KR101686146B1 - Copy Number Variation Determination Method Using Sample comprising Nucleic Acid Mixture - Google Patents
Copy Number Variation Determination Method Using Sample comprising Nucleic Acid Mixture Download PDFInfo
- Publication number
- KR101686146B1 KR101686146B1 KR1020150172212A KR20150172212A KR101686146B1 KR 101686146 B1 KR101686146 B1 KR 101686146B1 KR 1020150172212 A KR1020150172212 A KR 1020150172212A KR 20150172212 A KR20150172212 A KR 20150172212A KR 101686146 B1 KR101686146 B1 KR 101686146B1
- Authority
- KR
- South Korea
- Prior art keywords
- score
- chromosome
- chromosomes
- reference value
- sequence information
- Prior art date
Links
Images
Classifications
-
- G06F19/18—
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- G06F19/22—
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a method for determining the number of replica variants in a mixture of nucleic acids that are known to be different or different in the amount of one or more sequences of interest, A bioinformatic analysis method, and a statistical analysis method.
The mutation determination method according to the present invention can be used to determine the number of chromosome copy number variations that are related to or related to a medical condition of a fetus. The chromosomal copy number variation that can be determined according to the method of the present invention may be any one or more of the chromosomes 1-22, any one or more of the trisomes and the halo chromosomes of X and Y, the chromosome of the entire nucleic acid sequence, And / or redundancy of the fetus, which is useful for analyzing the gender and replica number variation of the fetus.
Description
More particularly, the present invention relates to a method for detecting sex and a copy number abnormality of a fetus, more specifically, extracting DNA from a biological sample of a mother, obtaining sequence information, and then performing normalization correction of the chromosomal region and randomizing the reference chromosome The present invention relates to a non-invasive fetal chromosome abnormality detection method using a method for detecting a fetal chromosome abnormality.
The prenatal diagnosis of fetal chromosomal abnormalities includes ultrasound, blood markers, amniocentesis, chorioamnion, and transcutaneous umbilical cord blood (Malone FD, et al., 2005; Mujezinovic F, et al. Ultrasonography and blood markers are classified as screening, and amniocentesis is classified as confirmed. Non-invasive methods, such as ultrasound and blood marker testing, are safe because they do not take direct samples of the fetus, but the sensitivity of the test drops below 80% (ACOG Committee on Practice Bulletins. The invasive amniocentesis, chorioamnion, and transcutaneous umbilical cord blood tests can confirm the fetal chromosomal abnormality, but there is a disadvantage that there is a possibility of fetal loss due to invasive medical treatment (Mujezinovic F, et al. 2007). In 1997, Lo et al. (1997) have used the fetal genetic material in maternal plasma and serum for the prenatal screening of fetal genetic material as a result of Y chromosome sequencing analysis (Lo YM, et al. The fetal genetic material in maternal blood is a part of the trophoblast cells that underwent apoptosis during the placental remodeling process and enters the maternal blood through the mass exchange mechanism. It is actually derived from the placenta and is called cff DNA (cell-free fetal DNA) do. cff DNA is found in most maternal blood from the 18th day of embryo transfer as early as the 37th day (Guibert J, et al. 2003). Since cff DNA has a short strand of 300 bp or less and exists in a small amount in maternal blood, a large-scale parallel base analysis technique using a next-generation nucleotide sequence analyzer (NGS) is used to detect it in fetal chromosome aberration detection. Non-invasive fetal chromosomal anomaly detection using large-scale parallel base analysis technology has detection sensitivity of 90-99% or more depending on the chromosome, but false-positive and false-negative results are 1-10% (Gil MM, et al., 2015).
The present inventors have made efforts to develop a method for detecting a fetal chromosomal abnormality with high sensitivity, low false positive and false negative results, and as a result, when performing normalization and chromosomal assignment of a fetal chromosome region randomly, High sensitivity and low false positive / false negative results can be obtained, and the present invention has been completed.
It is an object of the present invention to provide a method for non-invasively detecting gender and replica number abnormalities of a fetus.
It is another object of the present invention to provide an apparatus for non-invasively detecting gender and replica number abnormalities of a fetus.
It is yet another object of the present invention to provide a computer readable medium comprising instructions that are configured to be executed by a processor that detects gender and replica number anomalies of the fetus in the manner described above.
In order to accomplish the above object, the present invention provides a method for obtaining a DNA sample, comprising the steps of: a) extracting DNA from a biological sample of a mother to obtain sequence information; b) aligning the obtained sequence information to a reference genome database; c) calculating a Q-score with respect to sorted sequence information, and selecting only sequence information having a cut-off value or less; And d) gender and cloning of the fetus, including calculating the G-score for the selected sequence information and determining the gender and replica number of the fetus as compared to the reference chromosomal combination Thereby providing a method for detecting a number abnormality.
