WO2023193357A1 - Procédé de construction d'une banque de séquençage de méthylation de l'adn libre et son utilisation - Google Patents

Procédé de construction d'une banque de séquençage de méthylation de l'adn libre et son utilisation Download PDF

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WO2023193357A1
WO2023193357A1 PCT/CN2022/103499 CN2022103499W WO2023193357A1 WO 2023193357 A1 WO2023193357 A1 WO 2023193357A1 CN 2022103499 W CN2022103499 W CN 2022103499W WO 2023193357 A1 WO2023193357 A1 WO 2023193357A1
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dna
sequencing
methylation
biological sample
fragment
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曹云龙
谢晓亮
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昌平国家实验室
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • 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
    • C12Q2600/154—Methylation markers

Definitions

  • the present application relates to biotechnology, and more particularly to methods for epigenetic analysis of cell-free DNA.
  • Extracellular free DNA is generally a DNA fragment released from inside cells into body fluids or the environment during cell metabolism, exocytosis or apoptosis (apoptotic) and necrotic (necrotic).
  • Plasma, serum, urine, saliva, amniotic fluid and other materials contain trace amounts of free DNA, which reflect information such as genetic variation, disease, and aging in the body.
  • cell-free DNA extraction, library construction, sequencing and analysis are widely used in all aspects of clinical diagnosis and scientific research, such as cancer screening and diagnosis, prenatal diagnosis, and pre-implantation diagnosis. , aging research and other fields.
  • Epigenetic information carried by cell-free DNA such as DNA methylation/hydroxymethylomics signatures and segmentomics signatures, is increasingly considered to be valuable molecules for the detection, diagnosis and/or monitoring of various diseases such as cancer. mark.
  • DNA methylation is the phenomenon of methylation chemical modification of DNA in organisms. Its chemical essence is: on the fifth carbon atom of the pyrimidine ring of the cytosine base, an original hydrogen atom is replaced by Replaced with a methyl group to form 5-methylcytosine.
  • This kind of DNA modification affects many life processes such as gene expression and regulation, cell division and differentiation, cell metabolism and growth, etc. without changing the DNA sequence. It ultimately affects the growth and development, metabolism and reproduction of individual organisms, and the occurrence and development of diseases. and aging death, which is an epigenetic mechanism. If you need to perform high-throughput sequencing (Next Generation Sequencing, NGS) on methylation modifications on DNA sequences, you must first specifically convert cytosine with/without methylation into other bases in order to distinguish methylation Grooming.
  • NGS Next Generation Sequencing
  • Fragmentomics information is a collection of information obtained by analyzing the fragmentation characteristics of free DNA, such as the distribution characteristics of free DNA on the genome, fragment length and start and end positions, fragment end base characteristics, fragment uneven characteristics and other information. Studies have shown that free DNA tends to be cleaved at certain genomic regions or elements, so fragmentomics information can reflect the opening and closing of chromatin, nucleosome occupancy, transcription factor binding, epigenetic modification and other states. It is one of the key epigenomic information and a valuable molecular marker for monitoring and diagnosing various diseases such as cancer [1]. If you want to obtain fragment omics information during high-throughput sequencing, the integrity of the DNA fragments must be preserved.
  • cell-free DNA is formed by strong DNase degradation inside/outside the cell, it has the following characteristics that make it difficult to detect cell-free DNA:
  • the cfDNA content is small (for example, each milliliter of plasma contains only a few nanograms of cfDNA). This requires that the detection method must be adaptable to 1ng or even lower cfDNA input amounts, and it needs to have higher library construction efficiency to improve cfDNA detection sensitivity.
  • the cfDNA fragment is short (about 100bp to 200bp).
  • short DNA fragments are easily lost due to purification operations during the library construction process, and cannot withstand secondary fragmentation caused by chemical transformation;
  • short fragments cannot be constructed using transposase, random primers, disrupted ligation, and other whole-genome libraries.
  • Methods, the application of targeted PCR and other targeted amplification library construction methods also have certain difficulties and limitations.
