CN117305466B - Detection method capable of identifying single base methylation state - Google Patents

Detection method capable of identifying single base methylation state Download PDF

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CN117305466B
CN117305466B CN202311607291.6A CN202311607291A CN117305466B CN 117305466 B CN117305466 B CN 117305466B CN 202311607291 A CN202311607291 A CN 202311607291A CN 117305466 B CN117305466 B CN 117305466B
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CN117305466A (en
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余丽萍
储俊东
吴强
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Naonda Nanjing Biological Technology Co ltd
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Abstract

Provided herein is a detection method capable of recognizing a single base methylation state, comprising: 1) Constructing a DNA library by using the DNA molecules in the sample, and obtaining a methylation library after transformation; 2) Performing hybrid capture on the methylation library to construct a targeted methylation library; and 3) sequencing the targeted methylation library and comparing to the base sequence of the untransformed DNA molecule to determine the methylation status of the DNA molecule, wherein the hybridization capture is performed in a hybridization system comprising at least one single stranded oligonucleotide blocking reagent having a sequence selected from the group consisting of: tn, (GT) p and (TA) q, wherein n is an integer from 15 to 18, p is an integer from 6 to 8, and q is an integer from 6 to 8. Kits for detecting the methylation state of a DNA molecule in a sample are also provided herein.

Description

Detection method capable of identifying single base methylation state
Technical Field
The present application relates to a method of detecting the methylation state of a DNA molecule in a sample, and to a kit for detecting the methylation state of a DNA molecule in a sample.
Background
DNA methylation is associated with many physiological, pathological factors, carrying a large amount of biological information. The occurrence and progression of cancer is accompanied by changes in the pattern of DNA methylation, including DNA hypomethylation of reverse transcription elements, centromeres and proto-oncogenes, and methylation of key gene regulatory elements associated with gene suppression (e.g., overlapping regions of distal enhancers and promoter transcription initiation). Most CpG sites in the normal genome carry 5mC, while the distal enhancer element and CpG island region are resistant to the activity of methyltransferase DNMT, and the silencing of oncogene underexpression and DNA damage repair genes is an important mechanism for carcinogenesis. Cancer cells are primarily characterized by an overall range of unmethylated genetic modifications, rather than abnormal methylation sites within enhancer and promoter regions. This change in methylation profile results in inhibition of tumor suppressor gene expression, accompanied by an increase in protooncogene expression, thereby further promoting tumor development and progression, and therefore detection of gene methylation is more suitable as a marker for cancer detection.
In terms of detection technology, traditional pyrosequencing is considered as a "gold standard" of methylation detection, and pyrosequencing technology is an enzyme cascade chemiluminescent reaction in the same reaction system catalyzed by 4 enzymes, and the reaction system of pyrosequencing technology consists of a reaction substrate, a single strand to be detected, a sequencing primer and 4 enzymes. The 4 enzymes are respectively: the reaction substrates of the DNA polymerase, the ATP sulfurylase, the luciferase and the apyrase are 5' -phosphosulfate (APS) and fluorescein, and the reaction system also comprises a DNA single strand to be sequenced and a sequencing primer. After annealing the primer and the template DNA, under the synergistic effect of 4 enzymes of DNA polymerase, ATP sulfurylase, luciferase and apyrase, the polymerization of each dNTP on the primer is coupled with the release of a primary fluorescence signal, and the purpose of measuring the DNA sequence in real time is achieved by detecting the release and the intensity of fluorescence. However, since the pyrosequencing template is based on a bisulfite converted sequence, it is difficult to design primers, and it is impossible to design appropriate primers for detection of a part of methylation sites, and the actual level of detection is greatly different for samples with low methylation levels (< 10%).
In addition, the targeted methylation detection technology based on liquid phase hybridization is a high-throughput and accurate methylation detection method. It can more accurately recognize methylation sites and obtain deeper methylation information than Whole Genome Bisulfite Sequencing (WGBS). However, the liquid phase hybridization capture procedure is very time consuming, requiring 2-4 days from nucleic acid extraction to capture library acquisition; meanwhile, the hybridization capture is complicated in reagent types and abnormal in operation flow, the technical requirements on operators are high, and the performance of the captured library can be influenced when any link in the middle is problematic. These links become key technical bottlenecks that limit the development of liquid phase hybridization capture.
Disclosure of Invention
In one aspect, provided herein is a method of detecting the methylation state of a DNA molecule in a sample, comprising:
1) Constructing a DNA library by using the DNA molecules in the sample, and obtaining a methylation library after transformation;
2) Performing hybrid capture on the methylation library to construct a targeted methylation library; and
3) Sequencing said targeted methylation library and comparing the base sequence of said DNA molecule that has not been transformed to determine the methylation status of said DNA molecule,
wherein the hybridization capture is performed in a hybridization system comprising at least one single stranded oligonucleotide blocking reagent having a sequence selected from the group consisting of: tn, (GT) p and (TA) q, wherein n is an integer from 15 to 18, p is an integer from 6 to 8, and q is an integer from 6 to 8.
In some embodiments, the single stranded oligonucleotide blocking reagent is a combination of single stranded oligonucleotide blocking reagents of sequences Tn and (GT) p.
