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WO2023018024A1 - Méthode de diagnostic de l'instabilité des microsatellites à l'aide d'un taux de variation de longueurs de séquence au niveau de locus microsatellites - Google Patents

Méthode de diagnostic de l'instabilité des microsatellites à l'aide d'un taux de variation de longueurs de séquence au niveau de locus microsatellites Download PDF

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WO2023018024A1
WO2023018024A1 PCT/KR2022/010138 KR2022010138W WO2023018024A1 WO 2023018024 A1 WO2023018024 A1 WO 2023018024A1 KR 2022010138 W KR2022010138 W KR 2022010138W WO 2023018024 A1 WO2023018024 A1 WO 2023018024A1
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microsatellite
region
length
diagnosing
sequences
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English (en)
Korean (ko)
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박승구
김동하
서성현
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Dxome Co Ltd
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Dxome Co Ltd
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/20—Supervised data analysis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/20—Sequence assembly
    • 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

Definitions

  • the present application relates to a method for diagnosing microsatellite instability using the rate of change in sequence length of a microsatellite region.
  • Microsatellite refers to a sequence in which six or fewer short DNA sequences are sequentially repeated throughout the entire genome of the human body, and are scattered with different repeat numbers and repeat patterns on all chromosomes. These are the number of repeat units produces multiple alleles of different lengths.
  • Microsatellite instability refers to the length diversity of increasing or decreasing short tandem repeat sequences that make up microsatellites. The length changes due to germline mutations caused by mismatch repair genes (MMR genes) such as hMSH2, hMLH1, hMSH6, hPMS1, and hPMS2, or replication errors due to promoter methylation.
  • MMR genes mismatch repair genes
  • MSI detection methods include extracting DNA from normal and tumor tissues, amplifying DNA through fluorescence-labeled polymerase chain reaction using five microsatellite markers recommended by the National Cancer Institute, and then capillary electrophoresis.
  • the "Multiplex fluorescence PCR amplification and capillary electrophoresis" analysis method which diagnoses MSI by confirming the sequence while detecting fluorescently labeled DNA by illuminating it with a laser shooting from the opposite side using capillary electrophoresis, and the MSI detection method based on NGS It is known.
  • Patent Document 0001 Patent Registration No. 10-1969971
  • the present application relates to a method for diagnosing microsatellite instability using the rate of change in sequence length of a microsatellite region based on NGS.
  • a first aspect of the present invention provides a method for diagnosing microsatellite instability of a single sample using the rate of change in the sequence length of a microsatellite region.
  • a second aspect of the present application provides a method for diagnosing microsatellite instability by comparing samples using the rate of change in sequence length of a microsatellite region.
  • the present invention uses a simple rate of change for calculation, it is possible to reduce the time required for diagnosis of microsatellite instability compared to conventional NGS-based tests, making it easier to establish a more effective treatment strategy for patients.
  • 1 is a diagram showing a method for diagnosing microsatellite instability of a single sample using the rate of change in sequence length of a microsatellite region.
  • FIG. 2 is a diagram showing a method for diagnosing microsatellite instability in pair comparison of samples using the rate of change in sequence length of the microsatellite region.
  • FIG. 3 is a diagram showing the basic principle of a method for diagnosing microsatellite instability using the rate of change in sequence length of a microsatellite region.
  • FIG. 4 is a view showing the results of comparing the microsatellite instability diagnosis method using the change rate of the sequence length of the microsatellite region and the microsatellite instability result values of the existing diagnosis method.
  • FIG. 5 is a diagram showing the results of comparing the microsatellite instability calculation speed of the microsatellite instability diagnosis method using the change rate of the sequence length of the microsatellite region and the existing diagnosis method.
  • the term “combination(s) of these” included in the expression of the Markush form means a mixture or combination of one or more selected from the group consisting of the components described in the expression of the Markush form, It means including one or more selected from the group consisting of the above components.
  • a first aspect of the present invention provides a method for diagnosing microsatellite instability of a single sample using the rate of change in the sequence length of a microsatellite region.
  • the present application includes the steps of searching for a microsatellite region; selecting a target microsatellite region; detecting sequences aligned to a single sample microsatellite region; selecting a sequence containing a microsatellite from among the sequences detected in each region; calculating the composition and length of microsatellites in the selected sequences; calculating a rate of change in the length of microsatellites detected in each microsatellite region; and diagnosing microsatellite instability using the calculated rate of change (see FIG. 1).
  • microsatellite used throughout the present specification refers to a repetitive part of a DNA base sequence, and usually 1 to 6 base pairs are repeated 5 to 50 times. It accounts for about 5% of the total human DNA and has a higher mutation rate than other DNA parts.
  • microsatellite instability used throughout the present specification is a variation in length that occurs as a short tandem repeat sequence constituting a microsatellite increases or decreases. It undergoes a recovery process through the repair process of the DNA mismatch repair gene (MMR gene). However, if malfunction occurs in repair due to germline mutation of the DNA mismatch repair gene or promoter methylation, changes in the length of the microsatellite occur. The occurrence of gene repair abnormalities can be diagnosed.
  • MSI is generally classified into two types according to international standards.
  • NCI National Cancer Institute
  • Somatic markers BAT-25, BAT-26, D2S123, D17S250, D5S346 were presented, and instability in two or more markers was measured as high-level microsatellite instability (MSI, MSI-H).
  • MSI microsatellite instability
  • RER+ replication error positive
  • microsatellite stable (MSS) (Boland et al., Cancer Res., 58:5248-57, 1998; Deokwoo Kim, Journal of Genetic Medicine 7:24-36, 2010).
  • rate of change used throughout the present specification means the maximum value of microsatellite lengths determined as true sequences, not sequencing errors, among microsatellite (repeat sequence unit) lengths found in the microsatellite region. is the value divided by the minimum value.
  • FASTQ used throughout the present specification means that a base sequence or protein amino acid sequence and quality score data are stored in an ASCII text format. In other words, it displays each sequence, including how reliable the sequence is and how accurate the information is.
  • genetic information used throughout the present specification refers to all genetic information possessed by an organism, and since all organisms, except RNA of some viruses, constitute genetic information with DNA, genetic information generally composed of DNA refers to information
  • the rate of change approaches 1 and each microsatellite length change is large.
  • the change rate is greater than 1, so microsatellite instability can be scored in the range of 1 to more than 1 change rate. Due to the nature of microsatellite sequence changes, infinite values do not appear.
  • the time required for diagnosing microsatellite instability can be reduced.
  • a second aspect of the present invention provides a method for diagnosing microsatellite instability by pairwise comparison of samples using the rate of change in the sequence length of the microsatellite region.
  • Content overlapping with the first aspect of the present application is also applied to the method of the second aspect of the present application.
  • the present application includes the steps of searching for a microsatellite region; selecting a target microsatellite region; detecting sequences aligned to the same microsatellite region in the normal sample and the cancer sample; merging sequences detected in the same microsatellite regions of paired samples; selecting a sequence containing a microsatellite from among the sequences detected in each region; calculating the composition and length of microsatellites in the selected sequences; calculating a rate of change in the length of microsatellites detected in each microsatellite region; and diagnosing microsatellite instability using the calculated rate of change (see FIG. 2).
  • the time required for diagnosing microsatellite instability can be reduced because sequences detected in the same microsatellite region of a normal sample and a cancer sample are merged and calculated at once.
  • microsatellite instability (MSI) test using a single sample was performed through the following steps.
  • MSI sites Sites expected to be MSI sites in the genome were searched for. Most MSI sites are sites with simple repetitive sequences, and all sites were searched for from start to finish for each chromosome. At this time, all possible repetitive sequences were predicted and patterned, and all patterns were searched in the chromosome.
  • FASTQ sequence reads sequence-aligned to the selected MSI predicted site were selected.
  • Selected FASTQ sequence reads were used in a non-sequence aligned state (original FASTQ sequence read).
  • original FASTQ sequence reads those with MSI predicted sites at both ends were removed to clarify the beginning and end of the MSI predicted sites.
  • Rate of change maximum value of microsatellite (repeat unit) length in the microsatellite region excluding repeats due to sequencing errors / minimum value of microsatellite (repeat sequence unit) length in the microsatellite region excluding repeats due to sequencing errors
  • the change rate is 1 or greater than 1 when there is no change in length between microsatellites existing at the same location on the genome or when the length is similar. Due to the nature of microsatellite length changes, values greater than 1 do not extend to infinity.
  • the calculated statistic (rate of change) of MSI predicted regions significantly different from other MSI predicted regions was calculated as a percentage of the total MSI predicted regions.
  • MSI sites Sites expected to be MSI sites in the genome were searched for. Most MSI sites are sites with simple repetitive sequences, and all sites were searched for from start to finish for each chromosome. At this time, all possible repetitive sequences were predicted and patterned, and all patterns were searched in the chromosome.
  • Selected FASTQ sequence reads were used in a non-sequence aligned state (original FASTQ sequence read).
  • original FASTQ sequence reads those with MSI predicted sites at both ends were removed to clarify the beginning and end of the MSI predicted sites.
  • Rate of change maximum value of microsatellite (repeat unit) length in the microsatellite region excluding repeats due to sequencing errors / minimum value of microsatellite (repeat sequence unit) length in the microsatellite region excluding repeats due to sequencing errors
  • the change rate is 1 or greater than 1 when there is no change in length between microsatellites existing at the same location on the genome or when the length is similar. Due to the nature of microsatellite length changes, values greater than 1 do not extend to infinity.
  • the calculated statistic (rate of change) of MSI predicted regions significantly different from other MSI predicted regions was calculated as a percentage of the total MSI predicted regions.
  • the total number of microsatellite regions tested on the same server was divided by the computation time of the program according to each calculation method, and the time required to calculate a single microsatellite region was compared.
  • Single microsatellite area calculation time total analyzed microsatellite area / program calculation time (according to each calculation method)

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Abstract

La présente demande se rapporte à une méthode de diagnostic de l'instabilité des microsatellites à l'aide du taux de variation de longueurs de séquence au niveau de locus de microsatellites, et concerne une méthode de diagnostic de l'instabilité des microsatellites, comprenant les étapes de : recherche des locus microsatellites ; la sélection de locus de microsatellites cibles ; la détection de séquences alignées au niveau de locus microsatellites d'échantillon unique ; la sélection des séquences contenant un microsatellite parmi les séquences détectées au niveau de chaque locus ; le calcul des compositions et des longueurs des microsatellites au niveau des séquences sélectionnées ; le calcul du taux de variation des longueurs des microsatellites détectés au niveau de chaque locus microsatellite ; et le diagnostic de l'instabilité des microsatellites à l'aide du taux de variation calculé. Dans la présente demande, le taux de variation, qui est facile à calculer, est utilisé de sorte que le temps nécessaire pour diagnostiquer l'instabilité des microsatellites peut être inférieur à celui d'un test basé sur NGS classique, favorisant ainsi la mise en place d'une stratégie de traitement de patient plus efficace.
PCT/KR2022/010138 2021-08-10 2022-07-13 Méthode de diagnostic de l'instabilité des microsatellites à l'aide d'un taux de variation de longueurs de séquence au niveau de locus microsatellites Ceased WO2023018024A1 (fr)

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Citations (5)

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US20190169685A1 (en) * 2017-12-01 2019-06-06 Personal Genome Diagnostics Inc. Process for microsatellite instability detection
US20190206513A1 (en) * 2017-12-29 2019-07-04 Grail, Inc. Microsatellite instability detection
US20200024669A1 (en) * 2017-03-20 2020-01-23 Caris Mpi, Inc. Genomic stability profiling
KR20210052511A (ko) * 2018-08-31 2021-05-10 가던트 헬쓰, 인크. 무세포 dna에서의 미세부수체 불안정성 검출
KR20210092196A (ko) * 2018-09-14 2021-07-23 렉센트 바이오, 인크. 미소부수체 불안정성을 평가하기 위한 방법 및 시스템

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CN110621784B (zh) 2017-03-24 2024-06-11 海阳生物材料有限公司 使用双功能pna探针诊断微卫星不稳定性的方法和诊断微卫星不稳定性的试剂盒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200024669A1 (en) * 2017-03-20 2020-01-23 Caris Mpi, Inc. Genomic stability profiling
US20190169685A1 (en) * 2017-12-01 2019-06-06 Personal Genome Diagnostics Inc. Process for microsatellite instability detection
US20190206513A1 (en) * 2017-12-29 2019-07-04 Grail, Inc. Microsatellite instability detection
KR20210052511A (ko) * 2018-08-31 2021-05-10 가던트 헬쓰, 인크. 무세포 dna에서의 미세부수체 불안정성 검출
KR20210092196A (ko) * 2018-09-14 2021-07-23 렉센트 바이오, 인크. 미소부수체 불안정성을 평가하기 위한 방법 및 시스템

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