  • cfDNA is often severely damaged in its chemical structure. Damage to cfDNA comes from multiple processes: DNA damage caused by cellular physiological and pathological conditions; damage caused by nuclease digestion during cell death; sustained attack by active substances such as free nucleases in the body fluid environment (such as blood circulation) damage; damage caused by prolonged storage in plasma and other solutions, repeated freezing and thawing, etc. Studies have found that 97 to 98% of cfDNA has gaps, which are breaks in single-stranded phosphodiester bonds that occur on both strands of DNA [2]. Using the single-stranded library construction method, cfDNA will break at the gap after double-stranded denaturation and dissociation, resulting in fragments [3].
  • the existing DNA methylation sequencing library construction methods can be divided into the following four types: (1) double-stranded library construction method based on bisulfite conversion [4]; (2) single-stranded library construction method based on bisulfite conversion.
  • Library methods such as Swift Biosciences kit Methyl-Seq DNA Library Kit, Cat. No. 30024); (3) Double-stranded library construction method based on enzyme conversion method (such as New England Biolabs, Inc. EM-seq technology WO2017075436A1 and its commercial kit E7120; WO2019136413A1 TAPS technology; Cabemet technology of WO2021077415); (4) Single-stranded library construction method based on enzyme conversion method (such as CN114032287A).
  • enzyme conversion method such as CN114032287A
  • the large amount of DNA fragmentation and loss makes the bisulfite conversion method unable to meet the requirements of low starting amount of cfDNA library construction, and aggravates the short fragmentation and gap damage of cfDNA, so it is difficult to develop highly sensitive products based on the bisulfite conversion method.
  • Cell-free DNA methylation detection technology Cell-free DNA methylation detection technology.
  • DNA methylation sequencing technologies based on enzymatic conversion methods have gradually developed and matured, such as EM-seq technology, TAPS technology, and Cabernet technology.
  • the enzymatic transformation method abandons the harsh chemical treatment process and avoids fragmentation and degradation of DNA during transformation. It has higher library construction efficiency, higher sensitivity, and higher library uniformity.
  • the existing enzymatic conversion method patents and products cannot solve all the problems of free DNA methylation detection.
  • the minimum starting amounts of EM-seq technology (WO2017075436A1) and TAPS technology (WO2019136413A1) are 5 to 10 nanograms, which cannot perform high-sensitivity sequencing of free DNA;
  • Cabemet technology (WO2021077415) can Library construction with low starting amounts of DNA can even perform highly sensitive library construction on single cells, but transposase cannot be used to apply to short fragments of free DNA.
  • Cabemet technology has not yet provided a solution.
  • double-stranded libraries are used to build libraries.
  • the gap will be smoothed out by the end repair process, which does not affect sequencing, and has not been taken seriously. It has not been taken seriously in traditional bisulfite methylation sequencing because the use of single-strand library construction will cause cfDNA to be broken and lost at the gap, but the methylation sequencing data itself will not have errors.
  • the conventional method based on the new enzyme conversion method is double-stranded library construction, and severe methylation erasure has indeed been observed, resulting in severe methylation loss.
  • this article provides a method for preprocessing biological samples used to construct sequencing libraries, which includes:
  • the sequencing library is used to obtain DNA methylation information data of the biological sample after sequencing or to obtain DNA methylation information and fragment omics information data of the biological sample.
  • the length of the DNA fragment is between 50 bp and 1000 bp; preferably, between 100 bp and 500 bp; more preferably, between 100 bp and 350 bp.
  • the method further includes fragmenting the DNA molecules contained in the biological sample prior to step S1 to generate the DNA fragments.
  • the ligase has 3',5'-phosphodiester bond catalytic activity.
  • the ligase is selected from the group consisting of HiFi-Taq ligase, T4 DNA ligase, Taq DNA ligase, E. coli DNA ligase, and T7 DNA ligase.
  • the biological sample is selected from animal fluids, cell culture fluids or natural biological samples, including but not limited to peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph fluid, alveolar lavage fluid , amniotic fluid, blastocoel fluid, cell culture fluid, embryo culture fluid, microbial culture medium, soil leachate and bone meal leachate.