The present invention also provides a method for detecting a DNA fragment, comprising: a decoding unit for extracting DNA from a biological sample of a mother to decode sequence information; An alignment unit for aligning the decoded sequence to a standard chromosome sequence database; A quality control unit for calculating a Q-score with respect to sorted sequence information reads and selecting only sequence information having a cut-off value or less; (G-score) for the selected sequence information and comparing the sex and the number of copies of the fetus with the reference chromosome combination, Thereby providing a duplication number abnormality detecting apparatus.
The present invention also provides a computer readable medium comprising instructions that are configured to be executed by a processor that detects a gender and a number of copies of a fetus, the method comprising: a) extracting DNA from a biological sample of a mother, Obtaining; b) aligning the obtained sequence information to a reference genome database; c) calculating a Q-score for the ordered sequence information reads, and selecting only sequence information that is less than or equal to a cut-off value; And d) calculating the G-score for the selected sequence information and comparing the gender and replica number of the fetus with the reference chromosome combination to determine the gender and replica of the fetus The instructions being configured to be executed by a processor that detects a number or more.
The method of distinguishing sex and chromosome duplication number of fetus according to the present invention not only improves the accuracy of gender discrimination by using Next Generation Sequencing (NGS), but also detects sex chromosomes such as XO, XXX, and XXY It is possible to increase the detection accuracy and to increase the commercial utilization. Therefore, the method of the present invention is useful for prenatal diagnosis which can determine early abnormality due to abnormality of fetal sex chromosomes.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a whole flow chart for detecting the gender and the number of copies of the embryo of the present invention. Fig.
FIG. 2 is a diagram illustrating the correction results before and after the GC correction by the LOESS algorithm during the QC process of the read data.
FIG. 3 is a diagram illustrating the correction results before and after the correction of the Coefficient of Variation (CV) value by the LOESS algorithm during the QC process of the read data.
FIG. 4 is a schematic diagram comparing the G-score values calculated in the normal group with the chromosomal abnormal group according to the method of the present invention.
Unless otherwise defined, 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. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.
In the present invention, the sequence analysis data obtained from the sample is normalized, and based on the reference value, the combination of the reference chromosomes is randomly permutated to determine the G-score difference Of the total number of chromosomes of the reference chromosomes were compared with each other to obtain a high sensitivity and low false positive / false negative.
That is, in one embodiment of the present invention, the DNA extracted from maternal blood is sequenced, the quality is managed using the LOESS algorithm, the G-score is calculated, and then the normal population and the subject chromosome The reference chromosome combinations are randomly assigned until the absolute value of the G-score difference satisfies the maximum value. Based on the determination, the reference value of the G-score is determined. A method of determining that the number of copies of the subject chromosome is abnormal is developed (Fig. 1)
Thus, the present invention, in one aspect,
a) extracting DNA from a biological sample of a mother to obtain sequence information;
b) aligning the obtained sequence information to a reference genome database;
c) calculating a Q-score for the sorted sequence information and selecting only sequence information having a cut-off value or less; And
d) calculating the G-score for the selected sequence information and comparing the sex chromosomal combination with the reference chromosome combination to determine the gender and replica number variation of the fetus; And more particularly to a method for detecting a number abnormality.
In the present invention, when the selected sequence information is chromosome 13, the combination of reference chromosomes is not limited thereto, but may be chromosomes 4 and 6. If the selected sequence information is chromosome 18, The
In the present invention,
The step a)
(i) The mixture of fetal and maternal nucleic acids is amniotic fluid obtained by amniocentesis, villus obtained by chorionic villi sampling, percutaneous umbilical blood sampling In umbilical cord blood, spontaneous miscarrying fetus tissue, or human peripheral blood, obtained by the method of the present invention;
(ii) removing proteins, fats, and other residues using a salting-out method, a column chromatography method, and a beads method in a collected fetal and parent nucleic acid mixture to obtain a purified nucleic acid;
(iii) producing a single-end sequencing or pair-end sequencing library for purified nucleic acids or nucleic acids that have been enzymatically cleaved, disrupted, or randomly fragmented by the hydroshear method;
(iv) reacting the constructed library to a next-generation sequencer; And
(v) acquiring the sequence information of the nucleic acid in a next-generation gene sequence checker.
