WO2014133369A1 - Method and apparatus for diagnosing fetal aneuploidy using genomic sequencing - Google Patents

Method and apparatus for diagnosing fetal aneuploidy using genomic sequencing Download PDF

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WO2014133369A1
WO2014133369A1 PCT/KR2014/001704 KR2014001704W WO2014133369A1 WO 2014133369 A1 WO2014133369 A1 WO 2014133369A1 KR 2014001704 W KR2014001704 W KR 2014001704W WO 2014133369 A1 WO2014133369 A1 WO 2014133369A1
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chromosome
score
analysis target
section
sequence
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Korean (ko)
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박종화
김태형
김종수
박신기
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주식회사 테라젠이텍스
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • the present invention relates to a method for non-invasive fetal malformation prenatal diagnosis. More specifically, the present invention relates to a method and apparatus for diagnosing abnormality by analyzing sequencing information on genomic DNA extracted from mother's blood and accurately determining the fetal chromosome aneuploidy. will be.
  • Prenatal diagnosis' refers to the process of determining and diagnosing the disease of a fetus before the fetus is born. According to a recent domestic statistics, congenital malformations account for about 3% of all newborns, and about 20% of congenital malformations are due to chromosomal abnormalities. In particular, the well-known malformations of Down's syndrome account for about 26% of congenital malformations.
  • the interest in prenatal diagnosis is increasing day by day due to the increase in birth rate and development of various prenatal diagnosis equipment.
  • fetal malformations are suspected in maternal serum screening and ultrasonography.
  • Prenatal diagnosis can be divided into invasive and non-invasive diagnosis.
  • invasive diagnostics include chorionic villi sampling (CVS) performed between 10 and 12 weeks of gestation, and fetal chromosomes by measuring the concentration of AFP in amniotic fluid using immunoassay between 15 and 20 weeks of gestation. Amniocentesis to analyze, cordocentesis to extract fetal blood directly from the umbilical cord under ultrasound guidance between 18 and 20 weeks of gestation.
  • CVS chorionic villi sampling
  • fetal chromosomes by measuring the concentration of AFP in amniotic fluid using immunoassay between 15 and 20 weeks of gestation.
  • Amniocentesis to analyze cordocentesis to extract fetal blood directly from the umbilical cord under ultrasound guidance between 18 and 20 weeks of gestation.
  • invasive diagnostic methods such as the above may cause abortion, disease, or malformation by shocking the fetus during the examination, and non-invasive diagnostic methods have been developed
  • the pre-implantation genetic diagnosis method is a technique for selecting embryos without pre-implantation genetic defects using molecular genetic or cytogenetic techniques used in in vitro fertilization.
  • QF-PCR quantitative-fluorescent PCR
  • STRs short tandem repeats
  • DNA auto-sequence analyzer is a rapid screening test to measure the amount of fluorescence amplified DNA.
  • a chromosomal microarray (CMA) method and the like are known which accumulate and examine a mapped DNA sequence mapped on a glass slide in order to find a copy number change.
  • next-generation sequencing technologies are also used in the prenatal diagnosis area.
  • NGS next-generation sequencing
  • the mother's blood contains about 10% of the genome of the fetus
  • prenatal diagnostic methods are known to separate fetal cells from the mother's blood and analyze the chromosomes.
  • Korean Patent Application No. 2010-7003969 discloses a method for diagnosing fetal chromosome aberration using massively parallel genomic sequencing.
  • U.S. Patent No. 8195415 also discloses a method of quantitatively analyzing the mapping of DNA obtained from maternal blood for a specific length for each chromosome.
  • the present invention analyzes genomic DNA (genomic DNA) extracted from the blood of pregnant mothers of normal fetuses and chromosome aneuploids (Filter and Read Depth for the GC content) Filter) and Z-score CutOff values, and check the abnormality of Z-score values based on the Z-score CutOff values using the extracted parameters for the subjects to be diagnosed.
  • genomic DNA genomic DNA
  • chromosome aneuploids Feter and Read Depth for the GC content
  • Filter Z-score CutOff values
  • the 'section' may be set in units of 300kb on genomic DNA.
  • the step of removing a portion of low confidence in the sequence information from the analysis target a method for removing mismatch parts, the removal of the read (read) sequence parts attached to various sites Method, and methods for removing redundant reads from PCR.
  • the diagnostic method may be a method for diagnosing whether a fetus is at least one chromosome selected from the group consisting of chromosomes 13, 18 and 21 of the fetus.
  • the diagnostic method according to the present invention has the advantage of being easy to harm the mother or the fetus in that it can be analyzed using the mother's blood sample. It was also confirmed that even a small amount of fetal chromosome can make a very accurate diagnosis. Therefore, the method of the present invention can be usefully used as a prenatal diagnosis method that can determine whether the abnormality due to abnormalities in the fetal chromosome early.
  • FIG. 1 is a flow chart showing a diagnostic method of the present invention.
  • step 3 is a detailed flowchart of step 3) of a diagnostic method according to an embodiment of the present invention.
  • step 3 is a detailed flowchart of step 1) of a diagnostic method according to an embodiment of the present invention.
  • FIG. 4 is a plot illustrating the quality of base sequences and the distribution of the base sequences by type of data generated through sequencing.
  • Figure 5 shows the results of confirming the read thickness distribution (depth distribution) for genomic DNA set to 300kb intervals according to an embodiment of the present invention.
  • FIG. 6 is a result of analyzing the GC content.
  • the figure on the left shows the distribution on the entire DNA, and the figure on the right shows an enlarged portion of the high frequency (darker one). Dark areas are areas of high frequency.
  • FIG. 7 is a diagram showing a method for determining the feasibility of chromosomes 13, 18 and 21 of a fetus by the method of the present invention with respect to genomic DNA set to 300 kb in accordance with an embodiment of the present invention. .
  • FIG. 8 is a table showing the accuracy of the diagnostic method of the present invention.
  • the result of the karyotype analysis was compared with the results of fetuses diagnosed by chromosome 18 (4 in chromosome 18 and 7 in chromosome 21).
  • Four patients (100%) and seven (100%) of chromosome 21 were diagnosed as 74 (100%).
  • FIG. 9 is a block diagram of a diagnostic apparatus of the present invention.
  • the present invention relates to a method for diagnosing chromosomal aberration of a fetus by analyzing genomic DNA extracted from blood of pregnant mothers of a normal fetus.
  • the method of extracting genomic DNA (genomic DNA) from the blood sample of step 1) can be widely used known DNA extraction method.
  • a library is prepared for samples that have passed quality control standards through qualitative and quantitative analysis of samples.
  • the amplified adapter-ligated lirary is amplified, the amplified sample is purified, and tested using a Bioanalyzer.
  • the library that has passed the quality inspection creates an ISP using One-Touch2 device for template prep.
  • Once the ISP for sequencing has been made install the Sequencing 200kit V2 on Proton and load the ISP made on the PI chip. After placing the chip in the device, perform chip check to confirm that there is no problem with the chip and reagents, and proceed with sequencing. In the Monitor Tab of the Torrent Browser, check if Loading, Live ISPs, Library ISPs are normal.
  • Step 3) comprises: 3-1) checking whether the analysis target is determined for each section set for the sequence information, and removing the sequence section identified as an inappropriate sequence section from the analysis target; And 3-2) setting the portion corresponding to the reference range as an analysis target by examining the GC content of the remaining sequence section portion (FIG. 2).
  • Step 3-1) is a step of removing a portion of low reliability of the sequence information from the analysis target by checking the thickness distribution of the reads by a predetermined interval, but is not limited thereto.
  • the interval may be set in units of 300 kb.
  • the reason for setting the 300 kb as above is to filter by using a nucleotide sequence GC ratio. This is because it can form a group of ratios and is easy for statistical analysis.
  • the sequence section to be removed may include multiple overlapping sequences and PCR duplicated reads, and the reason for removing the multiple overlapping sequences may be a repetitive sequence region.
  • the reason for this is that the PCR duplication reads are removed, and the reason for the amplification process is necessary for sequencing.