  • animal fluids including but not limited to peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph fluid, alveolar lavage fluid , amniotic fluid, blastocoel fluid, cell culture fluid, embryo culture fluid, microbial culture medium, soil leachate and bone meal leachate.
  • the DNA fragments include, but are not limited to, extracellular cell-free DNA, extranuclear cell-free DNA, fragmented DNA, for example, extracellular cell-free DNA from a cancer subject.
  • the DNA fragment content in the sample is no more than 10 ng, or no more than 1 ng, such as 0.1 ng.
  • the DNA methylation includes, but is not limited to, methylation and/or hydroxymethylation of cytosine.
  • this article provides a method for constructing a sequencing library, which includes:
  • the sequencing library is used to obtain DNA methylation information data of the biological sample after sequencing or to obtain DNA methylation information and fragment omics information data of the biological sample.
  • step 2) further includes:
  • a linker sequence Preferably, part or all of the cytosine in the linker sequence is methylated cytosine and/or hydroxymethylated cytosine;
  • cytosine in the DNA fragment into uracil through enzymatic conversion, which is recognized as thymine in subsequent amplification and sequencing, and methylated cytosine is recognized as cytosine in subsequent amplification and sequencing.
  • pyrimidine Convert cytosine in the DNA fragment into uracil through enzymatic conversion, which is recognized as thymine in subsequent amplification and sequencing, and methylated cytosine is recognized as cytosine in subsequent amplification and sequencing.
  • step 2) further includes:
  • the enzymatic conversion method adopts EM-seq conversion method or TAPS conversion method.
  • the method further comprises adding carrier DNA to the biological sample after step a).
  • the carrying DNA can be any DNA that does not include the linker sequence.
  • the fragment size of the carrying DNA is 100-500 bp.
  • this article provides a sequencing library obtained by the above method.
  • this article provides a method for determining the position of a single-stranded gap in a DNA fragment contained in a biological sample, which includes:
  • this article provides a method for identifying health conditions in subjects, which includes:
  • the DNA methylation information data of the biological sample or the DNA methylation information data and fragment omics information data of the biological sample are obtained according to the sequencing data, and are compared with the normal DNA methylation information data and/or fragments in the population.
  • the omics information data are compared to determine the health status of the subject.
  • the above methods provided in this article can be used for epigenetic analysis of extracellular cell-free DNA.
  • the methods presented here may also be used in the fields of genomics, medicine, diagnostics, and epigenetics research.
  • Figure 1 is a schematic flow chart of the methylation library construction method provided in this article.
  • Figure 2 is a schematic flow chart of the EM-seq conversion method.
  • FIG. 3 is a schematic flow chart of the TAPS conversion method.
  • Figure 4 shows the library methylation bias analysis results of methylation library construction of human peripheral blood cell-free DNA using DNA Ligase treatment method (solid line) and no treatment method (dashed line).
  • solid line DNA Ligase treatment method
  • dashed line no treatment method
  • Figure 5 shows the fragment distribution pattern of free DNA, in which there is a peak with a period of about 170 bp.
  • Figure 6 shows the distribution of library fragments generated by different library construction methods. Capillary gel electrophoresis was performed with the same input amount of library DNA, and the distribution of DNA fragments in the range of 1 to 6000 bp was analyzed. The results show that the library of the method of the present invention (above) exhibits periodic DNA fragment peak spectra, which is consistent with the characteristics of peripheral blood free DNA; and Swift Biosciences, Methyl-Seq DNA Library Kit (picture below) has only one main peak, no periodic peaks, and contains a large number of non-library impurity peaks.
  • DNA ligase refers to an enzyme used to repair single-stranded nicks in double-stranded DNA molecules.
  • DNA ligases usually have 3',5'-phosphodiester bond catalytic activity. In one example, the DNA ligase can catalyze the formation of a phosphodiester bond between the 5' phosphate group and the 3' hydroxyl group at the gap. DNA ligases usually do not add new nucleotides at gaps.
  • DNA polymerase herein refers to an enzyme that, in the presence of a primer, can use one DNA strand as a template to add dNTPs (deoxyribonucleotides) to the 3’ end of the primer to synthesize its complementary strand.