In the present invention, the next-generation sequencer includes, but is not limited to, the Hiseq system of Illuminator Company, the Miseq system of Illuminator Company, the genome of Illuminator Co., Analyzer (GA) system, 454 FLX from Roche Company, SOLiD system from Applied Biosystems Company, LifeTechnology Company's ion torrent system.
In the present invention, the alignment step may be performed using the BWA algorithm and the GRch38 sequence, but not limited thereto.
In the present invention, the step c)
(i) identifying regions of each aligned nucleic acid sequence;
(ii) specifying a sequence satisfying a mapping quality score and a reference value of the GC ratio;
(iii) calculating the fraction of chromosome N (ChrN) of any of the above identified sequences in Case 1 by the following formula 1;
Equation 1:
(i) calculating the Z-score of the region of chromosome N by the following equation 2;
Equation 2:
(ii) The standard deviation of the Z-score for the remaining chromosomal regions except for the Z-score of the region corresponding to chromosome 13, 18, and 21 in any case 1 is referred to as Q-score ); And
(iii) determining a reference value of the Q-score, determining that the calculated Q-score value exceeds the reference value, and reproducing the sequence information of the corresponding sample;
And a step of determining whether or not the image is displayed.
In the present invention, in the step of specifying the region of the nucleic acid sequence of the step (i), the region of the nucleic acid sequence may be 20 kb to 1 MB, though not limited thereto.
In the present invention, the mapping quality score of step (ii) may vary depending on the desired criteria, but may be preferably 15-70, more preferably 50-70, most preferably It can be 60 points.
In the present invention, the GC ratio in the step (ii) may be varied depending on a desired standard, but is preferably 20 to 70%, and most preferably 30 to 60%.
In the present invention, the reference value of the step (vi) may be 4, preferably 3, and most preferably 2.
In the present invention, the case group refers to a sample for detecting sex and chromosome duplication number of a fetus, and the reference group means a group of reference chromosomes that can be compared, such as, but not limited to, a standard chromosome sequence database .
In the present invention, it is preferable that the step (d)
(i) randomly selecting reference chromosomes from chromosomes 1 to 22;
(ii) calculating a fraction value of an arbitrary chromosome N by the following equation 3;
Equation 3:
(iii) calculating the G-score of the chromosome N of the arbitrary case 1 by the following equation 4;
Equation 4:
(iv) repeating the above steps (i) to (iii) to select a chromosome combination that maximizes the G-score difference between the normal and abnormal groups; And
(v) The G-score is calculated using the chromosomal combination obtained in the above step (iv). If the calculated G-score is less than the reference value, the copy number is determined to be decreased. ;
And a step of determining whether or not the image is displayed.
In the present invention, the step (iv) may be repeatedly performed 100 times or more, preferably 1,000 times or more, and most preferably 100,000 times or more.
In the present invention, the reference value of the G-score in the step (v) may be any value as long as it is calculated from a normal chromosome, but is preferably -2 or 2, and most preferably -3 or 3 .
In the present invention, the step of determining the gender of the fetus in the step (d)
(i) the step (i) to (iv) of determining the number of copies above is performed in a reference group of a mother with 46, XX or 46, XY of the fetal karyotype to determine a G-score reference value for the X and Y chromosomes ; And (ii) comparing the G-score for the X and Y chromosomes in any case with the reference value to determine sex.
In the present invention, the G-score reference value for the X and Y chromosomes is not limited thereto, but may be -2 or 2, and most preferably -3 or 3. The G- The X chromosome is determined to be three or more if the score is less than or equal to the reference value and the Y chromosome is determined to be one or more if the G-score for the Y chromosome is equal to or greater than the reference value .
In the present invention, when the number of the Y chromosome is one or more, the fetal fraction of the X chromosome is calculated by the
Equation 5:
Equation 6:
Equation 7:
According to another aspect of the present invention, there is provided a method for screening a biological sample, comprising: a decoding unit for extracting DNA from a biological sample of a mother to decode sequence information; An alignment unit for aligning the decoded sequence to a standard chromosome sequence database; A quality control unit for calculating a Q-score with respect to sorted sequence information reads and selecting only sequence information having a cut-off value or less; (G-score) for selected sequence information (reads), and comparing the sex and the number of copies of the fetus with that of the reference chromosome, Sex and a copy number abnormality detecting apparatus.
