the total genome, with the 20-megabase sequence with the smallest variability being the identified normalization sequence for chromosome 5 (13000014-33000033 bp) See Table 8) and used to calculate the dose for the sequence of interest in the test sample (see Table 9). Table 8 provides the sequence capacity for the sequence of interest (810000082-103000103bp) for chromosome 11 in the test sample produced as the ratio of the sequence tag mapped to the sequence of interest and the sequence tag mapped to the identified normalization sequence. Figure 10 shows the sequence capacity (o) for the sequence of interest in the seven eligible samples and the sequence capacity of the corresponding sequence in the test sample (). The mean is expressed by a solid line, and the calculated threshold value for the positive diagnosis of partial perfusion set at 5 standard deviations from the mean is indicated by a dotted line. Diagnosis of partial perfusion was based on sequence capacity in the test sample below the set threshold. The test sample was confirmed to have a deletion q21-q23 on chromosome 11 by nucleation.

Thus, in addition to confirming chromosomal integrity, the method of the present invention can be used to confirm partial immunity.

Table 8

order Chr11 Eligible Standardization Sequence, Capacity and Variability for: 81000082-103000103 (Eligible Sample n = 7)

Table 9

The sequence capacity (test sample 11206) for the sequence of interest (81000082-103000103) on chromosome 11

Example  5

Proof of Detection of Isolation

The sequencing data obtained for the samples depicted in Examples 2 and 3 and shown in Figures 2-6 were further analyzed to illustrate the sensitivity of the method in successfully identifying the acceptability in the maternal sample. Standardized chromosome capacities for chromosomes 21, 18, 13 X and Y were analyzed as a distribution over the mean standard deviation (Y-axis) and shown in FIG. The normalized chromosome used is shown as denominator (X-axis).

FIG. 11A is a graph showing the relationship between the standard deviation of the chromosomal capacity from the mean to the chromosome 21 dose in the unfermented sample (O) and the 21st trisomy 21 sample (T21; DELTA) when chromosome 14 is used as the standardization chromosome for chromosome 21 FIG. FIG. 11B shows a comparison of chromosomal capacity versus standard deviation from the mean for chromosome 18 doses in unfermented samples (O) and 18 trisomy 18 samples (T18; .DELTA.) When chromosome 8 was used as a standardization chromosome for chromosome 18 FIG. FIG. 11C shows a comparison of the unmodified sample (O) and the 13th trisomyhedge sample (O) using the average sequence tag density of the group of chromosomes 3, 4, 5, and 6 as a standardized chromosome to determine the chromosome capacity for chromosome 13 Lt; RTI ID = 0.0 &gt;(T13; &lt; / RTI &gt; FIG. 11D shows an average (average) value of chromosome X capacity in chromosome X when chromosome 4 is used as a standard chromosome for chromosome X in the unsterioned female sample (O), unaltered male sample (DELTA), and X- Lt; / RTI &gt; shows the distribution of the chromosome capacity relative to the standard deviation. Fig. 11E shows a case where an undistorted male sample (O), an undamaged female sample (DELTA), and an unmodified female sample (?) Are used when the average sequence tag density of the group of chromosomes 1-22 and X is used as a standardized chromosome to determine the chromosome capacity for chromosome Y , And the standard deviation from the mean for the chromosome Y dose in the X monosodium chromosome sample (+).

These data indicate that trisomy 21, trisomy 18, and trisomy 13 could be clearly distinguished from untouched (normal) samples. The X monosomy chromosome sample has a chromosome X capacity that is clearly lower than the chromosome X dose of the unaltered female sample (FIG. 11d) and a chromosome Y capacity that is clearly lower than the chromosome Y dose of the unaltered male sample (FIG. 11e) .

Thus, the methods provided are sensitive and specific in determining the presence or absence of chromosomal aberrations in maternal blood samples.

Example  6

From maternal blood Acellular cell  fetus DNA Massively parallel DNA  Determination of Fetal Chromosomal Abnormalities Using Sequencing: Training Set 1 and  Irrelevant test set 1

The study was conducted by an appropriate local clinical researcher at 13 US clinical sites between April 2009 and July 2010, under the human subject protocol approved by the institutional audit committee (IRB) of each institution. Prior to participating in the study, written consent was obtained from each subject. The protocol was designed to provide blood samples and clinical data to support the development of noninvasive prenatal genetic diagnostic methods. Pregnant women over the age of 18 could be eligible for inclusion. For patients who underwent clinically indicated CVS or amniocentesis, blood was collected prior to performing the procedure and the fetal karyotype results were also collected. Peripheral blood samples (2 tubes or a total of ~ 20 mL) were collected from all subjects on an acid citrate dextrose (ACD) tube (Becton Dickinson). The source of all samples was identified and an anonymous patient ID number was assigned. Blood samples were transferred from the temperature controlled transport container provided in the study to the laboratory overnight. The elapsed time between collection and sample receipt was recorded as part of the sample factor.

The regional research facilitator entered clinical data related to the patient's current pregnancy and medical history into the CRF using an anonymous patient ID number. Cytogenetic studies of fetal karyotypes from invasive prenatal procedure samples were performed at each local laboratory and the results were also recorded in the study CRF. All data obtained on the CRF was entered into the clinical database of the laboratory. Cell free plasma was obtained from individual blood tubes utilizing a two-step centrifugation process within 24-48 hours of a venous puncture sample. Plasma from a single blood tube was sufficient for sequencing analysis. Cell-free DNA was extracted from plasma free of cells using a QIAamp DNA blood mini kit (Qiagen) according to the manufacturer's instructions. Since the length of a cell-free DNA fragment is known to be about 170 base pairs (bp) (Fan et al., Clin Chem 56: 1279-1286 [2010]), DNA fragmentation was not required prior to sequencing.

For training set samples, cfDNA was prepared using a standard manufacturer's protocol with the Illumina Dielectric Analyzer IIx machine (http://www.illumina.com/), sequencing library production (cfDNA ligation and universal adapter ligation) and Prognosys Biosciences, Inc. for sequencing. (La Jolla, CA). 36 base pairs of single-ended leads were obtained. Upon completion of the sequencing, all base call files were collected and analyzed. For the test set samples, a sequencing library was prepared and sequencing was performed on an Illumina Dielectric Analyzer IIx machine. Sequencing library production was performed as follows. The full-length protocol described is essentially a standard protocol provided by Illumina, and only purification of the amplified library is different from the Illumina protocol: the Illumina protocol instructs the amplified library to be purified using gel electrophoresis, &Lt; / RTI &gt; uses magnetic beads in the same purification step. NEBNext ™ DNA Sample for Illumina® essentially according to the manufacturer's instructions To prepare a primary sequencing library using Prep DNA Reagent Set 1 (Part No. E6000L; New England Biolabs, Ipswich, MA) 2 ng of purified cfDNA was used. All steps, except the final purification of the adapter-ligated product, carried out with Agencourt magnetic beads and reagents instead of the purification column, were carried out using a protocol involving NEBNext ™ reagents for sample preparation for a sequenced genomic DNA library using Illumina® GAII Lt; / RTI &gt; The NEBNext ™ protocol was essentially provided by Illumina available at grcf.jhml.edu/hts/protocols/11257047_ChIP_Sample_Prep.pdf.