In some embodiments, the single stranded oligonucleotide blocking reagent comprises one or more locked or bridged nucleobases, preferably 5-8 locked or bridged nucleobases.
In some embodiments, the hybridized capture is performed using a capture probe in the form of μcaler comprising a target-specific sequence, a first probe-binding sequence located 5 'to the target-specific sequence, and a second probe-binding sequence located 3' to the target-specific sequence, the first probe-binding sequence being at least partially complementary to the second probe-binding sequence such that when two or more capture probes in the form of μcaler bind their respective target sequences in an adjacent manner, complementary binding can be formed therebetween by the first probe-binding sequence and the second probe-binding sequence.
In some embodiments, the first and second probe binding sequences are 8-30nt in length.
In some embodiments, the target-specific sequence is 20-80nt in length.
In some embodiments, the concentration of each of the single stranded oligonucleotide blocking reagents in the hybridization system is 100 μm.
In some embodiments, the capture probe is used in the hybridization system in an amount of 1 fmol/rxn.
In some embodiments, the hybridization system is further supplemented with a linker blocking reagent and a Human Cot DNA.
In some embodiments, the DNA molecule is from genomic DNA, plasma episomal DNA, or FFPE samples.
In another aspect, provided herein is a hybridization capture kit comprising a capture probe and at least one single stranded oligonucleotide blocking reagent, wherein the sequence of the oligonucleotide blocking reagent is selected from the group consisting of: tn, (GT) p and (TA) q, wherein n is an integer from 15 to 18, p is an integer from 6 to 8, and q is an integer from 6 to 8.
In some embodiments, the single stranded oligonucleotide blocking reagent is a combination of single stranded oligonucleotide blocking reagents of sequences Tn and (GT) p.
In some embodiments, the single stranded oligonucleotide blocking reagent comprises one or more locked or bridged nucleobases, preferably 5-8 locked or bridged nucleobases.
In some embodiments, the capture probe is a μcaler format probe comprising a target-specific sequence, a first probe-binding sequence located 5 'to the target-specific sequence, and a second probe-binding sequence located 3' to the target-specific sequence, the first probe-binding sequence being at least partially complementary to the second probe-binding sequence such that when two or more of the μcaler format probes bind their respective target sequences in an adjacent manner, complementary binding can be formed therebetween by the first probe-binding sequence and the second probe-binding sequence.
In some embodiments, the first and second probe binding sequences are 8-30nt in length.
In some embodiments, the target-specific sequence is 20-80nt in length.
In some embodiments, the hybrid capture kit further comprises a linker blocking reagent and a Human Cot DNA.
The methylation detection method and the kit provided herein can improve the capture mid-target rate, improve the sequencing depth, enhance the detection stability, and are suitable for mixed hybridization detection of multiple samples.
Drawings
Fig. 1: construction of methylation library and preparation procedure of targeted methylation capture library.
Fig. 2: the traditional targeted methylation-captured sequencing was compared to the methylation-captured sequencing procedure of the present application.
Fig. 3: the performance differences of the traditional hybrid capture method and the hybrid capture method of the application are compared.
Fig. 4: methylation levels detected in different samples.
Fig. 5: cpG site coverage depth comparison at different probe usage levels.
Fig. 6: the CpG site coverage depth of different groups was compared.
Fig. 7: the CpG site coverage depth of different groups was compared.
Detailed Description
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The term "or" refers to a single element of a list of selectable elements unless the context clearly indicates otherwise. The term "and/or" means any one, any two, any three, any more, or all of the listed selectable elements.
The terms "comprising," "including," "having," and similar referents used herein do not exclude the presence of unrecited elements. These terms also include cases consisting of only the recited elements.
"DNA molecule" refers herein to a deoxyribonucleic acid, a polymer of deoxyribonucleotides. It may be in double-stranded form, single-stranded form, or, for a population of DNA molecules, may include double-stranded DNA molecules, and may also include single-stranded DNA molecules. The DNA molecule may be of any length, for example, genomic DNA, DNA fragments or extracellular free DNA. Free DNA refers to DNA that is free outside of the cell or nucleus and can be extracted from a variety of biological materials including, but not limited to, body fluids, in vitro cell culture fluids, natural environments, and the like: peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph, etc. The free DNA can be obtained by extraction and purification. "DNA fragment" refers herein to a shorter DNA molecule, for example having a length of between 50bp and 700 bp, for example 100 bp and 500 bp, in particular 100 bp to 350 bp. The DNA fragments contained in a biological sample are typically heterogeneous, i.e. of unequal length, and accordingly, the above-mentioned length may refer to the length average of these DNA fragments. These DNA fragments may have different sequences, for example from different regions of the genome of the same organism, or even from different organisms. The DNA fragment may be single-stranded, blunt-ended or non-blunt-ended (with 3 'or 5' overhang).