In the present invention, when the selected sequence information is chromosome 13, the combination of reference chromosomes is not limited thereto, but may be chromosomes 4 and 6. If the selected sequence information is chromosome 18, The
In the present invention, the above-described detoxification unit is preferably constructed such that (i) the fetal and maternal nucleic acid mixture is amniotic fluid obtained by amniocentesis, villus obtained by chorionic villi sampling, A sample collection obtained from umbilical cord blood obtained by percutaneous umbilical blood sampling, spontaneous miscarrying fetus tissue or human peripheral blood; (ii) a nucleic acid obtainable by removing the proteins, fats, and other residues using a salting-out method, a column chromatography method, or a beads method in a collected fetal and parent nucleic acid mixture, and obtaining a purified nucleic acid; (iii) a library production section for producing single-end sequencing or pair-end sequencing libraries for purified nucleic acids or nucleic acids that have been enzymatically cleaved, disrupted, and randomly fragmented by the hydroshear method; (iv) a next-generation gene sequencer that reacts the constructed library to a next-generation sequencer; And (v) a sequence information acquiring unit for acquiring sequence information of a nucleic acid in a next generation gene sequence checker.
In the present invention, the next-generation sequencer includes, but is not limited to, the Hiseq system of Illuminator Company, the Miseq system of Illuminator Company, the genome of Illuminator Co., Analyzer (GA) system, 454 FLX from Roche Company, SOLiD system from Applied Biosystems Company, LifeTechnology Company's ion torrent system.
In the present invention, the alignment unit may be performed using the BWA algorithm and the GRch38 sequence, but not limited thereto.
In the present invention, the quality management unit
(i) a region specifying unit specifying an area of each aligned nucleic acid sequence;
(ii) a sequence specifying unit which specifies a sequence satisfying a mapping quality score and a reference value of the GC ratio;
(iii) a chromosomal fraction calculating unit for calculating a fraction of the chromosome N in any of the above-identified sequences in Case 1 by the following equation 1;
Equation 1:
(iv) a Z-score calculating unit for calculating a Z score of a chromosome N region by the following equation 2; Equation 2:
(iv) The standard deviation of the Z-score for the remaining chromosomal regions except the Z-score for the chromosome 13, 18, (Q-score) calculating unit for calculating a Q-score; And
(v) determining a reference value of the Q-score, determining that the calculated Q-score value is below the reference value, and reproducing the sequence information of the corresponding sample;
And a control unit.
In the present invention, in the region specifying section, the region of the nucleic acid sequence may be 20 kb to 1 MB, though not limited thereto.
In the present invention, the mapping quality score of the sequence specifying unit may vary depending on a desired criterion, but may be preferably 15-70 points, and most preferably 60 points.
In the present invention, the GC ratio of the sequence specifying unit may be varied depending on the desired standard, but is preferably 20 to 70%, and most preferably 30 to 60%.
In the present invention, the reference value of the quality organizing unit may be 4, preferably 3, and most preferably 2.
In the present invention, the case group refers to a sample for detecting sex and chromosome duplication number of a fetus, and the reference group means a group of reference chromosomes that can be compared, such as, but not limited to, a standard chromosome sequence database .
In the present invention, the copy number variation determining unit for determining the number of copies and the number of copies of the copy number variation determining unit includes: (i) a random permutation unit for randomly selecting reference chromosomes from chromosome 1 to 22; (ii) a chromosomal fraction calculating unit for calculating a fraction value of an arbitrary chromosome N by the following equation 3;
Equation 3:
(iii) a G-score calculation unit for calculating the G-score of the chromosome N of the arbitrary case 1 by the following equation (4);
Equation 4:
(iv) a reference chromosome combination selection unit for selecting a chromosome combination that maximizes the G-score difference between the normal and abnormal groups by repeating the above (i) to (iii) apparatus; And (v) a G-score is calculated using a combination of reference chromosomes selected from the reference chromosome combination selection unit. If the calculated G-score value is less than the reference value, the number of copies is determined to be decreased. And a copy number variation determining unit that determines the number of copies to be increased.
In the present invention, the number of iterations of the optimum reference chromosome combination G-score calculation unit may be 100 or more, preferably 1,000 or more, and most preferably 100,000 or more.
In the present invention, the reference value of the G-score of the copy number variation determining unit may be any value as long as the reference value is a value calculated by a normal chromosome, but is preferably -2 or 2, and most preferably -3 or 3 .
In the present invention, the gender determination unit of the fetus in the gender and the copy number variation determining unit may be configured such that (i) the apparatus of (i) to (iv) of the copy number variation determining unit for determining the number of copies, 46, and XY to obtain a G-score reference value for the X and Y chromosomes; And (ii) a G-score for the X and Y chromosomes in any case with the reference value to determine sex.
In the present invention, the G-score reference value for the X and Y chromosomes is not limited thereto, but may be -2 or 2, and most preferably -3 or 3. The G- The X chromosome is determined to be three or more if the score is less than or equal to the reference value and the Y chromosome is determined to be one or more if the G-score for the Y chromosome is equal to or greater than the reference value .