  • DNA polymerase usually also has 3’-5’ exonuclease activity, which plays a proofreading role during synthesis, and 5’-3’ exonuclease activity, which plays a role in excision repair.
  • DNA fragment means here a short fragment of DNA, for example between 50 bp and 700 bp in length, for example between 100 bp and 500 bp, especially between 100 bp and 350 bp.
  • the DNA fragments contained in biological samples are usually heterogeneous, that is, of varying lengths.
  • the above-mentioned length may refer to the average length of these DNA fragments.
  • These DNA fragments may have different sequences, such as from different regions of the same organism's genome, or even from different organisms.
  • DNA fragments can be single-stranded nicked, blunt-ended or non-blunt-ended (with 3’ or 5’ overhangs).
  • Bount-ended DNA fragments refers to double-stranded DNA fragments without 3' overhangs or 5' overhangs at the ends.
  • DNA methylation refers to the modification of a cytosine base in a DNA molecule or DNA fragment to 5-methylcytosine (5mC).
  • DNA methylation in vertebrates generally occurs at CpG sites (i.e., the site immediately followed by guanine after cytosine in the DNA sequence). Cytosine is converted into 5-methylcytosine catalyzed by DNA methyltransferase. Most CpG sites in the human genome are methylated, but some specific regions, such as CpG islands rich in cytosine (C) and guanine (G), are usually not methylated. CpG methylation can affect the transcriptional activity of related genes.
  • methylation can inhibit tumor suppressor genes, while demethylation can stimulate the expression of certain oncogenes. In these cases, it may lead to cancer.
  • the incidence rate is lower than 5mC, a few cytosine bases are modified into 5-hydroxymethylcytosine (5hmC), 5-formylation (5fC), and 5-carboxylation (5caC).
  • 5hmC 5-hydroxymethylcytosine
  • 5fC 5-formylation
  • 5caC 5-carboxylation
  • DNA methylation information herein refers to information about the methylation status in DNA molecules or DNA fragments, including but not limited to, methylation sites, methylation levels, methylation patterns (5mC or 5hmC), etc. .
  • the "methylation level” in this article can also be called the “methylation degree”, which refers to the proportion (or frequency) of a specific methylation site in a sample that is modified by methylation.
  • methylation degree refers to the proportion (or frequency) of a specific methylation site in a sample that is modified by methylation.
  • There are several ways to detect whether a site is methylated Commonly used methods include chemical or enzymatic conversion methods, in which one of methylated cytosine and unmethylated cytosine is converted to uracil (U) or is essentially equivalent to uracil in a base-pairing manner. base (e.g.
  • the corresponding uracil is paired with adenine (A) as thymine (T), and the final result is the cytosine or methylated cytosine at the methylation site in the detection result (such as sequencing Result) is reflected in thymine.
  • A adenine
  • T thymine
  • the reference sequence can be a sequence from the same sample without transformation as described above, or a corresponding sequence in a healthy population.
  • DNA methylation information has been widely used in cancer (eg, lung cancer, breast cancer, liver cancer, colorectal cancer, etc.) screening and diagnosis, especially early screening and diagnosis.
  • Fragmentomics information in this article refers to a collection of information obtained by analyzing the fragmentation characteristics of free DNA in a sample, such as the distribution characteristics of free DNA on the genome, fragment length and start and end positions, fragment end base characteristics, Fragment unevenness characteristics and other information. Recently, some literature has reported its use in cancer (such as lung cancer) screening.
  • Enzymatic conversion method refers to the modification of cytosine or methylated cytosine by the catalytic action of a specific enzyme so that its methylated and unmethylated states can be distinguished in subsequent detection, or to distinguish between 5mC and 5mC. 5hmC.
  • enzymatic transformation methods including, for example, but not limited to, EM-seq transformation and TAPS transformation. These enzymes are usually used in combination with some chemical reagents in enzymatic conversion methods.
  • E-seq conversion method in this article refers to the technology developed by New England Biolabs to distinguish methylated cytosine from unmethylated cytosine through enzymatic conversion. It uses dioxygenase TET (including TET1, TET2, TET3, etc.) to modify 5mC and 5hmC into 5-carboxycytosine (5caC), and then uses cytidine deaminase (cytidine deaminase) to modify unmethylated cytosine Deamination and conversion to uracil. Cytidine deaminases used include members of the APOBEC protein family, such as APOBEC 3A.