"DNA methylation" refers herein to methylation modification of bases in a DNA molecule or DNA fragment, in particular cytosine to 5-methylcytosine (5 mC). DNA methylation in vertebrates typically occurs at CpG sites (i.e., sites in the DNA sequence immediately following cytosine by guanine) and is catalyzed by DNA methyltransferases to convert cytosine to 5-methylcytosine. Most CpG sites in the human genome have been methylated, but in certain specific regions, such as CpG islands rich in cytosine (C) and guanine (G), are typically unmethylated. CpG methylation can affect transcriptional activity of related genes, e.g., methylation can inhibit cancer suppressor genes, whereas demethylation can stimulate the expression of certain oncogenes, which can lead to the occurrence of cancer. In addition, although the incidence is less than 5mC, few cytosine bases are modified to 5-hydroxymethylcytosine (5 hmC), 5-formylation (5 fC), 5-carboxylation (5 caC). References herein to methylation may also refer to modifications as 5hmC, 5fC, 5caC unless the context indicates otherwise.
"cytosine base methylation state" or "DNA methylation state" refers herein to information about the methylation status in a DNA molecule or DNA fragment, including, but not limited to, the methylation site, the level of methylation, the manner of methylation (e.g., 5mC or 5 hmC), and the like. "methylation level," which may also be referred to as "degree of methylation," refers to the proportion (or frequency) of methylation modification of a particular methylation site in a sample. Whether a site is methylated can be detected in a number of ways. Common methods include chemical or enzymatic conversion methods in which one of methylated cytosine and unmethylated cytosine is converted to uracil (U) or a base substantially identical to uracil in base pairing (e.g., dihydrouracil, DHU). In a subsequent amplification process, the corresponding uracil is paired with adenine (A) as thymine (T), with the end result that the cytosine or methylated cytosine at the methylation site is represented as thymine in the detection result (e.g., sequencing result). By comparison with the reference sequence, it is possible to determine whether or not the cytosine in the DNA molecule or DNA fragment is methylated. The reference sequence may be a sequence from the same sample but not transformed as described above, or a corresponding sequence in a healthy population. The 5mC and 5hmC can also be distinguished by some means. Currently, DNA methylation information has been widely used in cancer (e.g., lung cancer, breast cancer, liver cancer, colorectal cancer, etc.) screening and diagnosis, particularly early screening and diagnosis. In addition, the identification of methylation status may also be used for non-diagnostic (or therapeutic) purposes, e.g., for scientific research, such as analysis of factors affecting methylation, methylation effects on gene function, or for detection of methylation standards, etc.
"Capture probe" or "hybridization Capture probe" refers herein to a single-stranded DNA fragment that is used to hybridize to a transformed target DNA molecule (or fragment thereof). In a preferred embodiment of the present application, the capture probes are probes in the form of μcaler (see chinese patent publication CN116083423a or CN114891859a for the design of μcaler probes, which are incorporated herein by reference in their entirety), such probes comprising a target-specific sequence, a first probe-binding sequence located 5 'to said target-specific sequence and a second probe-binding sequence located 3' to said target-specific sequence, the first probe-binding sequence being at least partially complementary to the second probe-binding sequence such that when two capture probes or more capture probes bind to said target sequence in an adjacent manner, complementary binding can be formed between adjacent capture probes by said first probe-binding sequence and said second probe-binding sequence. In a preferred embodiment, the first and second probe binding sequences are 8-30nt in length; the length of the target-specific sequence is 20-80nt. The use of a probe in the form of a μCaler has at least the benefit of significantly improving the binding capacity of the probe to the target, improving the efficiency of hybridization capture to the target region, improving overall coverage uniformity and stability over conventional capture probes (such as conventional 120 base probes as mentioned in the examples below). The capture probes may typically bear a detachable tag, such as biotin or the like, to isolate the target DNA molecule to which the probe is bound. The methylation status can be determined by sequencing the different target DNA molecules isolated (optionally PCR amplified) to obtain their sequence information.
"DNA library" as used herein refers to a library of sample DNA molecules that have undergone end repair and linker (e.g., methylated linker) ligation. Optionally, the DNA molecule may be fragmented prior to end repair to form 200-250bp DNA fragments. "methylation library" refers herein to a library of transformed DNA. "transformation" herein includes the chemical or enzymatic transformations mentioned above, the purpose of which is to distinguish between methylated and unmethylated cytosine bases. "targeted methylation library" refers herein to a collection of DNA molecules that are capable of binding to the capture probes used, obtained by subjecting the methylation library to further hybridization capture processing. Sequencing the targeted methylation library can achieve higher sequencing depth, uniformity, etc. with reduced sequencing costs.
"Single-stranded oligonucleotide blocking reagent", "methylation blocking reagent (MeBlocker)" or "blocking reagent" herein refers to a single-stranded oligonucleotide for specific binding to repetitive sequences generated after transformation that prevents binding of capture probes to these regions of high reproducibility, thereby improving hybridization capture efficiency. The single stranded oligonucleotide blocking reagents may have different nucleotide sequences, which may be used alone or in combination.
Provided herein is a novel targeted methylation library hybridization capture method, the flow of which comprises the preparation of a methylation library from the construction of the methylation library to the targeted methylation library, providing a detection method capable of recognizing the methylation state of a single base. The detection method comprises the following steps: the method comprises the steps of constructing a methylation library and carrying out hybridization capture on the novel targeted methylation library, wherein the hybridization capture of the novel targeted methylation library comprises sampling the methylation library, hybridizing the methylation library, capturing the methylation library, eluting the methylation library at normal temperature, amplifying the targeted methylation library and purifying the targeted methylation library, and the purified targeted methylation library is the targeted methylation sequencing library. The method can be used for detecting the single-base methylation state in the nucleic acid samples from various sources, shortens the experimental time, simplifies the experimental process and effectively improves the accuracy of the single-base methylation state.