In the present invention, when the number of the Y chromosome is one or more, the fetal fraction of the X chromosome is calculated by the
Equation 5:
Equation 6:
Equation 7:
In another aspect, the present invention provides a computer readable medium comprising instructions that are configured to be executed by a processor that detects a sex and a number of copies of a fetus, comprising: a) extracting DNA from a biological sample of a mother, Obtaining; b) aligning the obtained sequence information to a reference genome database; c) calculating a Q-score for the ordered sequence information reads, and selecting only sequence information that is less than or equal to a cut-off value; And d) calculating the G-score for the selected sequence information and comparing the gender and replica number of the fetus with the reference chromosome combination to determine the gender and replica of the fetus And more particularly to a computer readable medium comprising instructions configured to be executed by a processor detecting a number or more.
Example
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
Example 1. Extract DNA from maternal blood and perform next-generation sequencing
A total of 358 pregnant mothers were collected in 10 ml of EDTA tubes and were centrifuged at 1200g, 4 ° C, and 15 minutes for 2 hours. 16000g at 4 캜 for 10 minutes to separate the supernatant of the plasma except for the precipitate. For the separated plasma, cell-free DNA was extracted using QIAamp Circulating Nucleic Acid Kit, and 2-4 ng of DNA was made into a library to generate sequence information data on NextSeq instrument.
Example 2. Quality control of sequence information data
The following series of procedures were performed before preprocessing the nucleotide sequence information mixed with the maternal-fetal genetic material and calculating the z-score. After converting the Bcl file (including nucleotide sequence information) generated by the Next Generation Sequence Analyzer (NGS) into the fastq format, the fastq file was aligned with the reference chromosome Hg19 sequence using the BWA-mem algorithm. There is a possibility of errors in the alignment of the library sequence, so we have performed three steps to correct the errors. First, the duplicated library sequence is removed. Then, the sequence having no mapping quality score of 60 aligned among the library sequences sorted by the BWA-mem algorithm is removed. Finally, regions having a Mappability value of 0.75 or less are removed , And the number of library sequences sorted by chromosome GC ratio was corrected using the LOESS algorithm. After a series of procedures, we have created a bed file that has been calibrated for alignment errors.
In order to manage the quality of sequencing error, the following series of processes were performed. First, we calculate the relative fraction of each chromosome. For example, the relative fraction of chromosome 1 can be expressed as:
After calculating the relative fractions for all chromosomes, the Z score of chromosome N in case 1 can be expressed as:
The standard deviation of the Z-score for the remaining chromosomal regions can be expressed as a Q-score, except for the Z-score for the regions corresponding to chromosomes 13, 18 and 21.
Therefore, when the standard deviation value of the z-score value distribution of Case 1 exceeds 2, it is determined as QC-fail (sequencing error) and re-experimentation and data reproduction are performed. As a result of the above QC process, And the distribution of the read is constant as shown in FIG.
Example 3. G-score calculation using permutation and fetal sex / Number of copies Abnormal decision
The following procedure was performed to calculate the G-score. First, we calculate the relative fraction of the chromosomes of interest. For example, the relative fractions of a particular chromosome can be expressed as:
The relative fraction of such specific chromosomes may be expressed by the following equation (3).
Equation 3:
For all chromosomes, the G-score of subject A can be expressed as follows.
This G-score may be expressed by the following equation (4).
Equation 4:
The absolute value of the G-score difference of the chromosome N of the normal person and the subject A was obtained and the reference chromosome combination in which the absolute value satisfied the maximum value was determined by proceeding with randomization. When the random assignment was gradually increased and the results were compared, a large number of random assignment analyzes showed an improvement of more than 50% as shown in Table 1.
The reference chromosome combinations can be changed by optimization for each analysis, and combinations that are detected more than 5 times in 10 operations to determine G-score of chromosome 13, 18, 21, X, Y are shown in Table 2 .