  • the high repeat region has a higher depth than the point where it is 80 percentile (the portion where the order in ascending order of each value is 80% of the number of values) to remove the portion. We removed the higher values and, conversely, lower values below 20 percentile (the order in which each value is 20% of the number of values) in order to remove the portion where noise can occur.
  • the part without thickness is excluded from the analysis because most of the region is N region.
  • the step 3-2) by removing the remaining portion of the sequence section by examining the GC content a method for setting only the sequence section that satisfies the condition of 0.35 ⁇ GC content ⁇ 0.45 It can be performed as. Since the GC content of the human genome is known to be about 40%, only the sequence information close to the human GC content is analyzed to increase accuracy.
  • Step 4) is a step of setting the Z-score according to the following equation using the thickness value of the intervals set as the analysis targets for the entire autosome.
  • the Z-score value according to the present invention is a value using the mean and standard deviation of the lead thickness (depth) for each section calculated for the entire autosomal body. In other words, we created a Z-score population with a standard deviation of 1 and a mean of 0 over the whole range. Therefore, all values are standardized.
  • the diagnostic method according to the present invention can diagnose whether a fetus is at least one chromosome selected from the group consisting of chromosomes 13, 18 and 21 of the fetus.
  • Chromosome abnormality 13 is associated with Patau syndrome. Patau syndrome occurs in 1 out of 25,000 to 25,000 fetuses, and is known to die within one year due to severe congenital malformations of important organs such as the central nervous system and heart.
  • Chromosome abnormality 18 is associated with Edwards syndrome. Edwards syndrome occurs in about 1 in every 8,000 fetuses and occurs three to four times more frequently in girls. Severe malformations and mental retardation of many organs are known to die within 10 weeks.
  • Chromosome abnormality 21 is associated with Down syndrome. It appears in 1 out of 800 people and shows symptoms such as mental retardation, physical deformity and growth disorder. Life span is between 20 and 30 years old.
  • step 6) is a step of determining whether the chromosomal abnormality by the average Z-score calculated for each chromosome, two groups (normal group, chromosomal abnormal group) that can not be assumed to follow the normal distribution Since we cannot compare the difference in size through the mean, we analyzed the Mann-Whitney test, a nonparametric method that does not characterize the parameters. The Z-score cutoff set to the difference between the lowest Z-score values was applied to determine the chromosomal abnormality when the cutoff value was exceeded.
  • FIG. 4 is a plot showing the quality of base sequences and distribution of base sequences for data generated through sequencing in an embodiment of the present invention.
  • Table 1 shows the results of nucleotide sequence decoded nucleotide sequences for each decoded sample, which produced 7,478,574 Read on average, and the average total number of produced sequences was 961,605,947 bp and the average total number of mapped sequences was 7,417,179 Read, which is 99.18%.
  • the amount produced in proportion to the number of base sequences of the reference sequence produced an average of 0.32X base sequences for each sample, assuming that the number of base sequences of the reference sequence was 1X.
  • the thickness of the read is extracted with a slide window of 500 bp for each 300 kb section, and the thickness distribution on the genome is confirmed by a line plot (FIG. 5). ).
  • the GC content was calculated using a human reference sequence and represented by a Scatter plot (FIG. 6). 6, the darker the color, the higher the frequency corresponding to the portion. Since the average human is known to have a GC content of 40%, the analysis of the present invention was performed by selecting regions having a GC content of 35 to 45%.
  • the high repeat region has a high depth so that the point where 80 percentile (the order in which each value is in the ascending order is 80% of the number of values) is removed to remove the portion. We removed the higher values, and conversely, lower than 20 percentile (in which the order in ascending order of each value is 20% of the number of each value) to remove the parts that may cause noise for the analysis. Values were removed.
  • the average value and standard deviation of the whole autosome were obtained by using the thickness of each section set to 300kb, and then Z-score group was created with the average of 0 and the standard deviation of 1, and for each chromosome.
  • the average value of Z-score was obtained and used for diagnosis (FIG. 7). In FIG.
  • the diagnostic method of the present invention is a method that can determine chromosomal abnormality relatively accurately even when the thickness (depth) of the chromosome analysis result is low. This confirmed that the prenatal diagnosis related to the number of fetal chromosomes is possible through the method of the present invention.
  • the mapping unit 20 for mapping the read sequences to the human reference genome, the thickness distribution of the read sequences, and the GC content are identified for each interval and set as an analysis target.
  • Analysis target setting unit 30, Z-score calculation unit 40 for calculating a Z-score value by using the depth value of the section set as the analysis target, each chromosome for the calculated Z-scores
  • the average value calculation unit 50 for calculating the average value for each and a Z-score average value calculated for each chromosome includes a determination unit 60 for determining whether the chromosomal abnormality.

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Abstract

The present invention relates to a non-invasive method for prenatal diagnosis of fetal malformation. More specifically, the present invention relates to a method for diagnosing whether there is an abnormality by obtaining parameters through the analysis of the sequencing information on the genomic DNA extracted from the blood of a pregnant woman, and by accurately determining fetal aneuploidy by using the same. The diagnostic method according to the present invention does not harm a pregnant woman or a fetus and is simple in that it is possible to carry out analysis by using a blood sample of the pregnant woman. In addition, it has been confirmed that it is possible to carry out very accurate analysis with a small amount of fetal chromosome. Therefore, the method of the present invention can be useful as a prenatal diagnostic method capable of determining early on whether there is an abnormality through fetal numerical chromosomal abnormalities.

Description

유전체 서열분석을 이용한 태아 염색체 이수성의 진단 방법 및 장치Method and apparatus for diagnosing fetal chromosome aberration using genome sequencing
본 발명은 비침습적인 태아 기형 산전 진단 방법에 관한 것이다. 보다 구체적으로 본 발명은 산모의 혈액으로부터 추출한 게놈 DNA (genomic DNA)에 대한 시퀀싱(sequencing) 정보들을 분석하여 태아의 염색체 이수성(aneuploidy)을 정확히 판정함으로써 기형 여부를 진단하는 방법 및 이를 위한 장치에 관한 것이다.The present invention relates to a method for non-invasive fetal malformation prenatal diagnosis. More specifically, the present invention relates to a method and apparatus for diagnosing abnormality by analyzing sequencing information on genomic DNA extracted from mother's blood and accurately determining the fetal chromosome aneuploidy. will be.
'산전 진단'이란 태아가 태어나기 전 태아의 질병 유무를 판단 및 진단하는 과정을 말한다. 최근의 한 국내 통계자료에 따르면, 선천성 기형아가 전체 신생아의 약 3%에 이르며, 선천성 기형아 중 약 20%는 염색체 이상에 의한 것으로 보고되었다. 특히 널리 알려져 있는 다운증후군에 해당하는 기형아는 선천성 기형아의 약 26%에 이른다. 이러한 기형아 출산율의 증가와 여러 산전 진단 장비들의 개발로 인하여 산전 진단에 대한 관심은 날로 증가하고 있다. 특히, 만 35세 이상의 고령의 임산부, 염색체 이상이 있는 아이의 분만 경력이 있는 임산부, 부모 중 한 명에게서 염색체의 구조적 이상이 있는 경우, 유전질환의 가족력이 있는 경우, 신경관결손의 위험이 있는 경우, 모체혈청 선별검사와 초음파검사에서 태아기형이 의심되는 경우 등에는 산전 진단을 받을 필요가 있다.'Prenatal diagnosis' refers to the process of determining and diagnosing the disease of a fetus before the fetus is born. According to a recent domestic statistics, congenital malformations account for about 3% of all newborns, and about 20% of congenital malformations are due to chromosomal abnormalities. In particular, the well-known malformations of Down's syndrome account for about 26% of congenital malformations. The interest in prenatal diagnosis is increasing day by day due to the increase in birth rate and development of various prenatal diagnosis equipment. In particular, if a pregnant woman aged 35 years or older, a pregnant woman with a chromosomal abnormality, a structural abnormality of a chromosome in one of the parents, a family history of genetic disease, or a risk of neural tube defects In some cases, fetal malformations are suspected in maternal serum screening and ultrasonography.
산전 진단 방법은 크게 침습적 진단 방법과 비침습적 진단 방법으로 나누어 볼 수 있다. 침습적 진단 방법의 예로는, 임신 10 ~ 12주 사이에 시행하는 융모막검사(chorionic villi sampling, CVS), 임신 15 ~ 20주 사이에 면역분석법을 이용하여 양수 내 AFP의 농도를 측정함으로써 태아의 염색체를 분석하는 양수천자(amniocentesis), 임신 18 ~ 20주 사이에 초음파 유도하에 탯줄로부터 직접 태아 혈액을 추출하는 방법으로 시행하는 탯줄천자(cordocentesis) 방법 등이 있다. 그러나 위와 같은 침습적 진단 방법들은 검사 과정에서 태아에게 충격을 가하여 유산이나, 질병 또는 기형 등을 유발할 수 있으므로, 이러한 문제점들을 극복하기 위하여 비침습적 진단 방법들이 개발되고 있다. 예를 들어, 배아 착상 전 유전진단 방법은 체외수정에서 사용되는 분자유전학적 또는 세포유전학적 기술을 이용하여 자궁 내 착상 전 유전적 결함이 없는 배아를 선택하는 기술이다. 또한, 염색체 이수성(aneuploidy)을 신속히 진단하기 위한 QF-PCR (quantitative-fluorescent PCR) 형광 정량법은 염색체마다 특이적으로 존재하는 DNA의 짧은 염기서열 반복 표지자(short tandem repeats, STR)에 형광을 붙여 멀티플렉스(multiplex) PCR 법으로 증폭한 후 DNA 자동염기서열 분석기로 형광이 붙은 증폭된 DNA의 양을 측정하여 분석하는 신속 선별 검사방법이다. 또한, 복제수 변이(copy number change)를 찾아내기 위하여 유리 슬라이드 위에 맵핑한 DNA 서열(mapped DNA sequence)을 집적하여 검사하는 염색체 마이크로어레이 (chromosomal microarray, CMA) 방법 등이 알려져 있다.Prenatal diagnosis can be divided into invasive and non-invasive diagnosis. Examples of invasive diagnostics include chorionic villi sampling (CVS) performed between 10 and 12 weeks of gestation, and fetal chromosomes by measuring the concentration of AFP in amniotic fluid using immunoassay between 15 and 20 weeks of gestation. Amniocentesis to analyze, cordocentesis to extract fetal blood directly from the umbilical cord under ultrasound guidance between 18 and 20 weeks of gestation. However, invasive diagnostic methods such as the above may cause abortion, disease, or malformation by shocking the fetus during the examination, and non-invasive diagnostic methods have been developed to overcome these problems. For example, the pre-implantation genetic diagnosis method is a technique for selecting embryos without pre-implantation genetic defects using molecular genetic or cytogenetic techniques used in in vitro fertilization. In addition, QF-PCR (quantitative-fluorescent PCR) fluorescence quantification for rapid diagnosis of chromosome aneuploidy fluoresces the short tandem repeats (STRs) of DNA specific to each chromosome. After amplification by multiplex PCR method, DNA auto-sequence analyzer is a rapid screening test to measure the amount of fluorescence amplified DNA. In addition, a chromosomal microarray (CMA) method and the like are known which accumulate and examine a mapped DNA sequence mapped on a glass slide in order to find a copy number change.
한편, 시퀀싱 기술의 발달로 대규모의 유전체 정보를 해독하는 것이 가능해짐에 따라, 이러한 차세대 시퀀싱(Next-Generation Sequencing, NGS) 기술을 기반으로 한 유전체 분석 방법들이 산전 진단 영역에도 활용되고 있다. 특히, 모체의 혈액에는 태아의 유전체가 전체 유전체의 약 10% 수준으로 함유되어 있다는 사실이 알려져 있으며, 이를 이용하여 태아의 세포를 모체의 혈액에서 분리하여 그 염색체를 분석하려는 산전 진단 방법들이 알려져 있다. 이와 관련하여 대한민국특허출원 제2010-7003969호는 대규모 병렬 게놈 시퀀싱(massively parallel genomic sequencing)을 이용한 태아 염색체 이수성의 진단 방법에 관하여 개시하고 있다. 또한, 미국등록특허 제8195415호 역시 산모 혈액으로부터 수득한 DNA의 서열분석 결과를 각 염색체별로 특정 길이에 대해 맵핑(mapping)하여 정량분석하는 방법을 개시하고 있다. 상기 발명들은 대규모 병렬 게놈 서열분석 결과를 이용하여 태아 염색체 수의 이상을 판단한다는 점에서 본원 발명과 유사한 측면이 있지만, 구체적인 파라미터 값의 설정 방법에서 모두 본원 발명과는 차이가 있다.Meanwhile, as the development of sequencing technology enables the decoding of large-scale genome information, genome analysis methods based on these next-generation sequencing (NGS) technologies are also used in the prenatal diagnosis area. In particular, it is known that the mother's blood contains about 10% of the genome of the fetus, and prenatal diagnostic methods are known to separate fetal cells from the mother's blood and analyze the chromosomes. . In this regard, Korean Patent Application No. 2010-7003969 discloses a method for diagnosing fetal chromosome aberration using massively parallel genomic sequencing. In addition, U.S. Patent No. 8195415 also discloses a method of quantitatively analyzing the mapping of DNA obtained from maternal blood for a specific length for each chromosome. Although the above inventions have similar aspects to the present invention in determining the abnormality of fetal chromosome number using large-scale parallel genome sequencing results, all of them differ from the present invention in the method of setting specific parameter values.
본 발명은 산모의 혈액에 함유된 태아의 유전체를 이용하여 간편하게 태아의 염색체 이상 여부를 진단할 수 있는 방법 및 이를 위한 장치를 제공하는 것을 목적으로 한다.It is an object of the present invention to provide a method and apparatus for diagnosing chromosomal abnormalities of a fetus using a fetal genome contained in a mother's blood.
상기한 목적을 달성하기 위하여, 본 발명은 정상 태아 및 염색체 이수성을 가진 태아를 임신한 산모들의 혈액으로부터 추출한 게놈 DNA (genomic DNA)를 분석하여 진단방법에 대한 파라미터 (GC 함량에 대한 Filter 및 Read Depth에 대한 Filter) 및 Z-score CutOff 값을 추출하고, 진단 대상인 피험자를 대상으로 상기 추출된 파라미터를 사용하여 나온 Z-score값에 대해 Z-score CutOff 값을 기준으로 이상 여부를 확인하여 태아의 염색체 이수성을 진단하는 방법을 제공한다. In order to achieve the above object, the present invention analyzes genomic DNA (genomic DNA) extracted from the blood of pregnant mothers of normal fetuses and chromosome aneuploids (Filter and Read Depth for the GC content) Filter) and Z-score CutOff values, and check the abnormality of Z-score values based on the Z-score CutOff values using the extracted parameters for the subjects to be diagnosed. Provide a method of diagnosing aneuploidy.
구체적으로 상기 진단 방법은,Specifically, the diagnostic method,
임신한 여성 피험자로부터 태아의 유전체를 포함하는 혈액 시료를 채취하는 단계;Taking a blood sample comprising the genome of the fetus from a pregnant female subject;
상기 혈액 시료로부터 게놈 DNA (genomic DNA)를 추출하여 대규모 병렬 시퀀싱(massively parallel sequencing)을 수행하는 단계;Extracting genomic DNA from the blood sample and performing massively parallel sequencing;
상기 시퀀싱한 리드(read) 서열들을 사람 참조(reference) 유전체에 맵핑(mapping)하는 단계;Mapping the sequenced read sequences to a human reference genome;
상기 리드(read)들의 두께 분포(depth distribution)를 일정한 구간별로 확인하여 서열정보에 대한 신뢰도가 낮은 부분을 분석대상에서 제거하는 단계;Checking a thickness distribution of the reads by a predetermined section and removing a portion having low reliability of sequence information from an analysis target;
상기 제거하고 남은 서열 구간 부분에 대하여 추가로 GC 함량을 조사하여 신뢰도가 높은 부분을 분석 대상으로 설정하는 단계;Further investigating GC content with respect to the remaining portion of the sequence section to set a portion having high reliability as an analysis target;
전체 상염색체(autosome)에 대하여 상기 분석 대상으로 설정된 구간들의 두께(depth) 값을 이용하여 하기 식에 따른 Z-score를 계산하는 단계,Computing a Z-score according to the following equation using the depth value of the interval set as the analysis target for the entire autosome (autosome),
Figure PCTKR2014001704-appb-I000001
;
Figure PCTKR2014001704-appb-I000001
;
상기 각 구간에 대하여 계산된 Z-score들에 대하여 각 염색체별로 평균값을 구하는 단계; 및Obtaining an average value for each chromosome with respect to Z-scores calculated for each section; And
각 염색체별로 산출된 Z-score 평균값에 대하여 통계적 모델에 의한 Z-score CutOff 기준을 적용하여 ‘염색체 이상’여부를 판정하는 단계를 포함한다.And determining the chromosomal abnormality by applying the Z-score CutOff criterion by the statistical model to the Z-score average value calculated for each chromosome.
본 발명의 일실시예에 있어서, 상기 '구간'은 게놈 DNA 상에서 300kb 단위로 설정될 수 있다.In one embodiment of the present invention, the 'section' may be set in units of 300kb on genomic DNA.
본 발명의 일실시예에 있어서, 상기 서열정보에 대한 신뢰도가 낮은 부분을 분석대상에서 제거하는 단계는, 미스매치(mismatch) 부분을 제거하는 방법, 여러 부위에 붙는 리드(read) 서열 부분을 제거하는 방법, PCR에서 중복적인 리드를 제거하는 방법들로 이루어진 군에서 선택될 수 있다.In one embodiment of the present invention, the step of removing a portion of low confidence in the sequence information from the analysis target, a method for removing mismatch parts, the removal of the read (read) sequence parts attached to various sites Method, and methods for removing redundant reads from PCR.
본 발명의 일실시예에 있어서, 상기 GC 함량을 조사하여 신뢰도가 높은 부분을 분석 대상으로 설정하는 방법은, GC 함량을 조사하여 0.35 < GC 함량 < 0.45의 조건을 만족하는 서열 구간만 분석 대상으로 설정하는 방법으로 이루어질 수 있다.In one embodiment of the present invention, the method of setting the portion of high reliability by examining the GC content as an analysis target, only the sequence section satisfying the condition of 0.35 <GC content <0.45 by analyzing the GC content It can be done by the setting method.
본 발명의 일실시예에 있어서, 상기 진단 방법은 태아의 13번, 18번 및 21번 염색체로 이루어진 군에서 선택되는 어느 하나 이상의 염색체에 대한 이수성 여부를 진단하는 방법일 수 있다.In one embodiment of the present invention, the diagnostic method may be a method for diagnosing whether a fetus is at least one chromosome selected from the group consisting of chromosomes 13, 18 and 21 of the fetus.
본 발명에 따른 진단 방법은 산모의 혈액 샘플을 이용하여 분석이 가능하다는 점에서 산모나 태아에게 해를 가하지 않으며 간편하다는 장점이 있다. 또한 적은 양의 태아 염색체로도 상당히 정확한 진단이 가능함을 확인하였다. 따라서 본 발명의 방법은 태아의 염색체 수 이상으로 인한 기형 여부를 조기에 판단할 수 있는 산전 진단 방법으로 유용하게 이용될 수 있다.The diagnostic method according to the present invention has the advantage of being easy to harm the mother or the fetus in that it can be analyzed using the mother's blood sample. It was also confirmed that even a small amount of fetal chromosome can make a very accurate diagnosis. Therefore, the method of the present invention can be usefully used as a prenatal diagnosis method that can determine whether the abnormality due to abnormalities in the fetal chromosome early.
도 1은 본 발명의 진단 방법을 나타낸 흐름도이다.1 is a flow chart showing a diagnostic method of the present invention.
도 2는 본 발명의 실시예에 따른 진단 방법의 3) 단계의 상세 흐름도이다.2 is a detailed flowchart of step 3) of a diagnostic method according to an embodiment of the present invention.
도 3은 본 발명의 실시예에 따른 진단 방법의 1) 단계의 상세 흐름도이다.3 is a detailed flowchart of step 1) of a diagnostic method according to an embodiment of the present invention.
도 4는 시퀀싱을 통해 생성된 데이터(Raw Data)에 대해 염기서열의 퀄리티(Base Quality) 및 염기서열들의 종류별 분포를 나타낸 플롯(plot)이다. FIG. 4 is a plot illustrating the quality of base sequences and the distribution of the base sequences by type of data generated through sequencing.
도 5는 본 발명의 일실시예에 따라, 300kb 구간으로 설정된 게놈 DNA에 대한 리드(read) 두께 분포(depth distribution)를 확인한 결과를 나타낸다.Figure 5 shows the results of confirming the read thickness distribution (depth distribution) for genomic DNA set to 300kb intervals according to an embodiment of the present invention.
도 6은 GC 함량을 분석한 결과이다. 왼쪽 도면은 전체 DNA 상의 분포를 나타낸 것이고, 오른쪽 그림은 빈도수가 높은 부분(진하게 나타나는 부분)을 확대한 것이다. 진하게 나타나는 부분이 빈도수가 높은 영역이다.6 is a result of analyzing the GC content. The figure on the left shows the distribution on the entire DNA, and the figure on the right shows an enlarged portion of the high frequency (darker one). Dark areas are areas of high frequency.
도 7은 본 발명의 일실시예에 따라, 300kb 구간으로 설정된 게놈 DNA에 대하여 본 발명의 방법으로 태아의 13번, 18번 및 21번 염색체에 대한 이수성을 판정하는 방법과 그 결과를 보여주는 그림이다.FIG. 7 is a diagram showing a method for determining the feasibility of chromosomes 13, 18 and 21 of a fetus by the method of the present invention with respect to genomic DNA set to 300 kb in accordance with an embodiment of the present invention. .
도 8은 본 발명의 진단 방법의 정확성을 보여주는 표이다. 핵형분석을 통하여 진단된 태아에(염색체 18번 4명, 염색체 21번 7명) 대한 결과와 비교하였으며, 본 발명의 방법에 따라 진단한 결과 '염색체 이상(abnormal)'으로 판정된 수가 각각 염색체 18번 4명(100%), 염색체 21번 7명(100%) 이었으며, 정상으로 판정된 수가 74명(100%)로 진단되었다.8 is a table showing the accuracy of the diagnostic method of the present invention. The result of the karyotype analysis was compared with the results of fetuses diagnosed by chromosome 18 (4 in chromosome 18 and 7 in chromosome 21). Four patients (100%) and seven (100%) of chromosome 21 were diagnosed as 74 (100%).
도 9는 본 발명의 진단 장치의 구성도이다.9 is a block diagram of a diagnostic apparatus of the present invention.
본 발명은 정상 태아를 임신한 산모들의 혈액으로부터 추출한 게놈 DNA (genomic DNA)를 분석하여 태아의 염색체 이수성을 진단하는 방법에 관한 것으로서,The present invention relates to a method for diagnosing chromosomal aberration of a fetus by analyzing genomic DNA extracted from blood of pregnant mothers of a normal fetus.
1) 임신한 여성 피험자로부터 채취된 혈액 시료에서 게놈 DNA (genomic DNA)를 추출하여 병렬 시퀀싱(massively parallel sequencing)을 수행하는 단계;1) extracting genomic DNA from a blood sample taken from a pregnant female subject to perform massively parallel sequencing;
2) 시퀀싱된 리드(read) 서열들을 사람 참조(reference) 유전체에 맵핑(mapping)하는 단계;2) mapping the sequenced read sequences to the human reference genome;
3) 상기 리드(read) 서열들의 두께 분포(depth distribution) 및 GC 함량을 일정한 구간별로 확인하여 분석 대상으로 설정하는 단계;3) checking the thickness distribution and the GC content of the read sequences for a predetermined section and setting the target as an analysis target;
4) 전체 상염색체(autosome)에 대하여 상기 분석 대상으로 설정된 구간들의 두께(depth) 값을 이용하여 하기 식에 따른 Z-score를 계산하는 단계4) calculating the Z-score according to the following equation using the depth value of the intervals set as the analysis targets for the entire autosome;
Figure PCTKR2014001704-appb-I000002
;
Figure PCTKR2014001704-appb-I000002
;
5) 상기 계산된 Z-score들에 대하여 각 염색체별로 평균값을 산출하는 단계;5) calculating an average value for each chromosome for the calculated Z-scores;
6) 각 염색체별로 산출된 Z-score 평균값을 통계적 모델에 의한 Z-score CutOff 기준에 적용하여 ‘염색체 이상’ 여부를 판정하는 단계를 포함한다(도 1).6) determining the 'chromosome abnormality' by applying the Z-score mean value calculated for each chromosome to the Z-score CutOff criterion by the statistical model (FIG. 1).
본 발명의 일실시예에 있어서, 상기 1)단계의 혈액 시료로부터 게놈 DNA (genomic DNA)를 추출하는 방법은 공지의 DNA 추출 방법이 널리 사용될 수 있다. 대규모 병렬 시퀀싱(massively parallel sequencing)을 수행하는 방법으로는 시료의 정성분석 및 정량분석을 통하여 정도관리 기준을 통과한 샘플에 대하여 라이브러리 제작을 한다. 라이브러리를 제작하는 방법은 정도관리 기준을 통과한 샘플에 대해 단편화를 시킨다. 정도관리 기준을 통과한 샘플에 대해 단편화되어 있는 gDNA에 End Repair Enzyme을 이용하여 end repair된 DNA를 다시 Agencourt AMPure XP bead를 이용하여 정제 한 뒤, Ion Xpress™ Barcode가 붙은 A adaptor와 Ion P1 Adapter를 ligation하고 Nick Repair Polymerase로 Adaptor와 gDNA의 빈부분을 채워준 다음 정제한다. adapter-ligated lirary를 증폭하고, 증폭된 샘플을 정제한 뒤 Bioanalyzer를 이용하여 품질검사 한다. 품질검사를 통과한 라이브러리는 Template prep을 위해 One-Touch2 기기를 이용하여 ISP를 제작한다. Sequencing을 위한 ISP가 제작되었으면 Sequencing 200kit V2를 Proton에 설치하고 PI chip에 만든 ISP를 로딩한다. chip을 기기에 위치 시킨 후 Chip Check을 수행하여 chip 및 시약에 문제가 없음을 확인하고, 염기서열 해독을 진행한다. Torrent Browser의 Monitor Tab에서 Loading, Live ISPs, Library ISPs가 정상적인지에 대해 확인한다.In one embodiment of the present invention, the method of extracting genomic DNA (genomic DNA) from the blood sample of step 1) can be widely used known DNA extraction method. As a method of massively parallel sequencing, a library is prepared for samples that have passed quality control standards through qualitative and quantitative analysis of samples. The method of making a library fragments samples that have passed quality control criteria. After repairing the DNA repaired using the End Repair Enzyme to the fragmented gDNA using the Agencourt AMPure XP bead, the A adapter with Ion Xpress ™ Barcode and the Ion P1 Adapter Ligation and fill the blanks of the adapter and gDNA with Nick Repair Polymerase. The amplified adapter-ligated lirary is amplified, the amplified sample is purified, and tested using a Bioanalyzer. The library that has passed the quality inspection creates an ISP using One-Touch2 device for template prep. Once the ISP for sequencing has been made, install the Sequencing 200kit V2 on Proton and load the ISP made on the PI chip. After placing the chip in the device, perform chip check to confirm that there is no problem with the chip and reagents, and proceed with sequencing. In the Monitor Tab of the Torrent Browser, check if Loading, Live ISPs, Library ISPs are normal.
상기 3)단계는, 3-1) 서열 정보에 대해 설정된 구간별로 분석 대상 여부를 검사하여, 부적합한 서열 구간으로 확인되는 서열 구간을 분석대상에서 제거하는 단계; 및 3-2) 남은 서열 구간 부분에 대하여 GC 함량을 조사하여 기준 범위에 해당하는 부분을 분석대상으로 설정하는 단계를 포함한다(도 2).Step 3) comprises: 3-1) checking whether the analysis target is determined for each section set for the sequence information, and removing the sequence section identified as an inappropriate sequence section from the analysis target; And 3-2) setting the portion corresponding to the reference range as an analysis target by examining the GC content of the remaining sequence section portion (FIG. 2).
상기 3-1)단계는 리드(read)들의 두께 분포(depth distribution)를 일정한 구간별로 확인하여 서열정보에 대한 신뢰도가 낮은 부분을 분석대상에서 제거하는 단계로서, 이에 제한되는 것은 아니나, 본 발명의 일실시예에 있어서, 상기 구간은 300kb 단위로 설정될 수 있다 위와 같이 300kb로 설정한 이유는 염기서열 GC 비율을 이용하여 필터링하기 위함이며, 300kb 단위로 설정했을 때 염색체 내 두께(depth) 및 GC 비율의 집단을 형성할 수 있어 통계적인 분석에 용이하기 때문이다.Step 3-1) is a step of removing a portion of low reliability of the sequence information from the analysis target by checking the thickness distribution of the reads by a predetermined interval, but is not limited thereto. In one embodiment, the interval may be set in units of 300 kb. The reason for setting the 300 kb as above is to filter by using a nucleotide sequence GC ratio. This is because it can form a group of ratios and is easy for statistical analysis.
상기 3-1)단계에서 제거 대상의 서열 구간은 여러 군데(multi) 붙는 서열, PCR 중복 리드(PCR duplicated reads)가 포함될 수 있으며, 여러 군데(multi) 붙는 서열들을 제거하는 이유는 반복서열영역일 가능성이 크기 때문이며, 또 PCR duplication reads를 제거하는 이유는 시퀀싱을 하기 위해 증폭하는 과정이 필요한데 오류로 증폭이 더 많이 된 부분을 제거하기 위함이다. 또한, 반복이 많은 영역(high repeat region)은 두께(depth)가 높기 때문에 그 부분을 제거하기 위하여 80 percentile (각 값의 오름차순에서의 순서가 각 값의 개수에 대해 80%되는 부분) 되는 지점보다 높은 값들을 제거 하였고, 반대로 노이즈(noise)가 발생할 수 있는 부분을 제거하기 위하여 20 percentile(각 값의 오름차순에서의 순서가 각 값의 개수에 대해 20%되는 부분) 되는 지점보다 낮은 값들은 제거했고, 두께가 없는 부분은 N 영역(Region)이 대부분이기 때문에 분석에서 제외하였다. 통계적으로 분석하기 위해서는 어느 정도 편차가 고른 집단을 선택해야 유의한 결과가 나올 수 있다. 그러나 위의 경우는 너무 높게 나오는 부분이나 낮게 나오는 부분들이 평균이나 전체 값에 영향을 줄 수 있기 때문에 적당한 집단을 선택하기 위해 맵핑이 안 된 부분과 너무 높은 부분을 제거한 것이다.In the step 3-1), the sequence section to be removed may include multiple overlapping sequences and PCR duplicated reads, and the reason for removing the multiple overlapping sequences may be a repetitive sequence region. The reason for this is that the PCR duplication reads are removed, and the reason for the amplification process is necessary for sequencing. Also, the high repeat region has a higher depth than the point where it is 80 percentile (the portion where the order in ascending order of each value is 80% of the number of values) to remove the portion. We removed the higher values and, conversely, lower values below 20 percentile (the order in which each value is 20% of the number of values) in order to remove the portion where noise can occur. The part without thickness is excluded from the analysis because most of the region is N region. In order to analyze statistically, it is necessary to select a group with a certain degree of deviation so that a significant result can be obtained. However, in the above case, the parts that are too high or the parts that are too low may affect the average or the total value, so the unmapped and too high parts are removed to select a suitable group.
본 발명의 일실시예에 있어서, 상기 3-2)단계는 제거하고 남은 서열 구간 부분에 대하여 GC 함량을 조사하여, 0.35 < GC 함량 < 0.45의 조건을 만족하는 서열 구간만 분석 대상으로 설정하는 방법으로 수행될 수 있다. 사람의 유전체의 GC 함량은 약 40%인 것으로 알려져 있으므로 사람의 GC 함량에 가까운 서열정보들만을 분석의 대상으로 함으로써 정확도를 높이기 위함이다. In one embodiment of the present invention, the step 3-2) by removing the remaining portion of the sequence section by examining the GC content, a method for setting only the sequence section that satisfies the condition of 0.35 <GC content <0.45 It can be performed as. Since the GC content of the human genome is known to be about 40%, only the sequence information close to the human GC content is analyzed to increase accuracy.
상기 4) 단계는 전체 상염색체(autosome)에 대하여 상기 분석 대상으로 설정된 구간들의 두께(depth) 값을 이용하여 하기 식에 따른 Z-score를 설정하는 단계이다.Step 4) is a step of setting the Z-score according to the following equation using the thickness value of the intervals set as the analysis targets for the entire autosome.
Figure PCTKR2014001704-appb-I000003
Figure PCTKR2014001704-appb-I000003
본 발명에 따른 상기 Z-score 값은 상염색체 전체를 대상으로 계산한 구간별 리드 두께(depth)의 평균 및 표준편차를 이용한 값이다. 즉, 전체 영역에 대해서 표준편차가 1이고, 평균이 0인 Z-score 집단을 만든 것이다. 따라서 모든 값에 대한 표준화를 수행한 것이다.The Z-score value according to the present invention is a value using the mean and standard deviation of the lead thickness (depth) for each section calculated for the entire autosomal body. In other words, we created a Z-score population with a standard deviation of 1 and a mean of 0 over the whole range. Therefore, all values are standardized.
상기 본 발명에 따른 진단 방법은 태아의 13번, 18번 및 21번 염색체로 이루어진 군에서 선택되는 어느 하나 이상의 염색체에 대한 이수성 여부를 진단할 수 있다. 13번 염색체의 이상은 파타우 증후군(Patau syndrome)과 관련이 있다. 파타우 증후군은 태아 2만 ~ 2만 5천 명중 1명의 비율로 나타나며, 중추신경계 및 심장 등 중요 장기의 심한 선천성 기형을 나타내어 1년 이내 사망하는 것으로 알려져 있다. 18번 염색체 이상은 에드워즈 증후군(Edwards syndrome)과 관련이 있다. 에드워즈 증후군은 태아 8천 명당 1명꼴로 나타나며, 여아에게서 3~4배 정도 발생빈도가 높다. 여러 장기의 심한 기형 및 정신지체 장애를 나타내어 10주 이내 사망하는 것으로 알려져 있다. 21번 염색체 이상은 다운증후군(Down syndrome)과 관련이 있다. 800명 중 1명의 비율로 나타나며, 정신 지체, 신체 기형, 성장 장애 등의 증상을 나타낸다. 수명은 20세 ~ 30세 정도이다.The diagnostic method according to the present invention can diagnose whether a fetus is at least one chromosome selected from the group consisting of chromosomes 13, 18 and 21 of the fetus. Chromosome abnormality 13 is associated with Patau syndrome. Patau syndrome occurs in 1 out of 25,000 to 25,000 fetuses, and is known to die within one year due to severe congenital malformations of important organs such as the central nervous system and heart. Chromosome abnormality 18 is associated with Edwards syndrome. Edwards syndrome occurs in about 1 in every 8,000 fetuses and occurs three to four times more frequently in girls. Severe malformations and mental retardation of many organs are known to die within 10 weeks. Chromosome abnormality 21 is associated with Down syndrome. It appears in 1 out of 800 people and shows symptoms such as mental retardation, physical deformity and growth disorder. Life span is between 20 and 30 years old.
본 발명의 일실시예에 있어서, 상기 6) 단계는 염색체별로 산출된 Z-score 평균값으로 염색체 이상 여부를 판단하는 단계이며, 정규분포를 따른다고 가정할 수 없는 두 군(정상군, 염색체 이상군)은 평균을 통해 크기의 차이를 비교할 수 없기 때문에, 모수의 특성을 따지지 않는 비모수적인 방법인 순위합 검정 통계 모델(Mann-Whitney test)의 분석을 통하여 정상군의 Highest Z-score 값과 염색체 이상군의 Lowest Z-score 값의 차이로 설정된 Z-score 기준값(cutoff)을 적용하여, 기준값을 초과하는 경우 염색체 이상으로 판단하였다. In one embodiment of the present invention, step 6) is a step of determining whether the chromosomal abnormality by the average Z-score calculated for each chromosome, two groups (normal group, chromosomal abnormal group) that can not be assumed to follow the normal distribution Since we cannot compare the difference in size through the mean, we analyzed the Mann-Whitney test, a nonparametric method that does not characterize the parameters. The Z-score cutoff set to the difference between the lowest Z-score values was applied to determine the chromosomal abnormality when the cutoff value was exceeded.
이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.
<실시예 1> 산모의 혈액에서 추출한 gDNA의 염기서열해독Example 1 Base Sequence Decoding of gDNA Extracted from Mother's Blood
임신 12주 이상의 산모 85명으로부터 혈액 5ml를 채혈한 뒤, 1-1) 이를 원심분리시켜 혈장(plasma)만 분리하였다. 1-2) 상기 분리된 혈장을 용해한 후 QIAGEN DNA 미니 키트를 사용하여 gDNA를 추출한 후, 1-3) Life tech사의 라이브러리 제작 가이드라인에 따라 라이브러리를 제작하고 IPS를 생성하였다. 1-4) 그 후 Sequencing 200kit V2를 Proton 기기에 설치하고 PI chip에 만든 IPS를 로딩하고 염기서열 해독을 하였다(도 3). 도 4는 본 발명의 실시예에서 시퀀싱을 통해 생성된 데이터(Raw Data)에 대해 염기서열의 퀄리티(Base Quality) 및 염기서열들의 종류별 분포를 나타낸 플롯(plot)이다. 5 ml of blood was collected from 85 mothers over 12 weeks of gestation, followed by centrifugation to separate only plasma. 1-2) After dissolving the separated plasma was extracted gDNA using a QIAGEN DNA mini kit, 1-3) to produce a library according to the library production guidelines of Life tech company and to generate IPS. 1-4) Sequencing 200kit V2 was then installed in a Proton instrument, loaded with IPS made on the PI chip, and subjected to sequencing (FIG. 3). FIG. 4 is a plot showing the quality of base sequences and distribution of base sequences for data generated through sequencing in an embodiment of the present invention.
<실시예 2> 해독된 gDNA 서열의 참조 맵핑(reference mapping)Example 2 Reference Mapping of Decoded gDNA Sequences
참조 맵핑(reference에 mapping)을 통하여 여러 군데(multi) 붙는 서열들은 제거하였고, PCR Duplication으로 인한 오류를 제거하기 위하여 같은 위치에 맵핑되는 서열들에 대해서는 PCR Duplication으로 가정하고 서열을 제거하였다. 표 1은 염기서열 해독한 결과에 대한 내용으로서 해독된 각 샘플별 해독된 염기서열들은 평균 7,478,574 Read가 생산이 되었고, 평균 총 생산된 염기서열의 수는 961,605,947 bp, 평균 총 mapping된 서열들의 수는 7,417,179 Read, 그에 대한 비율은 99.18%이다. 또한 참조 서열의 염기서열 개수에 비례하여 생산된 양은 참조서열의 염기서열의 개수를 1X로 가정하였을 때, 각 샘플별 평균 0.32X 염기서열을 생산하였다.In order to eliminate errors due to PCR duplication, sequences attached to the reference were removed by mapping to the reference. In order to remove errors due to PCR duplication, sequences were mapped to the same position and PCR sequences were removed. Table 1 shows the results of nucleotide sequence decoded nucleotide sequences for each decoded sample, which produced 7,478,574 Read on average, and the average total number of produced sequences was 961,605,947 bp and the average total number of mapped sequences was 7,417,179 Read, which is 99.18%. In addition, the amount produced in proportion to the number of base sequences of the reference sequence produced an average of 0.32X base sequences for each sample, assuming that the number of base sequences of the reference sequence was 1X.
표 1
Figure PCTKR2014001704-appb-T000001
 Table 1
Figure PCTKR2014001704-appb-T000001
<실시예 3> 구간별 리드 두께(depth) 추출 및 GC 함량 계산Example 3 Lead Depth Extraction and GC Content Calculation for Each Section
실제로 게놈(genome) 상에 리드들이 골고루 붙었는지 확인하기 위하여, 300kb 구간별 slide window 500bp로 리드의 두께(depth)를 추출하고, line plot으로 게놈 상의 두께 분포(depth distribution)를 확인하였다(도 5). In order to check whether the leads are actually evenly attached to the genome, the thickness of the read is extracted with a slide window of 500 bp for each 300 kb section, and the thickness distribution on the genome is confirmed by a line plot (FIG. 5). ).
또한, GC bias를 확인하기 위하여, 사람 참조(reference) 서열을 이용하여 GC 함량을 계산하고 Scatter plot으로 나타내었다(도 6). 도 6에서 색이 진할수록 그 부분에 해당하는 빈도수(frequency)가 높다는 것을 의미한다. 보통 사람의 경우에는 GC 함량이 40%인 것으로 알려져 있기 때문에, GC 함량이 35 ~ 45%인 영역들을 선별하여 본 발명의 분석을 수행하였다. In addition, in order to confirm the GC bias, the GC content was calculated using a human reference sequence and represented by a Scatter plot (FIG. 6). 6, the darker the color, the higher the frequency corresponding to the portion. Since the average human is known to have a GC content of 40%, the analysis of the present invention was performed by selecting regions having a GC content of 35 to 45%.
추가로, 반복이 많은 영역(high repeat region)은 두께(depth)가 높기 때문에 그 부분을 제거하기 위하여 80 percentile (각 값의 오름차순에서의 순서가 각 값의 개수에 대해 80%되는 부분) 되는 지점보다 높은 값들을 제거 하였고, 반대로 분석에 대하여 노이즈(noise)가 발생할 수 있는 부분을 제거하기 위하여 20 percentile (각 값의 오름차순에서의 순서가 각 값의 개수에 대해 20%되는 부분) 되는 지점보다 낮은 값들은 제거하였다. In addition, the high repeat region has a high depth so that the point where 80 percentile (the order in which each value is in the ascending order is 80% of the number of values) is removed to remove the portion. We removed the higher values, and conversely, lower than 20 percentile (in which the order in ascending order of each value is 20% of the number of each value) to remove the parts that may cause noise for the analysis. Values were removed.
<실시예 4> 본 발명의 Z-score 계산에 따른 진단결과Example 4 Diagnostic Results According to Z-score Calculation of the Present Invention
<4-1> 본 발명에 따른 Z-score의 계산<4-1> Calculation of Z-score according to the present invention
300kb로 설정한 각 구간별 리드의 두께(depth)를 이용하여 상염색체(autosome) 전체의 평균값과 표준편차를 구한 후, 평균이 0, 표준편차가 1인 Z-score 집단을 만들었고, 각 염색체별로 Z-score의 평균값을 구하여 진단에 사용하였다(도 7). 도 7에서, 정규분포를 따른다고 가정할 수 없는 두 군(정상군, 염색체 이상군)은 평균을 통해 크기의 차이를 비교할 수 없기 때문에, 모수의 특성을 따지지 않는 비모수적인 방법인 순위합 검정 통계 모델인 Mann-Whitney test를 통하여 Z-score Cutoff를 추출하였고, 추출된 Z-score Cutoff보다 높은 값을 가지게 되면 ‘염색체 이상’으로 판단하였다. 이와 같은 설정은 순위합 통계 모델인 Mann-Whitney test를 통하여 선정된 값이므로, 정상군과 염색체 이상으로 확인된 환자의 수가 많아질수록 더욱 정확한 진단이 가능해 질 것이다. 다만, 본 발명의 실시예에서는 전체 샘플 수가 85이므로 이에 해당하는 값에 한해서이다. The average value and standard deviation of the whole autosome were obtained by using the thickness of each section set to 300kb, and then Z-score group was created with the average of 0 and the standard deviation of 1, and for each chromosome. The average value of Z-score was obtained and used for diagnosis (FIG. 7). In FIG. 7, the two groups (normal group, chromosomal abnormal group) that cannot be assumed to follow the normal distribution cannot be compared in size by means of a mean, and therefore, a non-parametric method of ranking parameters, which is a nonparametric method of determining the characteristics of parameters The Z-score Cutoff was extracted through the Mann-Whitney test, and when it had a higher value than the extracted Z-score Cutoff, it was judged as 'chromosome abnormality'. Since this setting is selected through the Mann-Whitney test, a ranking sum statistical model, the more accurate the number of patients identified as normal and chromosomal abnormalities, the more accurate the diagnosis will be. However, in the embodiment of the present invention, since the total number of samples is 85, only the value corresponding thereto is used.
<4-2> 진단결과<4-2> Diagnosis result
위와 같은 방법으로 27명의 피험자에 대하여 본 발명의 진단 방법을 테스트해 본 결과, 13번 염색체 수 이상(T13)에 대해서는 이상자가 없었기 때문에 판별을 할 수 없었고, 18번 염색체 수 이상(T18)에 대해서는 총 4명의 이상자 중 4명을 확진으로 진단하였다. 마지막으로 21번 염색체 수 이상(T21)은 총 7명의 이상자 중 7명을 확진 진단하였다(도 8).As a result of testing the diagnostic method of the present invention in 27 subjects as described above, no abnormalities were found for chromosome 13 (T13), and no discrimination was possible. Four out of four abnormalities were diagnosed with confirmation. Finally, the number 21 chromosome abnormality (T21) confirmed the diagnosis of 7 out of 7 total abnormalities (Fig. 8).
위와 같은 결과들은 본 발명의 진단 방법이 염색체 분석 결과 리드의 두께(depth)가 낮은 경우에도 비교적 정확하게 염색체 이상을 판단할 수 있는 방법임을 보여준다. 이로써 본 발명의 방법을 통하여 태아 염색체 수와 관련된 산전 진단이 가능하다는 것을 확인하였다.The above results show that the diagnostic method of the present invention is a method that can determine chromosomal abnormality relatively accurately even when the thickness (depth) of the chromosome analysis result is low. This confirmed that the prenatal diagnosis related to the number of fetal chromosomes is possible through the method of the present invention.
도 9는 본 발명에 따른 진단 장치의 구성도로 임신한 여성 피험자로부터 채취된 혈액 시료에서 게놈 DNA (genomic DNA)를 추출하여 병렬 시퀀싱(massively parallel sequencing)을 수행하는 시퀀싱부(10), 시퀀싱된 리드(read) 서열들을 사람 참조(reference) 유전체에 맵핑(mapping)하는 맵핑부(20), 상기 리드(read) 서열들의 두께 분포(depth distribution) 및 GC 함량을 일정한 구간별로 확인하여 분석 대상으로 설정하는 분석대상 설정부(30), 상기 분석 대상으로 설정된 구간들의 두께(depth) 값을 이용하여 Z-score 값을 산출하는 Z-score 산출부(40), 상기 산출된 Z-score들에 대하여 각 염색체별로 평균값을 산출하는 평균값 산출부(50) 및 각 염색체별로 산출된 Z-score 평균값을 확인하여 염색체 이상여부를 판단하는 판단부(60)를 포함한다. 9 is a sequencing unit 10 for extracting genomic DNA from a blood sample taken from a pregnant female subject and performing massively parallel sequencing according to the configuration of a diagnostic apparatus according to the present invention, and sequenced reads. The mapping unit 20 for mapping the read sequences to the human reference genome, the thickness distribution of the read sequences, and the GC content are identified for each interval and set as an analysis target. Analysis target setting unit 30, Z-score calculation unit 40 for calculating a Z-score value by using the depth value of the section set as the analysis target, each chromosome for the calculated Z-scores The average value calculation unit 50 for calculating the average value for each and a Z-score average value calculated for each chromosome includes a determination unit 60 for determining whether the chromosomal abnormality.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.
[부호의 설명][Description of the code]
10 : 시퀀싱부10: sequencing unit
20 : 맵핑부20: mapping unit
30 : 분석대상 설정부30: analysis target setting unit
40 : Z-score 산출부40: Z-score calculation unit
50 : 평균값 산출부50: average value calculation unit
60 : 판단부60: judgment unit
100 : 진단 장치100: diagnostic device

Claims (10)

1) 임신한 여성 피험자로부터 채취된 혈액 시료에서 게놈 DNA (genomic DNA)를 추출하여 병렬 시퀀싱(massively parallel sequencing)을 수행하는 단계;1) extracting genomic DNA from a blood sample taken from a pregnant female subject to perform massively parallel sequencing;
2) 시퀀싱된 리드(read) 서열들을 사람 참조(reference) 유전체에 맵핑(mapping)하는 단계;2) mapping the sequenced read sequences to the human reference genome;
3) 상기 리드(read) 서열들의 두께 분포(depth distribution) 및 GC 함량을 일정한 구간별로 확인하여 분석 대상으로 설정하는 단계;3) checking the thickness distribution and the GC content of the read sequences for a predetermined section and setting the target as an analysis target;
4) 전체 상염색체(autosome)에 대하여 상기 분석 대상으로 설정된 구간들의 두께(depth) 값을 이용하여 하기 식에 따른 Z-score 값을 구하는 단계4) Obtaining a Z-score value according to the following formula using the depth value of the intervals set as the analysis targets for the entire autosome
Figure PCTKR2014001704-appb-I000004
;
Figure PCTKR2014001704-appb-I000004
;
5) 상기 Z-score 값에 대하여 각 염색체별로 Z-score 평균값을 산출하는 단계;5) calculating a Z-score average value for each chromosome with respect to the Z-score value;
6) 각 염색체별로 산출된 Z-score 평균값을 확인하여 염색체 이상여부를 판단하는 단계를 포함하는 태아 염색체 이수성의 진단을 위한 정보제공방법.6) A method of providing information for diagnosing fetal chromosome aberration comprising determining a chromosome abnormality by checking an average value of Z-score calculated for each chromosome.
제1항에 있어서,The method of claim 1,
상기 1) 단계는, Step 1),
(a) 채취된 혈액을 원심분리하여 혈장을 분리하는 단계;(a) centrifuging the collected blood to separate plasma;
(b) 분리된 상기 혈장을 용해하여, gDNA를 추출하는 단계;(b) dissolving the separated plasma to extract gDNA;
(c) 추출된 상기 gDNA에 대한 라이브러리를 제작하고 IPS를 생성하는 단계;(c) preparing a library for the extracted gDNA and generating an IPS;
(d) 생성된 상기 IPS를 로딩하여, 염기서열을 해독하는 단계를 포함하는 것을 특징으로 하는 방법.(d) loading the generated IPS, and decoding the base sequence.
제1항에 있어서,The method of claim 1,
상기 3) 단계의 상기 구간은 게놈 DNA 상에서 300kb 단위로 설정된 것을 특징으로 하는 방법.Wherein said section of step 3) is set on a genomic DNA in units of 300 kb.
제1항에 있어서, The method of claim 1,
상기 3) 단계는,Step 3),
(a) 서열 정보에 대해 구간별로 분석 대상 여부를 검사하여, 부적합한 서열 구간으로 확인되는 서열 구간을 분석대상에서 제거하는 단계; 및 (a) checking whether the sequence information is analyzed for each section of the sequence information, and removing the sequence section identified as an inappropriate sequence section from the analysis target; And
(b) 남은 서열 구간 부분에 대하여 GC 함량을 조사하여 기준 범위에 해당하는 부분을 분석대상으로 설정하는 단계를 포함하는 것을 특징으로 하는 방법.(b) examining the GC content with respect to the remaining sequence section portion and setting the portion corresponding to the reference range as an analysis target.
제4항에 있어서,The method of claim 4, wherein
상기 (a) 단계는,In step (a),
미스매치(mismatch) 부분을 제거하는 방법, 여러 부위에 붙는 리드(read) 서열 부분을 제거하는 방법, PCR에서 중복적인 리드를 제거하는 방법들로 이루어진 군에서 선택되는 어느 하나 이상의 방법들로 수행되는 것을 특징으로 하는 방법.Is performed by any one or more methods selected from the group consisting of a method for removing mismatch portions, a method for removing a read sequence portion attached to multiple sites, and a method for removing redundant reads from PCR. Characterized in that the method.
제4항에 있어서,The method of claim 4, wherein
상기 (a) 단계는,In step (a),
반복이 많은 영역(high repeat region)에 대해서는 80 percentile 되는 지점보다 높은 값들을 분석대상에서 제거하고, 맵핑(mapping)이 적게 되는 영역에 대해서는 20 percentile 되는 지점보다 낮은 값들을 분석대상에서 제거하는 것을 특징으로 하는 방법.It removes values higher than 80 percentile from the analysis target for high repeat region and removes values lower than 20 percentile from analysis target for low mapping region. How to.
제4항에 있어서,The method of claim 4, wherein
상기 (b) 단계는Step (b) is
0.35 < GC 함량 < 0.45 을 만족하는 서열 구간만 분석 대상으로 설정하는 것을 특징으로 하는 방법.And a sequence section satisfying 0.35 <GC content <0.45 is selected for analysis.
제1항에 있어서,The method of claim 1,
상기 6) 단계는, Step 6) is
순위합 검정 통계 모델인 Mann-Whitney test를 통하여 설정된 기준값(cutoff)을 적용하는 것을 특징으로 하는 방법.A method of applying a cutoff set through the Mann-Whitney test, a rank sum test statistical model.
제1항 내지 제8항 중의 어느 한 항에 있어서,The method according to any one of claims 1 to 8,
상기 방법은 태아의 13번, 18번 및 21번 염색체로 이루어진 군에서 선택되는 어느 하나 이상의 염색체에 대한 이수성 여부를 진단하기 위한 것을 특징으로 하는 방법.The method is characterized in that for diagnosing whether the femorality of any one or more chromosomes selected from the group consisting of chromosomes 13, 18 and 21 of the fetus.
임신한 여성 피험자로부터 채취된 혈액 시료에서 게놈 DNA (genomic DNA)를 추출하여 병렬 시퀀싱(massively parallel sequencing)을 수행하는 시퀀싱부;A sequencing unit extracting genomic DNA from a blood sample collected from a pregnant female subject and performing massively parallel sequencing;
시퀀싱된 리드(read) 서열들을 사람 참조(reference) 유전체에 맵핑(mapping)하는 맵핑부;A mapping unit for mapping the sequenced read sequences to a human reference genome;
상기 리드(read) 서열들의 두께 분포(depth distribution) 및 GC 함량을 일정한 구간별로 확인하여 분석 대상으로 설정하는 분석대상 설정부;An analysis target setting unit configured to check the thickness distribution and the GC content of the read sequences for a predetermined section and set the analysis target;
상기 분석 대상으로 설정된 구간들의 두께(depth) 값을 이용하여 하기 Z-score를 산출하는 Z-score 산출부Z-score calculation unit for calculating the following Z-score using the depth value of the section set as the analysis target
Figure PCTKR2014001704-appb-I000005
;
Figure PCTKR2014001704-appb-I000005
;
상기 산출된 Z-score에 대하여 각 염색체별로 평균값을 산출하는 평균값 산출부; 및An average value calculator configured to calculate an average value for each chromosome with respect to the calculated Z-scores; And
각 염색체별로 산출된 Z-score 평균값을 확인하여 염색체 이상 여부를 판단하는 판단부를 포함하는 태아 염색체 이수성 진단 장치.Fetal chromosome aberration diagnostic apparatus including a determination unit for determining the chromosomal abnormality by checking the average Z-score calculated for each chromosome.
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