In some embodiments, a methylation blocking reagent (MeBlocker) is added to the hybridization capture system to increase the hybridization capture efficiency of the transformed library. The blocking reagent is composed of a nucleic acid sequence that is capable of specifically binding to the transformed repetitive sequence. The blocking reagent sequence may include 5-7 LNA or BNA modified bases that increase the binding efficiency of the sequence to the target. After conversion of some of the high GC regions, they become AT rich regions, and probes bind to these high repetition regions, capturing more non-target regions.
In some embodiments, the blocking reagent sequence is a contiguous T base, 15-18 in length; or a repetitive GT sequence, the number of repetitions being 6-8; or repeating TA bases, the number of the repeats being 6-8. The blocking reagent sequence may include 5-7 LNA or BNA modified bases.
In some embodiments, the hybridization capture system is optimized for the capture probe usage scheme, with a final methylation probe concentration of 1-2fmol, and an optimal concentration of 1fmol.
In some embodiments, the hybridization system comprises a capture probe, 1 XHyb Buffer, 1 XEnhance, 1ug Human Cot-1, and 100pmmol of a blocking linker sequence of a blocking linker and a Meblocking linker of 100 um.
In comparison with pyrosequencing, pyrosequencing is difficult to design due to primer design for sequences (low sequence complexity, repeated base-based) after BS transformation. The method is designed aiming at small Panel (< 10 kb), and can be designed aiming at methylation sites contained in promoter regions of different genes, so that the number of detected methylation sites is large, the flux is high, and the accuracy of methylation level is high.
Compared with the traditional targeted methylation hybridization capture sequencing, the detection method optimizes the experimental flow, shortens the experimental time, can finish the targeted methylation hybridization capture operation within 4 hours, shortens the system flow time by 14-15 hours, and can construct the targeted methylation hybridization capture library within one day. The detection method flow comprises the preparation of methylation library construction to targeted methylation capture libraries.
Construction of methylation library and preparation of targeted methylation Capture library the flow scheme for the preparation of the methylation-captured library is shown in FIG. 1. The process is suitable for constructing methylation libraries of genomic DNA, plasma free DNA or FFPE samples. The flow is briefly described as follows:
construction of methylation library: the genome DNA is broken into 200-250bp DNA fragments by using an ultrasonic breaking mode; end repair, methylation linker ligation, conversion of linker ligation products using a bisulfite conversion module, PCR amplification using an amplification enzyme capable of recognizing U bases (e.g., KAPA HiFi HotStart Uracil Mix), and quantification of methylation libraries using a Qubit quantification reagent are performed on genomic DNA, plasma free DNA, or FFPE samples.
Preparation of targeted methylation-captured libraries: selecting a methylation library meeting the requirements, performing hybridization, streptavidin magnetic bead capturing and eluting, performing PCR amplification after eluting, performing magnetic bead purification on the amplified product, and sequencing the purified product by using an Illumina Nova-Seq sequencer. Compared with the traditional targeted methylation hybridization capture sequencing, the method optimizes the experimental flow and shortens the experimental time.
The invention is further illustrated by the following specific examples.
Example 1:
using the detection method, the targeted methylation hybridization capture is carried out by designing a conventional 120-base probe, and the advantages and disadvantages of different detection methods are compared (see FIG. 2 for experimental process).
(1) Construction of methylation libraries
The genome DNA is broken into 200-250bp DNA fragments by using an ultrasonic breaking mode; 50ng of the samples after disruption were taken, and a methylation library was constructed using the NadPrep library construction kit (for Illumina) # 1002141. And (3) performing end repair, addition of A and methylation joint connection on the cut genomic DNA, and converting a joint connection product. The ligation product was transformed as a DNA library in the following examples, the transformation product was subjected to PCR amplification with a PCR amplification cycle number of 11, the amplification product was subjected to magnetic bead purification, and the purified product was sent to an Illumina Nova-Seq sequencer for whole genome sequencing.
(2) Methylation library quality inspection
Ensures that the transformation efficiency reaches the standard, and performs targeted methylation hybridization capture and detection methods.
(3) Traditional targeted methylation hybridization Capture
Performing hybridization capture by using NadPrep Hybrid Capture Reagents #1005102, taking 500ng of constructed methylation library, sequentially performing vacuum concentration, hybridization, streptavidin magnetic bead capture and library elution, performing PCR amplification after elution, performing magnetic bead purification on amplified products, and sequencing the purified products by using an IlluminaNova-Seq sequencer, wherein the conventional 120-base probe design is performed by referring to a liquid-phase hybridization capture method and a kit of China patent publication CN 114891859A.
(4) Detection method of the application
Taking 500ng of constructed methylation library, sequentially hybridizing the methylation library, capturing streptavidin magnetic beads and eluting the library, performing PCR amplification after eluting, purifying the amplified product by using magnetic beads, and sequencing the purified product by using an Illumina Nova-Seq sequencer, wherein the design of a mu Caler capture probe is performed by referring to a liquid phase hybridization capturing method and a kit thereof in Chinese patent publication CN 114891859A. The Human Cot DNA is applied to block repeated sequences in Human genome; mu Caller nanoblocks (for Illumina) were used to block the linker sequences and the linker sequences of the different sequencing platforms (mainly divided into Illumina or MGI platforms) were adjusted, only the test performance of the Illumina platform was shown here. And (3) combining the capture probe with the converted target sequence by using a mu Caler Methylation-beta Panel methylation hybridization, combining streptavidin magnetic beads with biotin of the probe, and capturing the target. MeBlocker is used to block the converted tandem repeat sequence; μhyb#1 and μhyb#2 provide optimal hybridization capture conditions for the hybridization capture buffer.
The following is the specific experimental procedure:
(a) Library hybridization (see Table 1 for components)
TABLE 1 library hybridization Components
。
* Including MeBlockerA: TTT+TTT+ TTT+TTT+ tt+tt (SEQ ID NO: 1); and MeBlockerB: GTG+TGT+GT+GTGTGT (+stands for LNA modified base) as the latter base (SEQ ID NO: 2). The concentration of the blocking reagent working solution of the MeBlockerA and MeBlockerB is 100 mu M.
Vortex mixing hybridization reaction mixture for more than 10 sec, instantaneous centrifugation, and then collect the reaction mixture to the bottom of PCR tube (avoid generating bubbles). The PCR tube was placed in a PCR instrument and the reaction procedure was started according to Table 2.
TABLE 2 hybridization procedure
。
(b) Magnetic bead cleaning
Streptavidin Beads was vortexed for 15 sec to ensure complete mixing.
Mix and wash n×25μ L Streptavidin Beads in 0.2 mL centrifuge tube (n is the total capture reaction number and n.ltoreq.5). Streptavidin Beads the solution was allowed to stand on a magnetic rack for about 2 min, and the supernatant was discarded using a pipette after the solution was completely clarified.
The centrifuge tube was removed from the magnet rack, 150. Mu.L of pre-heated Wash buffer 1 (5 XSSPE, 1% SDS) was added, and gently swirled and mixed more than 10 times.
The centrifuge tube was placed on a magnetic rack for about 2 min, and after the liquid was completely clarified, the supernatant was discarded using a pipette.
The centrifuge tube is removed from the magnetic rack, 150 mu L of preheated Wash buffer 1 is added, and the mixture is gently blown and sucked for more than 10 times.
The centrifuge tube was placed on a magnetic rack for about 2 min, and after the liquid was completely clarified, the supernatant was discarded using a pipette.
The centrifuge tube was centrifuged instantaneously and then placed on a magnetic rack for 10 sec, and Wash buffer 1 at the bottom of the tube was completely discarded by a 10. Mu.L tip.
The mixture was gently rinsed by resuspension Streptavidin Beads of n.8μl μhyb#1 for 10 times or more.
(c) Magnetic bead capture
2 hr, entering a capturing link after hybridization reaction, and maintaining the running state of the PCR instrument.
While keeping the PCR tube in the PCR apparatus, 8. Mu.L of the resuspended Streptavidin Beads was immediately added to each hybridization reaction solution, and the mixture was gently sucked and mixed for 10 times or more.
60. Incubating at 10 min.
After the incubation is finished, the PCR tube is taken off from the PCR instrument and placed on a magnetic rack for standing for 2 min, and after the liquid is completely clarified, the supernatant is discarded by using a pipette (the supernatant is removed as much as possible).
(d) Elution
The PCR tube of the previous step is removed from the magnetic rack, 150 mu L of preheated Wash Buffer 1 is added, and the mixture is gently blown and sucked for more than 10 times (avoiding generating bubbles).
The PCR tube was placed on a magnetic rack for 2 min, and after the liquid was completely clarified, the supernatant was discarded using a pipette.
Wash Buffer 2 (2 XSSPE, 0.1% SDS) was added, and the mixture was gently sucked and mixed for 10 times or more (room temperature operation, avoiding air bubbles). The reaction solution was transferred to a new PCR tube.
The PCR tube was placed in a PCR apparatus and incubated at 61℃for 15 min.
And after the incubation is finished, the PCR tube is taken off from the PCR instrument and placed on a magnetic rack for standing for 2 min, and after the liquid is completely clarified, the supernatant is discarded by using a pipette.
The PCR tube was removed from the magnetic rack, 150. Mu.L of preheated Wash Buffer 1 was added, and the mixture was gently sucked and mixed for more than 10 times (to avoid air bubbles).
The PCR tube was placed in a PCR instrument and incubated at 61℃for 3 min.
And after the incubation is finished, the PCR tube is taken off from the PCR instrument and placed on a magnetic rack for standing for 2 min, and after the liquid is completely clarified, the supernatant is discarded by using a pipette.
150. Mu.L of Wash Buffer 3 (0.1 XSSPE, 0.01% SDS) placed at room temperature was added to the PCR tube, and the mixture was gently sucked and mixed for 10 times or more.
The PCR tube was placed on a magnetic rack for 2 min, and after the liquid was completely clarified, the supernatant was discarded using a pipette.
And (3) placing the PCR tube on a magnetic rack after instantaneous centrifugation, and removing a small amount of residual liquid by using a 10 mu L suction head, wherein the magnetic bead is not sucked.
22.5 mu L Nuclease Free Water was added and the beads were gently flushed more than 10 times to resuspend.
(e) Hybrid Capture library PCR amplification
And taking out NadPrep 2X HiFi PCR Master Mix and NadPrep Amplification Primer Mix II, naturally melting on ice, mixing by using a pipette or a vortex mixer, and centrifuging instantly for standby.
And (3) configuring a PCR reaction system according to the table 3, and adding the configured PCR reaction system into the PCR reaction tube containing the magnetic beads in the step (d).
TABLE 3 PCR reaction System
。
The PCR tube was placed in a PCR apparatus and the amplification procedure was started as shown in Table 4, and the temperature of the hot cap was set at 105 ℃.
TABLE 4 PCR procedure
。
(f) Amplified library purification
Adding 50 mu L of NadPrep cube SP Beads into the PCR tube of the amplification reaction product, uniformly mixing by using a pipette or a vortex mixer, and incubating for 10 min at room temperature.
And (3) placing the PCR tube on a magnetic rack for standing for 5 min after instantaneous centrifugation, and sucking and discarding the supernatant by using a pipette after the liquid is completely clarified.
Slowly adding 150 mu L of 80% ethanol along the side wall of the PCR tube, taking care of not disturbing the magnetic beads, standing for 30 sec, and sucking and discarding the supernatant by using a pipette.
Slowly adding 150 mu L of 80% ethanol along the side wall of the PCR tube, taking care of not disturbing the magnetic beads, standing for 30 sec, and sucking and discarding the supernatant by using a pipette.
The PCR tube is placed on a magnetic frame after instantaneous centrifugation, a small amount of residual ethanol is removed by using a 10 mu L suction head, and the PCR tube is not sucked to magnetic beads;
the PCR tube cap was opened and allowed to stand at room temperature for about 2-5 min until ethanol was completely volatilized.
The PCR tube was removed from the magnetic rack, 20. Mu.L of TE Solution was added, and the beads were suspended uniformly using a pipette or vortex mixer and incubated for 5 min at room temperature.
After the PCR tube was centrifuged instantaneously for 2 min on a magnetic rack, after the liquid was completely clarified, the supernatant was carefully transferred to a new 0.2 mL PCR tube or 1.5 mL centrifuge tube for storage using a pipette, taking care not to suck the beads.
As a result, referring to FIG. 3, the experimental result is shown, the experimental process is optimized, the experimental time cost is saved, the target rate in the targeted methylation capturing library can be improved, the original depth and the depth after the duplication removal are improved, and meanwhile, the Fold 80 is far smaller than the conventional 120nt probe data.
Example 2:
using the detection methods of the present application, the accuracy of methylation level detection is compared to pyrosequencing
Construction of methylation library, library construction procedure see example 1, mixing 100%Human Methylated DNA with two standards of Non-Methylated DNA in a certain ratio, preparing samples with methylation levels of 0%, 2%, 10%, 50% and 100%, respectively taking 50ng samples with different methylation levels, detecting methylation levels of CpG sites in the target region by using the experimental scheme of the application, and taking 50ng samples with different methylation levels, and crossing the samples with different methylation levels to Shanghai Jielan Biotech Co., ltd for pyrosequencing, wherein each sample is subjected to 3 repetitions, and sample information is shown in Table 5.
Table 5 sample information formulated
。
(1) Hybridization capture and pyrophosphate sequencing covered target sequence information see table 6:
target sequence information covered by Table 6
。
(2) Methylation library quality inspection
See example 1.
(3) Magnetic bead cleaning
See example 1.
(4) Elution
See example 1.
(5) Hybrid Capture library PCR amplification
See example 1.
(6) Amplified library purification
See example 1.
Conclusion: the results are shown in FIG. 4. From the figure, the novel hybrid capture sequencing is closer to the theoretical methylation level of the sample than the pyrophosphoric acid sequencing detection data. For samples with methylation levels below 10%, the results of the pyrosequencing detection deviate from the theoretical value. The methylation detection protocol of the present application is applicable to detection of 0-100% level samples.
Example 3:
in liquid phase hybridization capture, probe concentration is one of the important factors affecting the capture effect. When the probe concentration is low, the chance of binding of the probe to the target sequence may be reduced, thereby affecting the capturing effect. In liquid phase hybridization, proper probe concentration may increase specificity and sensitivity, but too high a concentration may result in non-specific binding and increased background signal. Therefore, the balance of capture effect and background signal needs to be balanced when selecting probe concentration. Typically, the probe concentration is optimized experimentally to obtain the best capture effect.
(1) Construction of methylation libraries
Reference is made to example 1.
(2) Targeted methylation library construction
The method comprises the steps of performing hybridization capture by using a targeted methylation library construction mode, taking 500ng of constructed methylation library, sequentially performing methylation library hybridization, streptavidin magnetic bead capture and library elution, performing PCR amplification after elution, performing magnetic bead purification on amplified products, and sending the purified products to an Illumina Nova-Seq sequencer for sequencing, wherein the treatment of the demo Panel is shown in Table 7. The following is a specific procedure of the experiment:
TABLE 7 grouping information
。
(a) Library hybridization
Reference is made to example 1.
(b) Magnetic bead cleaning
Reference is made to example 1.
(c) Magnetic bead capture
Reference is made to example 1.
(d) Elution
Reference is made to example 1.
(e) Hybrid Capture library PCR amplification
Reference is made to example 1.
(f) Amplified library purification
Reference is made to example 1.
(3) Conclusion: as shown in Table 8, the optimal amount of probe used in the targeted methylation detection method of the present application should be 1 fmol/rxn. In several conditions tested, there was no difference in comparison of the basic QC data, the mid-target rates all reached above 40%, the contrast ratio (map availability) to the reference genome was as high as 98%, and the probe coverage depth of CpG sites was higher at probe usage of 1 fmol/rxn (fig. 5). The amount of probe used for targeted methylation sequencing in the present application is therefore recommended to be 1 fmol/rxn per probe.
TABLE 8 results of Probe usage experiments
。
Example 4:
the hybrid capture system captures a transformed library in a transformation mode including a Bisulfite (BS) transformation mode or an EM transformation mode, and the complexity of the transformed sequence is greatly reduced no matter which transformation mode is used. After the transformation of some high GC regions, the regions become AT rich regions, and other regions have continuous base T, so that hybridization capture probes have certain fault tolerance, and the probes can capture targets by having homology of more than 60% with target sequences, which is the advantage of the liquid phase hybridization capture technology. For the transformed library, probes will bind to these regions of high reproducibility, capturing more non-target regions, resulting in an increased proportion of off-target. And a blocking reagent is added into the hybridization capture system, so that the hybridization capture efficiency of the converted library is improved. The blocking reagent sequence is continuous T bases, and the length of the T bases is 15-18; or a repetitive GT sequence, the number of repetitions being 6-8; or repeating TA bases, the number of the repeats being 6-8. The sequence contains 4-7 LNA (locked nucleic acid) or BNA (bridged nucleic acid) modified bases. Such modified bases can increase the binding efficiency of the sequence to the target. The blocking sequence used in this example was MeBlockerA: TTT+TTT +TTT+ ttt+tt+tt; meBlockerB: GTG+TGT+GT+GTGTGT (+stands for LNA modified base) as the latter base. The blocking reagent working solution concentration was 100. Mu.M. In this example, the stability of the hybrid capture system was compared with or without adding a blocking sequence of MeBlockerA and MeBlockerB mixed in equal proportions, the final concentration of the single blocking sequence in the system being 100. Mu.M.
(1) Construction of methylation libraries
Reference is made to example 1.
(2) Targeted methylation library construction
Reference is made to example 1.
In this example, the demo panel is an autonomously designed hybridization probe comprising 620 CpG sites, 5.8. 5.8 kb in size.
In this embodiment, me-blockers (see Table 9) are used in some of the packets. The following is a specific procedure for this experiment:
table 9 Experimental grouping
。
* N means that Me-Blocker is not used
(a) Library hybridization
Reference is made to example 1.
(b) Magnetic bead cleaning
Reference is made to example 1.
(c) Magnetic bead capture
Reference is made to example 1.
(d) Elution
Reference is made to example 1.
(e) Hybrid Capture library PCR amplification
Reference is made to example 1.
(f) Amplified library purification
Reference is made to example 1.
(3) Conclusion: as shown in Table 10, in the target methylation detection method, the Me-Blocker can be added to improve the capturing targeting rate, the sequencing depth and the stability. In the 2 sets of conditions tested, there was no difference in comparison of the basic QC data, the data contrast ratio of the whole genome reached 99%, fold 80 (representing the average depth/depth at which more than 80% of the area was covered. The greater the Fold 80, the worse the uniformity, generally better within 2), was around 1.4. In terms of the target rate, the target rate in the experimental group to which Me-Blocker was added was about 5% higher than that in the control group, and the probe coverage depth of CpG sites was higher (FIG. 6). Therefore, the stability of the system can be improved by adding Me-Blockers in the targeted methylation sequencing experiments.
TABLE 10 results of Me-Blocker addition experiments
。
Example 5
Multiple sample hybrid hybridization in liquid phase hybridization capture (Multiplexed Hybridization) refers to a strategy for simultaneously performing hybridization capture on multiple samples. It has the following characteristics: saving cost and time, reducing technical differences, improving data comparability, etc. The targeted methylation detection method can simultaneously meet the mixed hybridization of 6 samples (the hybridization input amount is 6 x 500 ng), and the data is stable and reliable.
(1) Construction of methylation libraries
Reference is made to example 1.
(2) Targeted methylation library construction
Reference is made to example 1.
In this example, experimental grouping information is shown in table 11. The following is a specific procedure for this experiment:
TABLE 11 grouping of experiments
。
(a) Library hybridization
Reference is made to example 1.
(b) Magnetic bead cleaning
Reference is made to example 1.
(c) Magnetic bead capture
Reference is made to example 1.
(d) Elution
Reference is made to example 1.
(e) Hybrid Capture library PCR amplification
Reference is made to example 1.
(f) Amplified library purification
Reference is made to example 1.
(3) Conclusion: as shown in table 12, the targeted methylation detection methods of the present application perform well for hybridization of 6 independent samples. The comparison of QC data is basically no difference between the 6 independent samples in two hybridization modes; in 6 single miscellaneous samples, the target rate in two cases is 40%, the rest is more than 50%, slight technical difference is generated, and in the miscellaneous test, all samples can reach the middle target rate of more than 50%; and the contrast ratio with the reference genome and probe coverage depth of CpG sites, the two hybridization modes were highly consistent (fig. 7). The targeted methylation sequencing system is applicable to both single sample and multiple sample hybrid hybridization.
TABLE 12 results of experiments with impurities and hybridization
Advantages of one detection method provided herein that is capable of recognizing single base methylation status include, but are not limited to:
1. the time required is short. For example, the preparation of a conventional targeted methylation library requires 2-3 days, and the detection method can finish the preparation of the targeted library within one day, so that the time cost is greatly saved. Meanwhile, the detection method improves the target rate in the targeted methylation capturing library, the original depth and the depth after the duplication removal, and meanwhile, fold 80 is far smaller than the conventional 120nt probe data.
2. The accuracy of methylation level detection is high, and compared with 'gold standard' pyrosequencing, the detection method is closer to the theoretical methylation level of a standard substance and a configuration sample.
3. According to the method, the MeBlocker is added, so that the repeated sequence after conversion can be blocked, the capturing efficiency of a target is improved, and the capturing performance of the system on a small Panel is ensured to be more stable.
4. The method is suitable for single sample or multi-sample mixed hybridization, and the data performance of the multi-sample mixed hybridization is higher than the consistency of the results of the samples. The system is used for clinical queue research, and a multi-sample hybrid hybridization scheme can be adopted.

Claims (11)

1. A method of detecting the methylation state of a DNA molecule in a sample, comprising:
1) Constructing a DNA library by using the DNA molecules in the sample, and obtaining a methylation library after transformation;
2) Performing hybrid capture on the methylation library to construct a targeted methylation library; and
3) Sequencing said targeted methylation library and comparing the base sequence of said DNA molecule that has not been transformed to determine the methylation status of said DNA molecule,
wherein the hybridization capture is performed in a hybridization system comprising at least one single-stranded oligonucleotide blocking reagent which is a combination of single-stranded oligonucleotide blocking reagents of sequences Tn and (GT) p, wherein n is an integer from 15 to 18, p is an integer from 6 to 8,
the hybrid capture is performed using capture probes in the form of μCaller comprising a target-specific sequence, a first probe-binding sequence located 5 'to the target-specific sequence and a second probe-binding sequence located 3' to the target-specific sequence, the first probe-binding sequence being at least partially complementary to the second probe-binding sequence such that when two or more capture probes in the form of μCaller bind their respective target sequences in an adjacent manner, complementary binding can be formed between them by the first and second probe-binding sequences, the first and second probe-binding sequences being 8-30nt in length and the target-specific sequence being 20-80nt in length.
2. The method of claim 1, wherein the single stranded oligonucleotide blocking reagent comprises one or more locked or bridged nucleobases.
3. The method of claim 2, wherein the single stranded oligonucleotide blocking reagent comprises 4-7 locked or bridged nucleobases.
4. The method of claim 1, wherein the concentration of each of the single stranded oligonucleotide blocking reagents in the hybridization system is 100 μm.
5. The method of claim 4, wherein the capture probe is used in the hybridization system in an amount of 1 fmol/rxn.
6. The method of claim 1, wherein the hybridization system is further supplemented with a linker blocking reagent and a Human Cot DNA.
7. The method of claim 1, wherein the DNA molecule is from genomic DNA, plasma episomal DNA, or FFPE samples.
8. A hybridization capture kit comprising a capture probe and at least one single stranded oligonucleotide blocking reagent, wherein the at least one single stranded oligonucleotide blocking reagent is a combination of single stranded oligonucleotide blocking reagents of sequences Tn and (GT) p, wherein n is an integer from 15 to 18 and p is an integer from 6 to 8; the capture probes are μCaller-format probes comprising a target-specific sequence, a first probe-binding sequence located 5 'to the target-specific sequence, and a second probe-binding sequence located 3' to the target-specific sequence, the first probe-binding sequence being at least partially complementary to the second probe-binding sequence such that two or more μCaller-format probes are capable of complementary binding therebetween by the first and second probe-binding sequences when bound in an adjacent manner to their respective target sequences, the first and second probe-binding sequences being 8-30nt in length, and the target-specific sequence being 20-80nt in length.
9. The hybridization capture kit of claim 8, wherein the single stranded oligonucleotide blocking reagent comprises one or more locked or bridged nucleobases.
10. The hybridization capture kit of claim 9, wherein the single stranded oligonucleotide blocking reagent comprises 4-7 locked or bridged nucleobases.
11. The hybrid capture kit of claim 8, further comprising a linker blocking reagent and a Human Cot DNA.
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