In order to determine whether the chromosomes of interest in the test sample are chromosomally insoluble, the G-score range of the normal group is calculated and set. When an outlier which is outside the maximum value and the minimum value range of the normal group G-score is found, It is determined that the copy number of the corresponding chromosome has been added. If it is smaller than the minimum value of the normal group G-score, it is determined that the copy number of the corresponding chromosome has been lost As a result of comparing the chromosome aberration group (Trisomy 21, Trisomy 18, Trisomy 13) with the normal group by the above method, it was confirmed that the G-score maximum value / minimum value of the chromosome aberration group and the normal group did not match ). As shown in Table 3, when the reference values of the G-score for the chromosomal aberration are 3 (Trisomy 21), 2.55 (Trisomy 18), and 3.5 (Trisomy 13) Sensitivity and specificity were detected, and the lower 95% confidence interval of the specificity was found to be over 98%.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (16)
b) aligning the obtained sequence information to a reference genome database;
c) The Q-score for the sorted sequence information is calculated by the method of ci) to c-vi), and only the sequence information that is less than the cut-off value of the Q-score is selected ;
(ci) specifying the region of each aligned nucleic acid sequence;
(c-ii) specifying a sequence satisfying a mapping quality score and a reference value of the GC ratio;
(c-iii) calculating a fraction of chromosome N in any of the above-identified sequences in Case 1 by the following equation 1;
Equation 1:
(c-iv) calculating the Z-score of the chromosome N region by the following equation 2;
Equation 2:
(cv) The standard deviation of the Z-score for the remaining chromosomal regions except for the Z-score of the region corresponding to chromosome 13, 18, ); And
(c-vi) determining a reference value of the Q-score, determining that the calculated Q-score value is below the reference value of the Q-score, and reproducing the sequence information reads of the sample; And
d) The G-score for the selected sequence information is calculated by the method of di) to d-vii) and compared with the reference chromosomal combination, the gender and the number of copies of the fetus are determined Step
(di) randomly selecting reference chromosomes from chromosome 1 to 22;
(d-ii) calculating a fraction value of an arbitrary chromosome N by the following equation 3;
Equation 3:
(d-iii) calculating the G-score of the chromosome N of the case 1 by the following equation (4);
Equation 4:
(d-iv) repeating the above steps (di) to (d-iii) to select a chromosome combination that maximizes the G-score difference between the normal and abnormal groups;
(dv) The G-score is calculated using the chromosomal combination obtained in the step (d-iv). If the calculated G-score is less than the reference value of the G-score, Determining if the number of replications is increased;
(d-vi) The above (di) to (d-iv) steps are carried out in a reference group of a mother whose fetal karyotype is 46, XX or 46, XY to obtain a reference value of the G- score for the X and Y chromosomes step; And
(d-vii) determining the sex by comparing the G-score for the X and Y chromosomes in any case with the G-score reference value for the step d-vi);
Of fetal sex and the number of replica abnormalities
(i) The mixture of fetal and maternal nucleic acids is amniotic fluid obtained by amniocentesis, villus obtained by chorionic villi sampling, percutaneous umbilical blood sampling In umbilical cord blood, spontaneous miscarrying fetus tissue, or human peripheral blood, obtained by the method of the present invention;
(ii) removing proteins, fats, and other residues using a salting-out method, a column chromatography method, and a beads method in a collected fetal and parent nucleic acid mixture to obtain a purified nucleic acid;
(iii) producing a single-end sequencing or pair-end sequencing library for purified nucleic acids or nucleic acids that have been enzymatically cleaved, disrupted, or randomly fragmented by the hydroshear method;
(iv) reacting the constructed library to a next-generation sequencer; And
(v) obtaining the sequence information of the nucleic acid from a next-generation gene sequencer.
Equation 5:
Equation 6:
Equation 7:
An alignment unit for aligning the decoded sequence to a standard chromosome sequence database;
A quality control unit for calculating Q-score with respect to sorted sequence information by the following methods (i) to (vi), and selecting only sequence information of samples less than a cut-off value;
(i) identifying regions of each aligned nucleic acid sequence;
(ii) specifying a sequence satisfying a mapping quality score and a reference value of the GC ratio;
(iii) calculating a fraction of chromosome N in any of the above-identified sequences in Case 1 by the following equation 1;
Equation 1:
(iv) calculating the Z-score of the chromosome N region by the following formula 2;
Equation 2:
(v) The standard deviation of the Z-score for the remaining chromosomal regions except for the Z-score of the region corresponding to the chromosome 13, 18, and 21 in any case 1 is referred to as Q-score ); And
(vi) determining a reference value of the Q-score, determining that the calculated Q-score value is below the reference value of the Q-score, and reproducing the sequence information of the sample; And
G-score for selected sequence reads is calculated by the method of a) to g) and compared to the reference chromosome combination, gender and sex to determine the fetal sex and the number of replicates The number-
(a) randomly selecting reference chromosomes from chromosome 1 to 22;
(b) calculating a fraction value of an arbitrary chromosome N by the following equation (3);
Equation 3:
(c) calculating the G-score of the chromosome N in the case 1 by the following equation 4;
Equation 4:
(d) repeating the steps (a) to (c) to select a chromosome combination that maximizes the G-score difference between the normal and abnormal groups;
(e) The G-score is calculated using the chromosomal combination obtained in the step (d). If the calculated G-score is less than the reference value of the G-score, determining the number of copies to be increased in the case of chrhk;
(f) performing the steps (a) to (d) in a reference group of a mother having a fetal karyotype of 46, XX or 46, XY to obtain a G-score reference value for the X and Y chromosomes; And
(g) comparing the G-score for the X and Y chromosomes in any case with the G-score reference value in step (f) to determine gender;
Fetus gender and a duplication number abnormality detecting device
a) extracting DNA from a biological sample of a mother to obtain sequence information;
b) aligning the obtained sequence information to a reference genome database;
c) The Q-score for the sorted sequence information is calculated by the following methods ci) to c-vi), and only the sequence information having a cut-off value less than the Q-score is selected ;
(ci) specifying the region of each aligned nucleic acid sequence;
(c-ii) specifying a sequence satisfying a mapping quality score and a reference value of the GC ratio;
(c-iii) calculating a fraction of chromosome N in any of the above-identified sequences in Case 1 by the following equation 1;
Equation 1:
(c-iv) calculating the Z-score of the chromosome N region by the following equation 2;
Equation 2:
(cv) The standard deviation of the Z-score for the remaining chromosomal regions except for the Z-score of the region corresponding to chromosome 13, 18, ); And
(c-vi) determining a reference value of the Q-score, determining that the calculated Q-score value is below the reference value of the Q-score, and reproducing the sequence information reads of the sample; And
d) The G-score for the selected sequence information is calculated by the method of di) to d-vii) and compared with the reference chromosomal combination, the gender and the number of copies of the fetus are determined A processor for detecting the gender and the number of copies of the fetus,
(di) randomly selecting reference chromosomes from chromosome 1 to 22;
(d-ii) calculating a fraction value of an arbitrary chromosome N by the following equation 3;
Equation 3:
(d-iii) calculating the G-score of the chromosome N of the case 1 by the following equation (4);
Equation 4:
(d-iv) repeating the above steps (di) to (d-iii) to select a chromosome combination that maximizes the G-score difference between the normal and abnormal groups;
(dv) The G-score is calculated using the chromosomal combination obtained in the step (d-iv). If the calculated G-score is less than the reference value of the G-score, Determining that the number of replicas is increased when the reference value exceeds the reference value of the number of replicas;
(d-vi) obtaining the G-score reference value for the X and Y chromosomes by performing the steps (di) to (d-iv) in a reference group of a mother whose fetal karyotype is 46, XX or 46, XY ; And
(d-vii) comparing the G-score for the X and Y chromosomes in any case with the G-score reference value for the step d-vi) to determine the gender;
Wherein the instructions are configured to be executed by a computer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150172212A KR101686146B1 (en) | 2015-12-04 | 2015-12-04 | Copy Number Variation Determination Method Using Sample comprising Nucleic Acid Mixture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150172212A KR101686146B1 (en) | 2015-12-04 | 2015-12-04 | Copy Number Variation Determination Method Using Sample comprising Nucleic Acid Mixture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR101686146B1 true KR101686146B1 (en) | 2016-12-13 |
Family
ID=57575050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020150172212A KR101686146B1 (en) | 2015-12-04 | 2015-12-04 | Copy Number Variation Determination Method Using Sample comprising Nucleic Acid Mixture |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101686146B1 (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106709276A (en) * | 2017-01-21 | 2017-05-24 | 深圳昆腾生物信息有限公司 | Genovariation cause analysis method and system |
| KR20180098438A (en) * | 2017-02-24 | 2018-09-04 | 에스디지노믹스 주식회사 | Priority calculation method for copy number variation candidate |