  • the "TAPS (TET-assisted pyridine borane sequencing) conversion method” is somewhat similar to the EM-seq conversion method. It also modifies 5mC and 5hmC into 5-carboxycytosine (5caC) through enzymatic methods, but then it does not undergo deamination. Instead, the reducing agent pyridine borane is used to convert 5caC into dihydrouracil (DHU), while unmethylated cytosine remains unchanged. During subsequent replication or amplification, DHU is recognized by the polymerase as uracil and pairs with A. In this way, sites with 5mC and 5hmC modifications are detected as T, and C without modification is still detected as C.
  • 5mC and 5hmC can be further differentiated using DNA glycosyltransferase (GT) or KRuO .
  • GT DNA glycosyltransferase
  • KRuO KRuO .
  • the flow chart of the TAPS conversion method can be seen in Figure 3. More detailed information on the TAPS conversion method can be found in PCT Application Publication WO2019136413A, which is hereby incorporated by reference in its entirety.
  • Health status in this article refers to the subject's health status, including whether he or she suffers from a certain disease, the risk of suffering from a certain disease, whether he or she is suitable for a certain therapeutic agent or treatment method, the prognosis of the disease, etc.
  • Subject refers to an animal, such as a mammal, including but not limited to humans, rodents, apes, felines, canines, equines, bovines, porcines, sheep, goats , mammalian experimental animals, mammalian agricultural animals, mammalian sporting animals and mammalian pets.
  • the subject may be male or female and may be of any appropriate age, including infants, juveniles, young adults, adults, and geriatric subjects.
  • the subject is a patient.
  • the subject is a human, such as a human patient. The term is often used interchangeably with "patient,” "subject,” “subject,” etc.
  • a method for preprocessing samples to be constructed for sequencing library construction involves repairing single-stranded gaps in the DNA fragments contained in the sample, followed by conventional end repair.
  • DNA ligase can be used to repair the gap
  • DNA polymerase can be used to repair the ends.
  • gap repair is needed before routine end repair is because the inventor realized for the first time and confirmed through research that the existence of these single-stranded gaps is an important reason for the loss of methylation information, especially for extracellular free DNA (cfDNA). ), such as circulating tumor DNA (ctDNA).
  • a method for constructing a sequencing library which includes constructing a sequencing library after performing the above pre-processing on the sample.
  • the library construction process also includes using enzymatic conversion method to modify cytosine (C) or methylated cytosine (including 5mC and/or 5hmC), so that in the subsequent detection step, the methylated cytosine (C) can be modified. Differentiate between methylated and unmethylated cytosine or 5mC and 5hmC to form a DNA methylation sequencing library. After sequencing the sequencing library on a computer, the corresponding methylation information data can be obtained.
  • methylation information data can be obtained after sequencing, but also fragment omics information data can be obtained simultaneously.
  • this library construction technology is based on cfDNA double-stranded fragments for methylation NGS detection library construction, and after repairing the single-stranded gap, the methylation status is distinguished through enzymatic conversion, which basically does not damage the integrity of the original DNA fragments in the sample. It retains its original fragment omics information, including fragment length distribution characteristics, fragment length, etc.
  • the requirement of the method of the present invention on the content of DNA fragments in the sample can be further reduced until the DNA content is as low as 0.1ng, or even lower.
  • the position of a single-stranded gap in a DNA fragment contained in a biological sample is determined by differences in methylation information resulting from the addition or absence of a DNA ligase.
  • the gap position can be considered as a gap position range.
  • the gap position can be considered to be at the first position and the second position downstream of it. Between positions: The first position is the methylation position that can be detected with or without adding DNA ligase, and the second position is the position where methylation difference is detected (for example, adding DNA ligase can detect methylation, No methylation is detected without the addition of ligase).
  • the health status of the subject can be judged through the obtained methylation information.
  • the health status of the subject can be judged more accurately by combining the methylation information with the fragment omics information.
  • the method needs to be highly sensitive, and can efficiently construct NGS libraries for low-input samples as low as 1ng and below;
  • the method needs to preserve the integrity of the free DNA chain to ensure that the fragment omics information in the sequencing library is not destroyed;
  • the method provided by the present invention can be used for, but is not limited to, epigenetic analysis of extracellular free DNA, and can achieve the above goals.
  • the method may include one or more of the following features:
  • this method uses one or several specific DNA ligases, such as Taq DNA ligase, to repair the phosphodiester bond gaps on the double strands of the template DNA to prevent the breakage and loss of the DNA strand in the subsequent process. /or erasure of methylation information.
  • specific DNA ligases such as Taq DNA ligase
  • the 3' end of the library no longer produces severe erasure of methylation information, and the detected methylation rate returns to the average methylation rate. close to the conversion rate.
  • Free DNA refers to DNA free outside cells or nuclei. It can be extracted from a variety of body fluids, in vitro cell culture fluids, natural environments and other biological materials, including but not limited to: peripheral blood, plasma, serum, urine, feces, saliva , cerebrospinal fluid, lymph fluid, alveolar lavage fluid, amniotic fluid, blastocoel fluid, cell culture fluid, embryo culture fluid, microbial culture medium, soil leachate, bone meal leachate, etc. Free DNA can be obtained through extraction and purification.
  • the total amount of cell-free DNA used for downstream sequencing analysis can be as low as picogram or nanogram levels.
  • This step uses a specific ligase (Ligase), which can be but is not limited to any one or a combination of several repair reagents and/or DNA ligases: HiFi-Taq ligase, T4 DNA Ligase, Taq DNA Ligase, T7 DNA Ligase, E.coli DNA ligase, etc.
  • a specific ligase (Ligase)
  • HiFi-Taq ligase T4 DNA Ligase
  • Taq DNA Ligase Taq DNA Ligase
  • T7 DNA Ligase T7 DNA Ligase
  • E.coli DNA ligase etc.
  • DNA ligase and/or DNA repair reagents repair the gaps in the double strands of the template DNA to prevent the loss of DNA strand breaks and/or the erasure of methylation information in the subsequent process.
  • a (adenine) base is added to the end to facilitate adapter connection.
  • This step uses DNA ligase to ligate a specific sequence of DNA adapters with partial/all cytosine methylation or hydroxymethylation modification to both ends of the template DNA.
  • the DNA linker sequence can be labeled with biotin to facilitate subsequent purification.
  • sample barcodes and single DNA molecule barcodes can be introduced on the DNA linker sequence to mark the source of the DNA template, increase detection accuracy, and also allow samples from multiple sources to be mixed in subsequent steps.
  • carrier DNA with similar properties that is several times the amount of template DNA is added to the template DNA solution. This can be used in purification and other processes that are prone to DNA loss or degradation. This step becomes the main lost part, effectively avoiding the loss of template DNA.
  • carrier DNA does not contain a specific sequence of DNA adapter sequences, it will not be detected in subsequent library amplification and sequencing.
  • This step uses an enzymatic conversion method to specifically convert cytosine, methylated cytosine and/or hydroxymethylated cytosine on DNA, so that cytosine and the base of methylated and or hydroxymethylated cytosine are
  • the base pairing rules change and correspond to different bases during subsequent amplification and sequencing processes, thereby enabling differentiation.
  • This step can use the "EM-seq” technology invented by New England Biolabs.
  • TAPS technology can be used.
  • the library will be subjected to PCR amplification reaction, and the amplification primers contain sequences complementary to the above-mentioned adapter sequences and sequencer adapter sequences, and sample barcodes can also be introduced.
  • the primers can be complementary to the adapter sequences added at both ends of the template DNA to form the desired sequencing library during amplification.
  • the sequencing library is quantified and quality checked, and then delivered to a sequencer for sequencing.
  • DNA methylation information can be obtained through comparison with the reference genome. Since this method adds library adapters before methylation conversion and when the free DNA remains in a double-stranded state, the DNA fragment information is retained. Through comparison with the reference genome, the original DNA fragment length, fragment distribution, and Fragment omics information such as upstream and downstream breakpoint positions, terminal base patterns, etc.
  • the above analysis process can be referred to Figure 1.
  • DNA hydroxymethylation is less abundant than DNA methylation but has different biological significance.
  • Enriching target fragments of interest can save sequencing costs and improve the detection sensitivity of target genes.
  • step (6) of the above method overview using primers complementary to the adapters at both ends of the template DNA to perform PCR will amplify the entire template DNA; if you need to enrich the target gene fragment, you can use targeted PCR. amplification. Design a targeting primer containing a sequence complementary to the target gene fragment after methylation conversion, and put it into the PCR reaction system to amplify the target gene sequence.
  • Targeting primers may or may not contain some or all of the adapter sequences that are compatible with the sequencer.
  • the upstream and downstream primers can also be connected end-to-end and constructed on a piece of DNA to form a locked primer.
  • step (6) of the method overview section above hybridization capture enrichment can be performed before amplification or after amplification. It is necessary to design a probe containing a sequence complementary to the target gene fragment after methylation conversion.
  • Hybrid capture enrichment uses biotin-labeled single-stranded DNA/RNA probes mixed with the library. After thermal denaturation and renaturation, the library complementary to the target sequence will hybridize with the probe. The hybridized probe can be used with Capture using streptavidin magnetic beads and other methods to obtain an enriched library.
  • the methylation information downstream of the gap will be changed. By analyzing sequences where methylation has been erased/changed, the gap location can be determined.
  • the invention discloses a highly sensitive DNA methylation sequencing technology that can be used for DNA with single-strand gaps.
  • This technology first repairs DNA gap damage to ensure that DNA methylation signals will not be lost or misread.
  • this technology uses double-stranded DNA as a template to build libraries, overcoming the low efficiency of single-stranded library construction, and then uses it to greatly optimize
  • the adjusted enzymatic conversion method converts the methylation information into base pairing rules, and cooperates with the protection of the template DNA during the reaction and purification process of the carrier DNA.
  • a very low starting amount of DNA is used as a template to obtain high quality.
  • highly sensitive methylation information sequencing library while retaining DNA fragment omics information.
  • Minimum starting amount of DNA as low as 0.1ng or even lower;
  • Library output can be increased by 4 times
  • the method of the present invention can be applied to various liquid biopsy diagnostic products such as early cancer screening, cancer gene detection, prenatal diagnosis, preimplantation diagnosis, genetic diagnosis, etc., and also provides strong technical support for scientific research in related fields.
  • This embodiment uses peripheral blood cfDNA as an example to describe the sequencing library preparation process.
  • Extraction of cfDNA can be performed using any standard means in the art.
  • Sonicated Spike-in DNA is a mixture of whole-genome CpG methylated pUC19 DNA (NEB E7122) and whole-genome CpG unmethylated lambda DNA (NEB E7123) in equal volumes, broken to 300bp using ultrasonic waves and then diluted to 0.2ng/ ⁇ L for later use.
  • Carrier DNA The preparation method of Carrier DNA is as follows: use ultrasonic wave to break lambda DNA (NEB N3011) to 300bp and then dilute it to 25ng/ ⁇ L for later use.
  • Enzymatic Methyl-seq Conversion Module (NEB, E7125L) kit is used for enzymatic conversion. Including TET oxidation reaction and glycosylation protection of DNA, DNA thermal denaturation, and APOBEC deamination reaction. After the APOBEC deamination reaction, no purification is performed and the next reaction is carried out directly.
  • PCR primers with library adapters and Index tags to the liquid after the APOBEC deamination reaction in the previous step, for example, add 5 ⁇ L of Multiplex Oligos for Illumina primer pair. Then add 2x Q5U master mix (NEB M0597L) equal to the volume of existing liquid. Mix briefly and centrifuge, then proceed on a PCR machine:
  • paired-end sequencing is performed on an Illumina sequencer. Sequencing off-machine data were compared and analyzed using Bismark software (in order to avoid bases with unevenly distributed methylation rates, the methylation rates of the first ⁇ 10 bases of Read1 and the first ⁇ 40 bases of Read2 will be different. Included in the methylation analysis results), genome-wide methylation data were obtained.
  • the library After bioinformatics analysis, the library’s distribution characteristics on the genome, fragment length, start and end positions, 5’ end base distribution characteristics and other information are fragment omics information.
  • Sequencing results contain tens of millions to billions of pieces of the above information, and the sequencing volume varies depending on the sequencing instrument and chip selection.
  • Control group no DNA ligase treatment (as a control group, water was used instead of DNA ligase)
  • the template DNA was purified by magnetic beads and used Enzymatic Methyl-seq Conversion Module (NEB, E7125L) kit performs enzymatic conversion processing.
  • the transformed DNA was used for PCR library amplification using 2.Q5U master mi. (NEB M0597L).
  • the constructed library was subjected to paired-end 150bp sequencing on an Illumina sequencer, and Bismark software was used for comparison and analysis.
  • the methylation bias (M-bias) of the library was statistically analyzed along the 5' to 3' direction of the library fragment. , plot the average methylation rate at each read position to evaluate whether the methylation level of the library is average. The results are shown in Figure 4.
  • Example 3 Fragmentomics information can be obtained while obtaining methylation information
  • the DNA output after library construction was quantified by Qubit.
  • the library yield of the technical method of the present invention was 83.4ng, which was much higher than the yield of the library constructed by the Swift method;
  • the sequencing library of the technical method of the present invention The alignment rate (align rate) is 70%, which is nearly twice as high as the 44% of the swift library, indicating that the library is more effective;
  • the duplication rate (duplication rate) of the sequencing library constructed by this method is 15%, which is significantly lower than that of the swift library.
  • the 22% duplication rate of the library indicates that there are fewer duplicate fragments and better quality in the library; this method retains fragment omics information (see Figures 5 and 6).
  • the Agilent pulsed field automated electrophoresis fragment analysis system (Agilent Femto Pulse) was used to analyze the DNA fragments before library construction. The results are shown in Figure 5.
  • the free DNA itself has a characteristic peak spectrum with a period of about 170 bp, which is a typical Fragmentation characteristics caused by nucleosome occupancy; use the Agilent 5200 Fragment Analyzer System to analyze the fragments after the library construction.
  • the results show (see Figure 6), compared with the library constructed with the Swift kit , the sequencing library constructed by the library construction sequencing method provided by the present invention contains the fragmented distribution of free DNA characteristics, which can better analyze and obtain fragment omics data.
  • This invention points out for the first time that the severe methylation loss problem caused by double-stranded library construction is caused by single-strand gaps and terminal single-strand deletions.
  • the terminal single-strand deletion is an irreversible loss of original information; while single-strand gaps cause Methylation was wiped out across most reads.
  • the principle is: in the double-stranded library construction step, the double-stranded DNA template needs to be end-repaired first to form a DNA double-stranded with a blunt end or a 3’-A sticky end, and then the adapter sequence is connected.
  • the DNA synthase used in end repair has the activity of synthesis from the nick (Extension from Nick).

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Abstract

La présente invention concerne un procédé de construction d'une banque de séquençage de méthylation de l'ADN libre et son utilisation. Selon le procédé, la méthylation de l'ADN et la fragmentomique peuvent être détectées en même temps. Le procédé de la présente invention comprend les étapes suivantes : lors de l'établissement d'une banque de séquençage, réparation d'une lacune de liaison phosphodiester sur un ADN double brin, connexion de l'ADN double brin à une séquence de liaison, et établissement d'une banque de séquençage de méthylation de l'ADN selon un procédé de transformation enzymatique. Le procédé de la présente invention peut préserver l'intégrité de l'ADN double brin, et empêcher la rupture des brins d'ADN, la perte d'informations fragmentomiques dans la banque de séquençage, et l'effacement de la modification de méthylation de l'ADN en aval de la lacune dans le processus de réparation terminale conduisant à l'acquisition d'informations inexactes sur la méthylation de l'ADN.
PCT/CN2022/103499 2022-04-08 2022-07-02 Procédé de construction d'une banque de séquençage de méthylation de l'adn libre et son utilisation WO2023193357A1 (fr)

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