The excess packing of about 2 ng of purified cfDNA fragment contained in 40 [mu] l was incubated with 5 [mu] l of 10X phosphorylation buffer, 2 [mu] l of deoxynucleotide solution mix (10 mM each dNTP) provided in NEBNext ™ DNA Sample Prep DNA Reagent Set 1, , 1 [mu] l of a 1: 5 dilution of DNA polymerase I, 1 [mu] l of T4 DNA polymerase, and 1 [mu] l of T4 polynucleotide kinase were incubated in a 200 [mu] l microfuge tube for 30 min in a thermocycler Lt; / RTI &gt; to &lt; RTI ID = 0.0 &gt; phosphorylated &lt; / RTI &gt; smooth ends according to the NEBNext® End Repair Module. The samples were cooled to 4 ° C and purified using the QIAQuick column provided in a QIAQuick PCR purification kit (QIAGEN Inc., Valencia, Calif.) As follows. 50 μl of the reaction product was transferred to a 1.5 ml microfuse tube and 250 μl of Qiagen buffer PB was added. The resulting 300 μl was transferred to a QIAquick column, which was centrifuged at 13,000 RPM for 1 minute in microfuses. The column was washed with 750 [mu] l of Qiagen buffer PE and resuspended. Residual ethanol was removed by further centrifugation at 13,000 RPM for 5 minutes. The DNA was eluted by centrifugation in 39 [mu] l of Qiagen buffer EB. DA tailing of 34 평 of smooth-ended DNA was performed using 16 의 of dA-tailing master mix containing Klenow fragment (3 'to 5' exon minus) (NEBNext ™ DNA Sample Prep DNA Reagent Set 1) Lt; RTI ID = 0.0 &gt; 37 C &lt; / RTI &gt; for 30 minutes according to the NEBNext dA-tailing module. The sample was cooled to 4 캜 and purified using the column provided on the MinElute PCR purification kit (QIAGEN Inc., Valencia, Calif.) As follows. 50 μl of the reaction product was transferred to a 1.5 ml microfuse tube and 250 μl of Qiagen buffer PB was added. Was transferred to a MinElute column, which was centrifuged at 13,000 RPM for 1 minute in a microfuse. The column was washed with 750 [mu] l of Qiagen buffer PE and resuspended. Residual ethanol was removed by further centrifugation at 13,000 RPM for 5 minutes. The DNA was eluted by centrifugation in 15 [mu] l of Qiagen buffer EB. Ten microliters of the DNA eluate was mixed with 1 μl of a 1: 5 dilution of Illumina Dielectric Adapter Oligmix (Part No. 1000521), 15 μl of 2X Quick ligation reaction buffer, and 4 μl of Quick T4 DNA ligase Lt; 0 &gt; C for 15 minutes according to the NEBNext Quick ligation module. The sample was cooled to 4 &lt; 0 &gt; C and purified using the MinElute column as follows. 150 microliters of Qiagen buffer PE was added to 30 mu l of the reaction and the total volume was transferred to a MinElute column and centrifuged at 13,000 RPM for 1 minute in microfuses. The column was washed with 750 [mu] l of Qiagen buffer PE and resuspended. Residual ethanol was removed by further centrifugation at 13,000 RPM for 5 minutes. The DNA was eluted by centrifugation in 28 [mu] l of Qiagen buffer EB. A 23 microliter adapter-lined DNA eluate was added to the NEBNext ™ DNA sample using the Illumina genomic PCR primers (Part Nos. 100537 and 1000538) provided in Prep DNA Reagent Set 1 and the Phusion HF PCR master mix, Followed by 18 cycles of PCR (98 [deg.] C for 30 seconds, 98 [deg.] C for 10 seconds, 65 [deg.] C for 30 seconds, and 18 cycles of 72 [deg.] C for 30 seconds; last extension for 5 minutes at 72 [deg.] C and maintained at 4 [ The amplified product was purified using the Agencourt AMPure XP PCR purification system (Agencourt Bioscience Corporation, Beverly, Mass.) According to the manufacturer's instructions available at www.beckmangenomics.com/products/AMPureXPProtocol_000387v001.pdf. The Agencourt AMPure XP PCR purification system removes unincorporated dNTPs, primers, primer dimers, salts and other contaminants, and retrieves ampicrons greater than 100 bp. The purified and amplified products were eluted from Agencourt beads in 40 [mu] l of Qiagen EB buffer and the size distribution of the library was analyzed using an Agilent DNA 1000 kit (Agilent Technologies Inc., Santa Clara, Calif.) For 2100 bioanalyzer. For both the training and test sample sets, 36 base pairs of single-ended leads were sequenced.

Data analysis and sample classification

Sequence leads of 36 bases in length were aligned for the human genome assembly hg18 obtained from the UCSC database (http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/). Alignment was performed utilizing a Bowtie short lead aligner (version 0.12.5) that allowed up to two base mismatches during alignment (Langmead et al., Genome Biol 10: R25 [2009]). Only leads clearly mapped to a single dielectric location were included. The lead-mapped genomic region was counted and included in the chromosomal dose calculation (see below). Sequence tags from boys and girls excluded from analysis the regions on the Y chromosome mapped without any distinction (in particular, base 0 to base 2 x 10 ^6, base 10 x 10 ⁶ to base 13 x 10 ^6, and base 23 x 10 &lt; ⁶ &gt; to the end of chromosome Y).

Sequencing variation in progression and progression in the chromosomal distribution of sequence leads can mask the effect of fetal insensitivity on the distribution of mapped sequence regions. To correct for such a variation, the chromosome dose was calculated as a coefficient of the mapped region for a given interest chromosome normalized to the coefficients observed on a given normalized chromosome sequence. As previously described, standardized chromosomal sequences can consist of a single chromosome or a group of chromosomes. Considering each normal chromosome as a potential denominator in the ratio of the coefficients using the chromosomes of interest of the inventors of the present invention, the normalized chromosome sequence is first identified as a denatured sample having a diploid karyotype for the chromosomes 21, 18, 13 and X of interest It was identified in the subset of samples in the training set of samples that are eligible samples. In the sequencing process, we selected a denominator chromosome, that is, a normalized chromosome sequence that minimized the variation of the chromosomal capacity between sequences. Each of the chromosomes of interest was determined to have a separate normalized chromosomal sequence (denominator) (Table 10). No single chromosome could be identified as a normalized chromosome sequence for chromosome 13 because no chromosome was determined as to reduce the variability of the capacity of chromosome 13 across the sample, that is, the variability of NCV values for chromosome 13 T13 &lt; / RTI &gt; functionality. Chromosomes 2-6 were randomly selected and tested for their ability as a group to mimic the behavior of chromosome 13. The group of chromosomes 2-6 was shown to substantially reduce the variability of the dose for chromosome 13 in the training samples and was therefore selected as the normalized chromosome sequence for chromosome 13. As described above, the variability of the chromosome capacity for chromosome Y is greater than 30 regardless of which single chromosome is used as the standard chromosome sequence in determining chromosome Y capacity. The group of chromosomes 2-6 was chosen as the standardized chromosome sequence for chromosome Y since it has been shown to substantially reduce the variability of dose for chromosome Y in the training samples.

The chromosomal capacity for each of the chromosomes of interest in the eligible sample provides a measure of the variation in the total number of sequence tags mapped for each of the chromosomes of interest relative to each of the remaining chromosomes. Thus, an eligible chromosome dose can identify a group of chromosomes or chromosomes, that is, a normalized chromosome sequence, that will have the mutation closest to the mutation of the interest chromosome in the sample and serve as an ideal sequence for normalization values for further statistical evaluation.

The chromosomal capacity of all samples in the training set, that is, qualified and altered samples, also serves as a criterion for determining thresholds when determining the integrity of the test sample as described below.

Table 10

Standardized chromosome sequence to determine chromosome capacity

For each chromosome of interest in each sample of the test set, the normalization value was determined and used to determine the presence or absence of the biotyping. Normalization values were calculated as chromosome doses that could be further calculated to provide a normalized chromosome value (NCV).

Chromosome capacity

For the test set, chromosome capacity was calculated for each of the interest chromosomes, 21, 18, 13, X and Y for all samples. As provided in Table 10 above, the chromosome capacity for chromosome 21 was calculated as the ratio of the number of tags in the test sample mapped to chromosome 21 in the test sample and the number of tags in the test sample mapped to chromosome 9; The chromosome capacity for chromosome 18 was calculated as the ratio of the number of tags in the test sample mapped to chromosome 18 in the test sample and the number of tags in the test sample mapped to chromosome 8; The chromosome capacity for chromosome 13 was calculated as the ratio of the number of tags in the test sample mapped to chromosome 13 in the test sample and the number of tags in the test sample mapped to chromosome 2-6; The chromosome capacity for chromosome X was calculated as the ratio of the number of tags in the test sample mapped to chromosome X in the test sample and the number of tags in the test sample mapped to chromosome 6; The chromosome capacity for chromosome Y was calculated as the ratio of the number of tags in the test sample mapped to chromosome Y in the test sample and the number of tags in the test sample mapped to chromosome 2-6.

Standardized chromosome value

Using the chromosome dose for each of the interest chromosomes in each test sample and the average of the corresponding chromosome dose determined in the eligible sample of the training set, the normalized chromosome value (NCV) was calculated using the following equation:

및

는 j번째 염색체 용량에 대한 각기 추정 트레이닝 세트 평균 및 표준 편차이고,

는 시험 샘플 i에 대해 관찰된 j번째 염색체 용량이다. 염색체 용량이 정상적으로 분포된 경우, NCV는 용량에 대한 통계적 z-점수와 같다. 변질되지 않은 샘플로부터의 NCV의 분위-분위 플롯에서 선형성으로부터 어떠한 유의한 이탈도 관찰되지 않는다. 추가로, NCV에 대한 정규성의 표준 시험은 정규성의 귀무 가설을 거부하지 못했다. In this formula,

And

Is an estimated training set mean and standard deviation for each j &lt; th &gt;

Is the jth chromosome capacity observed for test sample i. If the chromosomal capacity is normally distributed, the NCV is equal to the statistical z-score for the dose. No significant deviations from linearity are observed in the quartile-quart plot of the NCV from the unaltered sample. In addition, the standard test of normality for NCVs failed to reject the null hypothesis of normality.

For the test set, the NCV was calculated for each of the interest chromosomes, 21, 18, 13, X and Y for all samples. To ensure a safe and valid classification scheme, conservation boundaries were selected for the binomial classification. In order to classify the chromosomal status of the chromosomes normally, NCV> 4.0 was required to classify the altered chromosomes (ie, chromosomal), and NCV <2.5 was required to classify unmodified chromosomes. Samples with normal chromosomes with an NCV of 2.5 to 4.0 were classified as " no-call ".

In the test, sex chromosomal classification was performed by sequential use of NCV for both X and Y as follows:

1. If NCV Y is> -2.0 standard deviation from the mean of the male sample, the sample was classified as male (XY).

2. If NCV Y is <-2.0 standard deviation from the mean of the male sample and NCV X is> -2.0 standard deviation from the mean of the female sample, the sample was classified as female (XX).

3. If NCV Y is <-2.0 standard deviation from the mean of the male sample and NCV X is <-3.0 standard deviation from the mean of the female sample, then the sample was classified as X null chromosome, Turner syndrome.

4. If the NCV does not meet any of the above criteria, the sample was classified as "no call" for sex.

result

Research group Demographic Graphics

A total of 1,014 patients were enrolled between April 2009 and July 2010. Patient demographics, invasive procedure types, and karyotypic results are summarized in Table 11. The average age of the study participants was 35.6 years (ranging from 17 to 47 years) and the gestational age ranged from 6 weeks 1 to 38 weeks 1 day (average 15 weeks 4 days). The overall incidence of abnormal fetal chromosomal karyotype was 6.8% and the T21 incidence was 2.5%. Of the 946 subjects with singleton pregnancy and karyotype, 906 (96%) showed at least one clinically recognized risk factor for fetal insemination prior to the prenatal procedure. Even with the elimination of the maternal age of the elderly as the only symptom, the data show a very high false positive rate for current screening tests. Ultrasound findings of increased fetal neck circumference, cystic lymphangioma, or other structural congenital anomalies by ultrasound may best predict abnormal karyotypes in these cohorts.

Table 11

patient Demographic Graphics

* Includes fetal outcome from multiple pregnancies, ** Evaluated and recorded by clinician

Abbreviation: AMA = age of the elderly, NT = fetal neck circumference

The distribution of the various ethnic backgrounds depicted in these study groups is also presented in Table 11. Overall, in this study, 63% of the patients were white, 17% Hispanics, 6% Asians, 5% multiples, and 4% Blacks. It was recognized that racial diversity differs markedly from region to region. For example, 60% Hispanics and 26% white subjects were registered in one area, but no Hispanic subjects were enrolled in all three clinics in the same state. As expected, there were no discernible differences observed in our results for different races.

Training Data Set 1

The training set study selected 71 samples from the initial sequential accumulation of 435 samples collected between April 2009 and December 2009. In this first series of subjects, all subjects with altered fetuses (abnormal karyotypes) were included in the random selection and numbering of the unchanged subjects with sequencing and appropriate samples and data. The clinical characteristics of the training set patients were consistent with the overall study demographics presented in Table 11. The gestational age range of the samples in the training set ranged from 10 weeks 0 to 23 weeks 1 day. 38 received CVS, 32 received positive amniocentesis, and 1 patient did not receive the specified invasive procedure (unchanged karyotype 46, XY). Seventy percent of the patients were Caucasian, 8.5% Hispanic, 8.5% Asian, and 8.5% multiples. Six sequenced samples were removed from the set for training purposes: four samples from a twin pregnant subject (discussed further below), one sample with contaminated T18 during manufacture, and one sample with fetal karyotype 69 , One sample with XXX, and 65 samples for training set.

The number of unique sequence regions (ie, tags identified as having unique sites in the genome) was changed from 2.2M to 13.7M in the early phase of the training set study due to improved sequencing techniques over time. To monitor for any potential changes in chromosomal capacity in excess of this 6-fold range at the only site, different undigested samples were advanced at the beginning and end of the study. During the first 15 undisturbed sample runs, the average number of unique sites was 3.8M and the average chromosome capacities for chromosome 21 and chromosome 18 were 0.314 and 0.528, respectively. During the last 15 undisturbed sample runs, the average number of unique sites was 10.7 M and the average chromosome capacities for chromosome 21 and chromosome 18 were 0.316 and 0.529, respectively. There was no statistical difference between chromosome 21 and chromosome 18 over time in the training set study.

The training set NCV for chromosomes 21, 18 and 13 is shown in FIG. The results shown in FIG. 12 are consistent with the assumption of normality that approximately 99% of the diploid NCV will be within +2.5 standard deviation of the mean. Of these sets of 65 samples, eight samples with clinical karyotypes indicate that T21 has an NCV in the range of 6-20. Four samples with clinical karyotypes representing fetal T18 had NCVs ranging from 3.3 to 12 and two samples with karyotypes indicative of fetal 13 (T13) had a NCV of 2.6 to 4. The diversity of NCV in altered samples is due to their dependence on the percentage of fetal cfDNA in each sample.

Similar to normal chromosomes, mean and standard deviation for sex chromosomes were established in the training set. Sex chromosome thresholds allowed 100% identification of boys and girls in the training set.

Test Data Set 1

After establishing chromosome capacity averages and standard deviations from the training set, a test set of 48 samples was selected from a total of 575 samples collected from January 2010 to June 2010. One of the samples from the twin pregnancies was removed from the final analysis leaving 47 samples in the test set. Individual manufacturing samples for sequencing and operating the equipment were not disclosed for 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, which was higher than the overall procedural demographic, but also similar to the training set. 50% of the subjects were Caucasian, 27% Hispanics, 10.4% Asians and 6.3% Blacks.

In the test set, the number of unique sequence tags varied from about 13M to 26M. For unaltered samples, the chromosomal capacities for chromosome 21 and chromosome 18 were 0.313 and 0.527, respectively. The test set NCV for chromosome 21, chromosome 18 and chromosome 13 is shown in FIG. 13 and the classification is given in Table 12.

Table 12

Test set classification data

* MX is a chromosome of X chromosome without evidence of Y chromosome

In the test set, 13/13 subjects with clinical karyotypes representing fetal T21 were correctly identified to have an NCV in the range of 5-14. An 8/8 subject with a karyotype representing fetal T18 was correctly identified to have an NCV in the range of 8.5-22. In this test set, a single sample with a karyotype classified as T13 was classified as a no.

In the case of the test data set, it was correctly confirmed that all male samples contained complex karyotype, 46, XY + marker chromosomes (not identified by cytogenetics) samples (Table 3). Of the 20 female samples, 19 were correctly identified, and one female sample was classified as a no-call. For three samples in a test set with a karyotype of 45, X, two out of three were correctly identified as X-shaped chromosomes and one was classified as a no-call (Table 12).

Twins

Four of the samples initially selected for the training set and one of the samples in the test set were obtained in a twin pregnancy. The thresholds used here could be confounded by the different amounts of cfDNA expected in an environment of twin pregnancy. In the training set, the karyotype from one of the twin samples was monochromatin 47, XY + 21. The second twin sample was fraternal and amniocentesis was performed individually on the fetus. In this twin pregnancy, one fetus had a karyotype of 47, XY + 21, while the other had a normal karyotype of 46, XX. In both of these cases, the acellular classification based on the method discussed above classifies the sample as T21. The other two twin pregnancies in the training set were correctly classified as unmodified for T21 (all twins showed a diploid karyotype for chromosome 21). For the twin pregnancy sample in the test set, the karyotype was established only for twin B (46, XX) and the algorithm was correctly categorized as not altered for T21.

conclusion

The data indicate that massively parallel sequencing can be used to determine multiple abnormal fetal karyotypes from the blood of a pregnant woman. These data demonstrate that 100% accurate classification of samples with 21 trisomy and 18 trisomy can be identified using independent test set data. In the case of a fetus with an abnormal sex chromosomal karyotype, none of the samples were incorrectly classified as an algorithm of the method. Importantly, the algorithm was also well performed in determining the presence of T21 in two sets of twin pregnancies with one or more altered fetuses, which have never been presented in the past. Moreover, these studies not only represent a range of abnormal karyotypes likely to be seen in commercial clinical settings, but also precisely classify pregnancies that have not been altered by normal trisomy to address the unacceptably high false positive rate that remains in prenatal screening today A variety of samples from a variety of centers showing significance were examined. These data provide valuable insights into the vast possibilities of using this method in the future. Analysis of a unique subset of genome regions showed an increase in variance consistent Poisson coefficient statistics.

Data based on the discovery of Fan and Quake, which demonstrate the sensitivity of noninvasive prenatal determinations of maternal plasma from maternal plasma using massively parallel sequencing, are limited to counting statistics (Fan and Quake, PLos One 5, e10439 [2010]) . Because the sequencing information has been collected across the entire genome, the method can determine any number of isomeric or other number of copies, including insertions and deletions. The karyotype from one of the samples was a chromosome located between q21 and q23 observed as a ~ 10% reduction in the relative number of tags in the 25 Mb region starting at q21 when the sequencing data was analyzed at 500 kbase bins It had a small fruit of 11. In addition, in the training set, three of the samples had complex sex wounds due to a mosaic in cytogenetic analysis. These karyotypes are i) 47, XXX [9] / 45, X [6], ii) 45, X [3] / 46, XY [ ]. Sample ii showing some XY-containing cells was correctly classified as XY. Sample i (from the CVS procedure) and iii (from the amniocentesis), both of which showed a mixture of XXX and X cells by cytogenetic analysis (consistent with Mosaic Turner syndrome), were classified as nocor and X chromosomes, respectively .

In testing the algorithm, another interesting data point was observed to have an NCV of -5 to -6 for chromosome 21 in the case of a sample from the test set (FIG. 13). Although these samples were diploid in chromosome 21 by cytogenetics, the karyotype is chromosome 9; 47, XX + 9 [9] / 46, XX [6]. Since chromosome 9 is used in the denominator to determine chromosome capacity for chromosome 21 (Table 10), this lowers the overall NCV value. The ability of using standardized chromosomes to determine fetal 9 trisomy in these samples is demonstrated by the results provided in Example 7 below.

The conclusion of Fan et al. On the sensitivity of this method is that the algorithm used is only correct if it can account for any arbitrary or intentional bias introduced by the sequencing method. If the sequencing data is not adequately standardized, the resulting analysis will not meet the counting statistics. In their recent paper, Chiu et al. Have recognized that the measurement of chromosomes 18 and 13 using the massively parallel sequencing method is unclear and concluded that further studies are needed to use the method for T18 and T13 determinations (Chiu et al. , BMJ 342: c7401 [2011]). The method used in the paper by Chiu et al. Simply uses the number of sequence tags for the chromosome of interest, in this case, chromosome 21 standardized by the total number of tags in the sequencing process. The challenge of this approach is that the distribution of the tags for each chromosome can change between sequencing progresses, thus increasing the overall variation of the isotropic decision metric. In order to compare the results of the Chiu algorithm with the chromosomal capacity used in this example, test data for chromosomes 21 and 18 were reanalyzed using the method recommended by Chiu et al. As shown in Fig. Overall, a reduction in the range of NCV for each of chromosomes 21 and 18 was observed, as well as a decrease in the rate of crystallization for the 10/13 T21 and 5/8 T18 samples, utilizing the NCV threshold of 4.0 for the isomeric classification Has been accurately confirmed from the test set of the present inventors.

Ehrich et al. Also focused on T21 and used the same algorithm as Chiu et al. (Ehrich et al., Am J Obstet Gynecol 204: 205 e1-e11 [2011]). In addition, after observing the changes in the x-scoring metrics of the test set z-score metrics from the xref data, i.e. the training set, they were retrained for the test set to establish the classification boundary. This approach is feasible in principle, but it will be challenged to determine how many samples are actually required for training and how often it needs to be refined to ensure that the classification boundary is correct. One way to alleviate this problem is to include a control that measures the baseline and adjusts the scale for the quantitative behavior for each sequencing run.

Data obtained using this method indicate that massively parallel sequencing can determine multiple fetal chromosomal anomalies from plasma of pregnant women when the algorithm for standardizing chromosomal coefficient data is optimized. This method for quantification not only minimizes any and intentional variations in sequencing progression but also enables efficient classification of the entire genome, most notably across T21 and T18. Larger sample collections are required to test the algorithm for T13 determination. To this end, prospective, confidential, multi-site clinical studies are being conducted to further demonstrate the diagnostic accuracy of the method.

Example  7

Determination of the presence or absence of 5 or more different chromosomal aberrations in all chromosomes of individual test samples

In order to demonstrate the possibility of a method for determining the presence or absence of any chromosomal integrity in each of a set of maternal test samples (Test Set 1; Example 6), the systematically determined normalized chromosomal sequences were compared to undistorted samples of the training set (Training Set 1; Example 6) and used to calculate chromosomal capacity for all chromosomes in each test sample. The determination of the presence or absence of any one or more different complete fetal chromosomal integrations in each test and training set sample was established from the sequencing information obtained from a single sequencing run for each individual sample.

By calculating the single chromosome capacity for each of the chromosomes 1-22, X and Y, using the chromosome density, i.e. the number of sequence tags identified for each chromosome in each sample of the training set described in Example 6, A systematically determined normalized chromosome sequence consisting of a group of chromosomes or chromosomes was determined. The standardized chromosome sequences determined systematically for each of the chromosomes 1-22, X, and Y were determined by systematically calculating the chromosomal capacity for each chromosome using all possible combinations of chromosomes as the molecules. For example, in the case of chromosome 21 as a chromosome of interest, the chromosomal capacity is determined by the ratio of (i) the number of sequence tags obtained for chromosome 21 (the 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 is calculated so that all possible combinations of all chromosomes 1-20, 22, X and Y are used to determine all possible chromosome capacities for each of the interested chromosomes in each of the eligible (isomeric) samples of the training set 21, 22, X, and Y in all possible combinations of the remaining chromosomes (except chromosome 21), i.e. 1, 2, 3, 4, 5, etc., so that they can be used as normalized chromosome sequences (molecules) 1 + 22, 1 + X, and 1 + Y ;, 1 + 2 + 3, 1 + 2 + 4, 1 + 2 from 1 + 2, 1 + 3, 1 + 1 + 2 + 20 to 1 + 2 + 20, 1 + 2 + 22, 1 + 2 + X, and 1 + 2 + Y; 1 + 3 + 20, 1 + 3 + 22, 1 + 3 + X, and 1 + 3 + Y from 1 + 3 + 4, 1 + 3 + 5, 1 + 3 + 1 + 2 + 3 + 20, 1 + 2 + 3 + 22, 1 + 2 + 3 + X from 1 + 2 + 3 + 4, 1 + 2 + 3 + 5, 1 + 2 + 3 + Y and the like. The chromosomal capacity was determined in the same manner as chromosome 21 in all training samples, and the standardized chromosome sequence determined systematically for chromosome 21 was determined as a single chromosome or group of chromosomes, with the lowest variability across chromosome 21 across all training samples Capacity. The same analysis was repeated to determine a single chromosome or combination of chromosomes that would function as a standardized chromosome sequence determined systematically for each of the remaining chromosomes, including chromosomes 13, 18, X and Y, (Single chromosome or group of chromosomes) for all other interest chromosomes 1-12, 14-17, 19-20, 22, X and Y in the training samples. Thus, all chromosomes were treated as chromosomes of interest, and a systematically determined normalization sequence was determined for each chromosome in each of the unaltered samples in the training set. Table 13 provides a single chromosome or group of chromosomes identified as the standardized sequences systematically determined for each of the chromosomes 1-22, X, and Y of interest. As highlighted in Table 13, for some of the chromosomes of interest, the systematically determined normalized chromosome sequence was determined to be a single chromosome (e.g., chromosome 4 is the chromosome of interest), and for other interest chromosomes, It was determined to be a group of chromosomes (e.g., when chromosome 21 is a chromosome of interest).

Table 13

Standardized chromosome sequences systematically determined for all chromosomes

The mean, standard deviation (SD) and coefficient of variation (CV) for the systematically determined normalized chromosomal sequences determined for each of the all chromosomes are provided in Table 14.

Table 14

The mean, standard deviation, and coefficient of variation for all systematically determined normalized chromosome sequences

^a Exclude trichomes

^b girl

Variations in chromosome capacity across all training samples reflected by the value of CV demonstrate the use of a systematically determined standardized chromosome sequence to provide a large signal-to-noise ratio and dynamic range, As suggested, Lee's decision to be carried out with high sensitivity and high specificity.

To demonstrate the sensitivity and specificity of the method, chromosome capacities for all interest chromosomes 1-22, X and Y were determined in each sample of the training set and the corresponding systematically determined normalized chromosome sequence provided in Table 13 above &Lt; / RTI &gt; was determined in each of the samples of the test set described in Example 5.

Using the systematically determined normalized chromosome sequences for each of the chromosomes of interest, the presence or absence of any chromosomal integrity was determined in each of the samples in the training set and in each of the test samples, i.e., each sample was identified on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, . Sequence information, i.e. the number of sequence tags, was obtained for each sample of the training set, and for all chromosomes in each test sample, and the single chromosome capacity for each of the chromosomes in each training and test sample was determined from those determined in the training set Using the number of sequence tags obtained for the corresponding systematically determined normalized chromosomal sequences (Table 13). The chromosomal capacity for each chromosome in each training sample was determined using the number of sequence tags obtained in each training sample for the systematically determined normalized chromosome sequence and the chromosomal capacity for each chromosome in each training sample was determined for each test The chromosomal capacity for each chromosome was determined for each test sample using the number of sequence tags obtained in the sample. To ensure a safe and effective classification of the water-solubility, the same preservation boundary was selected as described in Example 6.

Training set results

A plot of chromosome capacity for chromosomes 21, 18, and 13 in a training set of samples using a systematically determined normalized chromosome sequence is provided in FIG. Eight samples with clinical karyotypes representing T21 had a NCV of 5.4 to 21.5 when using a systematic determined standard chromosomal sequence, a group of chromosomes 4 + 14 + 16 + 20 + 22. Four samples with clinical karyotypes representing T18 had a NCV of 3.3 to 15.3 when using a systematic determined standard chromosomal sequence, a group of chromosomes 2 + 3 + 5 + 7. Two samples with clinical karyotypes representing T13 had a NCV of 8.0 to 12.4 when using a systematic determined normalized chromosome sequence, a group of chromosome 4 + 5. The T21 sample of the training set is represented as the last 8 samples of chromosome 21 data (O); The T18 sample of the training set is represented as the last four samples of chromosome 18 data (?); The T13 sample of the training set is displayed as the last two samples of chromosome 13 data (&amp; squ &amp;).

This data indicates that the normalized chromosome sequence can be used to determine and accurately classify the complete fetal chromosomal integrity with high confidence. Since all samples with altered karyotypes have NCVs greater than 3, the likelihood that these samples are part of the undistorted distribution is less than about 0.1%.

Similar to ordinary chromosomes, a systematically determined normalized chromosome sequence (ie, a group of chromosomes 4 + 8) is used for chromosome X and a systematically determined normalized chromosome sequence (ie, a group of chromosomes 4 + 6) , All boys and girls in the training set were correctly identified. In addition, all five X-linked chromosome samples were identified. Figure 18A shows plots of NCV (X-axis) determined for the X chromosome and NCV (Y-axis) determined for the Y chromosome for each sample of the training set. All samples with karyotypes of X chromosomes have NCV values of less than -4.83. An X-linked chromosome sample with a karyotype that matches 45, X karyotype (complete or mosaic) has a Y NCV value close to zero as expected. The female sample is clustered around NCV = 0 in both X and Y cases.

Test set results

A plot of chromosome capacity for chromosomes 21, 18, and 13 in a test sample using the systematically determined related standardization chromosomal sequences is provided in FIG. When using a systematic determined standard chromosomal sequence (ie, a group of chromosomes 4 + 14 + 16 + 20 + 22), 13 out of 13 samples with clinical karyotypes representing T21 were correctly identified to have a NCV of 7.2 to 16.3 . Using the systematically determined normalized chromosomal sequences (ie, a group of chromosomes 2 + 3 + 5 + 7), all eight samples with clinical karyotypes representing T18 were found to have an NCV of 12.7 to 30.7. When using a systematic determined normalized chromosome sequence (ie a group of chromosome 4 + 5), only one sample with a clinical karyotype showing T13 was correctly identified to have an NCV of 8.6. The T21 sample of the test set is displayed as the last 13 samples of chromosome 21 data (O); The T18 sample of the test set is represented as the last 8 samples of chromosome 18 data (DELTA); The T13 sample of the test set is displayed as the last sample of chromosome 13 data (&amp; squ &amp;).

These data indicate that systematically determined normalized chromosome sequences can be used to determine and accurately classify different complete fetal chromosomal aids with greater confidence. Similar to the training set, all samples with altered karyotypes have an NCV of greater than 7, indicating that these samples are unlikely to be part of the undisturbed distribution (Figure 16).

Similar to ordinary chromosomes, a systematically determined normalized chromosome sequence (ie, a group of chromosomes 4 + 8) is used for chromosome X and a systematically determined normalized chromosome sequence (ie, a group of chromosomes 4 + 6) , All boys and girls in the training set were correctly identified. In addition, all three of the X-linked chromosome samples were identified. Figure 18B shows plots of NCV (X-axis) determined for the X chromosome and NCV (Y-axis) determined for the Y chromosome for each sample in the test set.

As described above, the method allows determination of the presence or absence of complete or partial chromosomal integrity of each of chromosome 1-22, X, and Y in each sample. In addition to determining complete chromosomal T13, T18, T21, and X chromosomes, the method determined the presence of chromosome 9 trisomy 1 in one of the test samples. For the chromosome 9 of interest, a sample with NCV of 14.4 was identified using a systematically determined normalized chromosomal sequence (i.e., a group of chromosomes 3 + 4 + 8 + 10 + 17 + 19 + 20 + 22) . This sample corresponded to the test sample of Example 6 which was considered to be impervious to chromosome 9 after an abnormally low dose calculation for chromosome 21 (for this, chromosome 9 was used as the standardized chromosome sequence in Example 6).

The data show that 100% of the samples with clinical karyotypes representing T21, T13 T18, T9 and X monochromes were correctly identified. Figure 19 shows a plot of NCV for each of chromosomes 1-22 in each of the 47 test samples. The median value of NCV was normalized to zero. The data indicate that the method of the present invention (including the use of systematically determined normalized chromosomal sequences) determined the presence of chromosomalities of all 5 types present in this test set with 100% sensitivity and 100% specificity, The method clearly demonstrates that any arbitrary sample can identify any complete chromosomal anomaly for any one of chromosome 1-22, X,

Example  8

Determination of the presence or absence of partial fetal chromosomal aberrations: Cat  eye syndrome ) Determination

DiGeorge syndrome (22q11.2 deletion syndrome), a disease caused by a defect in chromosome 22, causes insufficient development of several body systems. Medical problems commonly associated with DiJoorge syndrome include heart defects, insufficient immune system function, cleft palate, insufficient function of the parathyroid glands, and behavioral disorders. The number and severity of problems associated with DiJoorge syndrome varies greatly. Nearly everyone with DiJozzi syndrome needs specialist treatment in a variety of areas.

To determine the presence or absence of partial deletion of fetal chromosome 22, a blood sample is obtained by venipuncture to the host and cfDNA is prepared as described in the above examples. The purified cfDNA was ligated to the adapter and cluster amplified using the Illumina cBot cluster station. Massively parallel sequencing using a reversible dye terminator generates millions of 36bp leads. The sequence leads are aligned with respect to the human hgl9 reference dielectric and the unique mapped leads to the reference dielectric are counted as a tag.

A set of qualifying samples known to be diploid for chromosome 22, that is, a set of qualifying samples known to exist only in a diploid state of chromosome 22 or any portion thereof, is first sequenced and analyzed to generate, for each of the 1000 segments of the 3 megabase (Mb) Multiple sequence tags were obtained (except region 22q11.2). Assuming that the human genome contains about 3 billion bases (3Gb), 1000 segments of 3 Mb each roughly comprise the remainder of the genome. Each of the 1000 segments may function individually or as a group of segment sequences used to determine the normalized segment sequence for the 3Mb region of interest, i.e. 22q11.2. The segment capacity for the 3Mb region of 22q11.2 is calculated using the number of mapped sequence tags individually for all single 1000bp segments. In addition, all possible combinations of two or more segments are used to determine the segment capacity for the segment of interest in all eligible samples. A single 3Mb segment or a combination of two or more 3Mb segments generating the segment capacity with the smallest variability across the sample is selected as the standardized segment sequence.

The segment capacity is determined in each eligible sample using the number of sequence tags mapped for the segment of interest in each eligible sample. The mean and standard deviation of the segment capacity in all eligible samples are calculated and used to set a threshold at which the segment capacity determined in the test sample can be compared. Preferably, a normalized segment value (NSV) is calculated for all segments of interest in all eligible samples, and the threshold is set using this.

Subsequently, the number of tags mapped to the normalized segment sequence in the corresponding test sample is used to determine the capacity of the segment of interest in the test sample. The normalized segment value (NSV) is calculated for the segment in the test sample as described above and the NCV of the segment of interest of the test sample is compared to the threshold determined using the qualifying sample to determine the presence or absence of a deletion of 22q11.2 And determines the absence.

The test NCV < -3 indicates that the loss in the segment of interest, i.e. the partial deletion (22q11.2) of chromosome 22, is present in the test sample.

Example  9

II A stool for predicting outcomes for primary colorectal cancer patients DNA  exam

Approximately 30% of all patients with stage II colon cancer will recur and die from the disease. Patients with stage II recurrence of the disease showed a significant loss of chromosome 4, 5, 15q, 17q and 18q. In particular, loss of 4q22.1-4q35.2 in patients with stage II colon cancer was associated with worse outcomes. Determination of the presence or absence of such genetic alterations can be helpful 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) to determine the presence or absence of one or more chromosomal deletions in the 4q22.1 to 4q35.2 region in a patient with stage II colorectal cancer. Fecal DNA was prepared according to the method described in Chen et al., J Natl Cancer Inst 97: 1124-1132 [2005]; Plasma DNA was prepared according to the method described in the above examples. DNA is sequenced according to the NGS method described herein and sequence information for the patient (s) sample (s) is used to calculate the segment capacity for one or more segments spanning the 4q22.1 to 4q35.2 regions. Segment capacity is determined using standardized segment sequences that are determined a priori in a set of eligible stools and / or plasma samples, respectively. By calculating the segment capacity in the test sample (patient sample) and comparing the NSV for each of the segments of interest with the set of thresholds from the NSV in the set of qualified samples, one or more partial chromosome deletions within the 4q22.1 to 4q35.2 region The presence or absence of the &lt; / RTI &gt;

While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be utilized in the practice of the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of such claims and their equivalents be included therein.

Claims (32)

CLAIMS What is claimed is: 1. A method for determining the presence or absence of any four or more different complete fetal chromosomal aneuploidies for each of four or more chromosomes of interest in a maternal test sample comprising fetal and maternal nucleic acids through a computer processing system,
(a) obtaining sequence information for said fetal and parent nucleic acid in said sample using next generation sequencing (NGS);
(b) confirming the presence of a plurality of sequence tags for each of the four or more interest chromosomes using the sequence information, and identifying a plurality of sequence tags for the standard chromosome for each of the four or more chromosomes Wherein the four or more chromosomes of interest are selected from chromosomes 1-22, X and Y, and each of the normalized chromosomes for each of the four or more chromosomes of interest has a normal number of copies of the chromosome of interest Wherein each of the normalized chromosomes for each of the four or more chromosomes of interest is identified using the sequence information of the qualifying sample obtained from a subject known to contain cells,
(i) exhibits variability in the number of sequence tags mapped to standardized chromosomes most similar to variability in the number of sequence tags mapped to the chromosome of interest,
(ii) the variation between the chromosomal capacity and the distribution of the capacity between the chromosomes of interest in the qualifying sample, and the distribution of the chromosomal capacity for the interest chromosome in the test sample Providing a large statistical difference,
(i) and (ii);
(c) using the number of sequence tags identified for each of the four or more interest chromosomes and the number of sequence tags identified for each of the standardization chromosomes to determine a single chromosome capacity for each of the four or more chromosomes of interest Wherein step (c) comprises comparing the single chromosomal capacity for each of said chromosomes of interest to the number of sequence tags identified for each of said chromosomes of interest and the sequence tag identified for said normalized chromosome sequence for each of said chromosomes of interest As a ratio of the number of pixels to the number of pixels; And
(d) determining the presence or absence of four or more complete different fetal chromosomalemias in the sample by comparing each of the single chromosome capacities for each of the four or more interest chromosomes with a threshold for each of the four or more chromosomes of interest Lt; / RTI &gt; The method according to claim 1,
(i) calculating a sequence tag density ratio for each of the chromosomes of interest by calculating the ratio of 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 of the normalized chromosomes by calculating a ratio of the number of sequence tags identified for the normalized chromosome sequence to the length of each of the normalized chromosomes in step (b) ; And
(iii) calculating a single chromosomal capacity for each of the chromosomes of interest using the sequence tag density ratio calculated in steps (i) and (ii), wherein the chromosomal dose is a sequence tag density ratio for each of the chromosomes of interest And calculating a ratio of the sequence tag density ratio to the normalized chromosome sequence for each of the interest chromosomes. The method of claim 1, wherein any four or more of the chromosomes of interest selected from chromosomes 1-22, X, and Y comprise at least 20 chromosomes selected from chromosomes 1-22, X, and Y, and at least 20 different complete embryos Wherein the presence or absence of chromosomal integrity is determined. The method of claim 1, wherein any four or more of the chromosomes of interest selected from chromosomes 1-22, X, and Y are all chromosomes 1-22, X, and Y and the complete fetus of all chromosomes 1-22, X, Wherein the presence or absence of chromosomal integrity is determined. The method according to claim 1, wherein the normalized chromosome sequence is a single chromosome selected from chromosomes 1-22, X, and Y. The method according to claim 1, wherein the normalized chromosome sequence is a group of chromosomes selected from chromosomes 1-22, X, and Y. CLAIMS What is claimed is: 1. A method for determining the presence or absence of any one or more different complete fetal chromosomal aberrations for one or more chromosomes of interest in a maternal test sample comprising fetal and maternal nucleic acids through a computer processing system,
(a) obtaining sequence information for said fetal and parent nucleic acid in said sample using next generation sequencing (NGS);
(b) identifying the presence of a plurality of sequence tags for each of the one or more chromosomes of interest using the sequence information, and identifying the presence of a plurality of sequence tags for the normalized segment sequence for each of the one or more chromosomes of interest Wherein said one or more interest chromosomes are selected from chromosomes 1-22, X and Y, and wherein each of said normalized segment sequences for each of said one or more chromosomes of interest comprises a cell known to contain cells having a normal number of copies of the chromosome of interest Wherein each of the normalized segment sequences for each of the one or more chromosomes of interest is identified using the sequence information of the qualifying sample obtained from the sample,
(i) exhibits variability in the number of sequence tags mapped to the most similar standardization segment sequence closest to the variability in the number of sequence tags mapped to the chromosome of interest,
(ii) the variation between the chromosomal capacity and the distribution of the capacity between the chromosomes of interest in the qualifying sample, and the distribution of the chromosomal capacity for the interest chromosome in the test sample Providing a large statistical difference,
(i) and (ii);
(c) calculating a single chromosome capacity for each of said one or more interest chromosomes using the number of said sequence tags identified for each of said one or more interest chromosomes and the number of said sequence tags identified for said normalization segment sequence Wherein step (c) comprises comparing the single chromosome capacity for each of said chromosomes of interest with the number of sequence tags identified for each of said chromosomes of interest and the number of sequence tags identified against said normalization segment sequence for each of said chromosomes of interest As a ratio of &lt; RTI ID = 0.0 &gt; And
(d) determining the presence or absence of one or more different complete fetal chromosomalemias in the sample by comparing each of the single chromosome capacities for each of the one or more interest chromosomes with a threshold for each of the one or more chromosomes of interest / RTI &gt; 9. The method of claim 7, wherein said at least one interest chromosome selected from chromosomes 1-22, X, and Y comprises at least 20 chromosomes selected from chromosomes 1-22, X, and Y and comprises at least 20 different complete chromosomes Wherein the presence or absence of the imperative is determined. 9. The method according to claim 7, wherein said one or more of the chromosomes of interest selected from chromosomes 1-22, X, and Y are all chromosomes 1-22, X, and Y and complete chromosomes 1-22, X, Wherein the presence or absence of the imperative is determined. The method according to any one of claims 1, 2 and 7, wherein said different complete chromosomal aberrations are selected from complete chromosomal trisomes, complete chromosomal single chromosomes and complete chromosomes. The method of any one of claims 1, 2, and 7, wherein said different complete fetal chromosomal aberrations are selected from the group consisting of trisomy 2, trisomy 8, trisomy 9, trisomy 21, Wherein the method is selected from the group consisting of trisomy 3, trisomy 16, trisomy 18, trisomy 22, 47, XXY, 47, XXX, 47, XYY, and X chromosomes. A method according to any one of claims 1, 2, and 7, wherein steps (a) - (d) are repeated for test samples from different parental subject, And determining the presence or absence of different complete fetal chromosomal integrity. The method according to any one of claims 1, 2 and 7, further comprising calculating a normalized chromosome value (NCV), wherein the NCV is capable of measuring the chromosomal capacity in a set of qualifying samples Methods to relate to the average of chromosome capacity:

In this formula,

And

Is the respective estimated mean and standard deviation for the j &lt; th &gt; chromosome dose in the set of eligible samples,

Is the jth chromosome capacity observed for test sample i. CLAIMS What is claimed is: 1. A method for determining the presence or absence of different partial fetal chromosomal anomalies for one or more segments of one or more chromosomes of interest in a maternal test sample comprising fetal and maternal nucleic acids through a computer processing system,
(a) obtaining sequence information for said fetal and parent nucleic acid in said sample using next generation sequencing (NGS);
(b) identifying the presence of a plurality of sequence tags for each of the one or more segments of any one or more of the interest chromosomes using the sequence information, and identifying the presence of a plurality of sequence tags for each of the one or more segments of interest Wherein the one or more segments are selected from chromosomes 1-22, X and Y, and wherein the normalized segment sequence for each of the one or more segments of the one or more chromosomes of interest is a chromosome of interest Wherein the normalized segment sequence for each of the one or more segments of the one or more chromosomes of interest is identified using the sequence information of the eligible sample obtained from a subject known to contain cells having an average number of copies of
(i) exhibits variability in the number of sequence tags mapped to the most similar standardized segment sequence closest to the variability in the number of sequence tags mapped for one or more segments of one or more chromosomes of interest,
(ii) a distribution of chromosomal capacity for one or more segments of one or more of the interest chromosomes in the qualifying sample and a distribution of chromosomal capacity for one or more of the interest chromosomes in the test sample, Providing the largest statistical difference between distributions of chromosome capacity for one or more segments,
(i) and (ii);
(c) using the number of sequence tags identified for each of the at least one segment of any one or more of the interest chromosomes of interest and the number of sequence tags identified for the normalized segment sequence, Wherein step (c) comprises calculating a single segment capacity for each of the one or more segments of any one or more of the interest chromosomes by comparing a single segment capacity for each of the one or more segments of interest with any one As the ratio of the number of identified sequence tags for each of the above segments and the number of sequence tags identified for the normalized segment sequence for each of the one or more segments of any one or more of the interest chromosomes of interest; And
(d) comparing each of the single segment capacities for each of the one or more segments of any one or more of the interest chromosomes with a threshold for each of the one or more chromosome segments of interest on any one or more of the interest chromosomes, Determining the presence or absence of different partial fetal chromosomal integrity. 15. The method of claim 14, further comprising calculating a normalized segment value (NSV), wherein the NSV relates the segment capacity to an average of the corresponding segment capacity in a set of qualifying samples as follows:

In this formula,

And

Is the respective estimated mean and standard deviation for the jth segment capacity in the set of eligible samples,

Is the jth segment capacity observed for test sample i. 15. The method according to any one of claims 7 to 14, wherein the normalized segment sequence is a single segment of any one or more of chromosomes 1-22, X, and Y. 15. The method according to any one of claims 7 to 14, wherein the normalized segment sequence is a group of one or more segments of chromosomes 1-22, X, and Y. 15. The method of claim 14, wherein said different partial fetal chromosomal integrity is selected from partial redundancy, partial increase, partial insertion and partial deletion. 15. The method according to claim 14, wherein the partial fetal anemia is selected from the group consisting of a partial heterozygote of chromosome 1, a partial heterozygote of chromosome 4, a partial heterozygote of chromosome 5, a partial heterozygote of chromosome 7, A partial single chromosome of chromosome 15, a partial single chromosome of chromosome 17, a partial single chromosome of chromosome 18, and a partial chromosome of chromosome 22. 15. The method of claim 14, wherein steps (a) - (d) are repeated for test samples from different maternal subjects, and the method comprises determining the presence or absence of different partial fetal chromosomal integrity in each of said samples How to. 15. The method according to any one of claims 1, 7, and 14, wherein step (a) comprises sequencing at least a portion of the nucleic acid in the test sample to identify the sequence information for the fetal and parent nucleic acid molecule &Lt; / RTI &gt; 15. The method of any one of claims 1, 7, and 14, wherein the test sample is a maternal sample selected from blood, plasma, serum, urine, and saliva samples. 15. The method according to any one of claims 1, 7 and 14, wherein the nucleic acid is a mixture of fetal and maternal cell-free DNA molecules. 15. The method according to any one of claims 1, 7 and 14, wherein the sequence information is obtained by massively parallel sequencing using sequencing-by-synthesis with a reversible dye terminator. 15. The method according to any one of claims 1, 7 and 14, wherein the sequence information is obtained by sequencing-by-ligation. 15. The method according to any one of claims 1, 7 and 14, wherein obtaining the sequence information comprises amplification. 15. The method according to any one of claims 1, 7 and 14, wherein the sequence information is obtained by single molecule sequencing. CLAIMS What is claimed is: 1. A method for determining the presence or absence of any more than 20 different complete fetal chromosomal aberrations for each of more than 20 interest chromosomes in a maternal plasma test sample comprising a mixture of DNA molecules free of fetal and maternal cells through a computer processing system, The method comprises:
(a) sequencing at least a portion of said cell-free DNA molecules using next generation sequencing (NGS) to obtain sequence information for said fetal and maternal cell-free DNA molecules in said sample;
(b) confirming the presence of a plurality of sequence tags for each of the 20 or more interest chromosomes using the sequence information, and confirming the presence of a plurality of sequence tags for the standardized chromosome for each of the 20 or more chromosomes of interest Wherein the 20 or more chromosomes of interest are selected from chromosomes 1-22, X, and Y, and each of the normalized chromosomes for each of the 20 or more chromosomes of interest comprises a cell having a normal number of copies of the chromosome of interest , And each of the normalized chromosomes for each of the above 20 or more interest chromosomes is identified by using the sequence information of the eligible sample obtained from the subject known to be &
(i) exhibits variability in the number of sequence tags mapped to standardized chromosomes most similar to variability in the number of sequence tags mapped to the chromosome of interest,
(ii) the variation between the chromosomal capacity and the distribution of the capacity between the chromosomes of interest in the qualifying sample, and the distribution of the chromosomal capacity for the interest chromosome in the test sample Providing a large statistical difference,
(i) and (ii);
(c) determining a single chromosome capacity for each of the above 20 or more chromosomes of interest using the number of sequence tags identified for each of the 20 or more chromosomes of interest and the number of sequence tags identified for each of the standardization chromosomes Wherein step (c) comprises comparing the single chromosomal capacity for each of said chromosomes of interest with the number of sequence tags identified for each of said chromosomes of interest and the sequence tag identified for said normalized chromosome sequence for each of said chromosomes of interest Calculating the ratio of the number to the number; And
(d) determining the presence or absence of at least 20 different complete fetal chromosomalemias in the sample by comparing each of the single chromosome capacities for each of the at least 20 interest chromosomes with a threshold for each of the at least 20 interest chromosomes Lt; / RTI &gt; The method of claim 1, wherein the four or more chromosomes of interest are chromosomes 13,18, 21 and X,
(i) the normalized chromosome for chromosome 13 is at least one of chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8;
(ii) the normalized chromosome for chromosome 18 is at least one of chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12, and chromosome 14;
(iii) the normalized chromosome for chromosome 21 is at least one of chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14;
(iv) said normalized chromosome for chromosome X is at least one of chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8. 8. The method of claim 7, wherein the chromosomes of interest are chromosomes 13, 18, 21, and X,
(i) said normalized segment sequence for chromosome 13 is selected from at least one of chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8;
(ii) said normalized segment sequence for chromosome 18 is selected from at least one of chromosome 8, chromosome 2, chromosome 3, chromosome 5, chromosome 6, chromosome 12 and chromosome 14;
(iii) said normalized segment sequence for chromosome 21 is selected from at least one of chromosome 9, chromosome 1, chromosome 2, chromosome 11, chromosome 12, and chromosome 14;
(iv) said standardized segment sequence for chromosome X is selected from at least one of chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, and chromosome 8. delete delete

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