| WO2020022733A1 (en) * | 2018-07-27 | 2020-01-30 | 주식회사 녹십자지놈 | Whole genome sequencing-based chromosomal abnormality detection method and use thereof |
| WO2021034034A1 (en) | 2019-08-19 | 2021-02-25 | 주식회사 녹십자지놈 | Method for detecting chromosomal abnormality by using information about distance between nucleic acid fragments |
| WO2021107676A1 (en) * | 2019-11-29 | 2021-06-03 | 주식회사 녹십자지놈 | Artificial intelligence-based chromosomal abnormality detection method |
| WO2022097844A1 (en) * | 2020-11-04 | 2022-05-12 | 국립암센터 | Method for predicting survival prognosis of pancreatic cancer patients by using gene copy number variation information |
| WO2022250512A1 (en) * | 2021-05-28 | 2022-12-01 | 한국과학기술원 | Artificial intelligence-based method for early diagnosis of cancer, using cell-free dna distribution in tissue-specific regulatory region |
| WO2024158118A1 (en) * | 2023-01-26 | 2024-08-02 | 지놈케어 주식회사 | Method for detecting fetal copy number variations based on virtual positive and negative data |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150070111A (en) * | 2012-08-30 | 2015-06-24 | 프리마이타 헬스 엘티디 | Method of detecting chromosomal abnormalities |
-
2015
- 2015-12-04 KR KR1020150172212A patent/KR101686146B1/en active IP Right Grant
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20150070111A (en) * | 2012-08-30 | 2015-06-24 | 프리마이타 헬스 엘티디 | Method of detecting chromosomal abnormalities |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106709276A (en) * | 2017-01-21 | 2017-05-24 | 深圳昆腾生物信息有限公司 | Genovariation cause analysis method and system |
| KR20180098438A (en) * | 2017-02-24 | 2018-09-04 | 에스디지노믹스 주식회사 | Priority calculation method for copy number variation candidate |
| KR101957909B1 (en) | 2017-02-24 | 2019-03-15 | 에스디지노믹스 주식회사 | Priority calculation method for copy number variation candidate |
| WO2020022733A1 (en) * | 2018-07-27 | 2020-01-30 | 주식회사 녹십자지놈 | Whole genome sequencing-based chromosomal abnormality detection method and use thereof |
| WO2021034034A1 (en) | 2019-08-19 | 2021-02-25 | 주식회사 녹십자지놈 | Method for detecting chromosomal abnormality by using information about distance between nucleic acid fragments |
| WO2021107676A1 (en) * | 2019-11-29 | 2021-06-03 | 주식회사 녹십자지놈 | Artificial intelligence-based chromosomal abnormality detection method |
| JP7539985B2 (en) | 2019-11-29 | 2024-08-26 | ジーシー ジェノム コーポレーション | Artificial intelligence-based method for detecting chromosomal abnormalities |
| WO2022097844A1 (en) * | 2020-11-04 | 2022-05-12 | 국립암센터 | Method for predicting survival prognosis of pancreatic cancer patients by using gene copy number variation information |
| WO2022250512A1 (en) * | 2021-05-28 | 2022-12-01 | 한국과학기술원 | Artificial intelligence-based method for early diagnosis of cancer, using cell-free dna distribution in tissue-specific regulatory region |
| WO2024158118A1 (en) * | 2023-01-26 | 2024-08-02 | 지놈케어 주식회사 | Method for detecting fetal copy number variations based on virtual positive and negative data |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101686146B1 (en) | Copy Number Variation Determination Method Using Sample comprising Nucleic Acid Mixture | |
| US12116628B2 (en) | Rapid aneuploidy detection | |
| AU2015266665C1 (en) | Detecting fetal sub-chromosomal aneuploidies and copy number variations | |
| CN107077537B (en) | Detection of repeat amplification with short read sequencing data | |
| KR101795124B1 (en) | Method and system for detecting copy number variation | |
| EP3061021B1 (en) | Method for improving the sensitivity of detection in determining copy number variations | |
| JP5938484B2 (en) | Method, system, and computer-readable storage medium for determining presence / absence of genome copy number variation | |
| KR101817785B1 (en) | Novel Method for Analysing Non-Invasive Prenatal Test Results from Various Next Generation Sequencing Platforms | |
| KR20170125044A (en) | Mutation detection for cancer screening and fetal analysis | |
| KR101739535B1 (en) | Method for detecting aneuploidy of fetus | |
| KR20140023847A (en) | Noninvasive detection of fetal genetic abnormality | |
| CN111052249B (en) | Methods of determining predetermined chromosome conservation regions, methods of determining whether copy number variation exists in a sample genome, systems, and computer readable media | |
| KR102405245B1 (en) | Method for Detecting Chromosomal Abnormalities Based on Whole Genome Sequencing and Uses thereof | |
| CN110770341A (en) | Aneuploidy noninvasive prenatal screening method | |
| JP2019500901A (en) | Method for determining copy number anomalies in a sample containing a mixture of nucleic acids | |
| KR101881098B1 (en) | Method for detecting aneuploidy of fetus | |
| KR20230076686A (en) | Method for detecting aneuploidy of fetus based on synthetic data | |
| KR102519739B1 (en) | Non-invasive prenatal testing method and devices based on double Z-score | |
| JP2024534899A (en) | Methods and devices for non-invasive prenatal testing | |
| Kit | Text S1. Supplementary methods for sequence analysis, FDR correction, indel and TE insertion analysis. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant |