A prenatal diagnosis method based on nanopore methylation sequencing
Through nanopore methylation sequencing methods, sample gDNA was extracted and whole-genome sequencing was performed, which solved the problem of rapid results and accurate identification of mutation types in prenatal diagnosis, especially in the face of maternal contamination, and achieved efficient and accurate prenatal diagnosis results.
Patent Information
- Application Number
- CN202411815532.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-04-04
AI Technical Summary
Existing prenatal diagnostic methods present challenges in rapid outcomes and accurate identification of mutation types, especially in the face of maternal contamination.
Using nanopore methylation sequencing methods, the detection of various types of mutations and methylation information is achieved by extracting sample gDNA, end repair and adding A, combining and connecting with the linker, whole-genome sequencing and bioinformatics analysis, and then identifying parent source contamination.
It realizes rapid and accurate prenatal diagnosis, can easily detect various types of mutations and CNVs, and identify parental contamination through methylation information, ensuring the accuracy of the detection results.
Smart Images
Figure CN119753112A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of prenatal diagnosis methods and technologies, and more specifically, to a prenatal diagnosis method based on nanopore methylation sequencing. Background Art
[0002] Oxford Nanopore's core technology is nanopore sequencing technology. Different from previous sequencing technologies, it does not read the base sequence by detecting the color of light or fluorescent signals, but is a sequencing technology based on electrical signals. Protein nanopores are embedded in synthetic membranes and immersed in electrophysiological solutions, allowing ion currents to pass through the nanopores. When molecules such as DNA or RNA pass through the nanopores, they interfere with the current and cause characteristic changes in the current signal. In this process, the electrical signal is analyzed in real time to determine the base sequence of the DNA or RNA chain passing through the hole, and even the base modification information on the nucleic acid chain can be directly read.
[0003] Long read length, the ability to directly read methylation information without sulfite and enzyme treatment, and flexible sample loading for rapid results are the three major technical advantages of third-generation sequencing. These technical advantages make nanopore sequencing suitable for prenatal diagnosis scenarios in medical clinics. First of all, third-generation sequencing has the technical advantage of long read length, which can detect various types of mutations, including point mutations (SNVs), small insertions and deletions (InDels), large fragment structural variations (SVs), and copy number variations (CNVs). One sequencing platform can complete all mutation detections.
[0004] Secondly, prenatal diagnosis requires rapid results and timely clinical decision-making. Nanopore sequencing library construction is fast and convenient, and can be stopped at any time, so that sufficient sequencing data can be quickly obtained for analysis. In addition, in the prenatal diagnosis scenario, amniotic fluid samples are easily contaminated by maternal sources, which affects the accuracy of the test results. The difference in methylation information obtained by nanopore sequencing can effectively distinguish maternal DNA from fetal DNA, ensuring that the test results are accurate and reliable. Figure 1 Methylation aids in distinguishing maternal contamination.
[0005] The present invention is based on a long-read nanopore sequencing method, which can simultaneously obtain pathogenic mutations and methylation information of gene regions, and accurately identify complex mutation types, including point mutations, structural variations, and CNVs. Methylation information can also effectively identify maternal contamination, ensuring the accuracy of prenatal diagnosis results. Summary of the invention
[0006] In view of this, in order to solve the above problems, the present invention provides a prenatal diagnosis method based on nanopore methylation sequencing, comprising the following steps: extracting sample gDNA; repairing and adding A to the end of the sample gDNA to obtain the gDNA after adding A; connecting the gDNA after adding A to a connector combination to obtain a connection product; a library construction step, wherein the library construction step includes a whole genome library construction step; adding the sample sequencing library obtained by the connection product to the Oxford nanopore sequencing chip for whole genome sequencing; bioinformatics analysis, since the nanopore sequencing read length is longer, it can reach more than 100 times the second generation sequencing read length, and various types of mutations can be easily detected. One platform can achieve prenatal diagnosis of various mutations including CNV by sequencing once. Secondly, nanopore sequencing does not require enzyme treatment or sulfite treatment, and can directly detect the methylation modification information of nucleic acid molecules. Compared with the enzyme or sulfite method, nanopore sequencing detects methylation site information more comprehensively and faithfully, and has no effect on nucleic acids. The methylation information obtained can be used to identify maternal nucleic acid contamination, ensuring the accuracy of prenatal diagnosis results; and nanopore sequencing can be stopped at any time, the experimental operation is fast and convenient, the sample throughput is flexible, and the chip can be reused and mixed flexibly through cleaning. Prenatal diagnosis reports can be issued in one day, leaving valuable time for clinicians.
[0007] A prenatal diagnosis method based on nanopore methylation sequencing, characterized in that it comprises the following steps:
[0008] 1) Extract sample gDNA;
[0009] 2) repairing the ends of the sample gDNA and adding A to obtain A-added gDNA;
[0010] 3) connecting the gDNA after A addition to the adapter combination to obtain a connection product;
[0011] 4) Adding the sample sequencing library obtained by ligation products to the Oxford Nanopore sequencing chip;
[0012] 5) placing the Oxford Nanopore sequencing chip from step 4) on a machine for whole genome sequencing;
[0013] 6) Bioinformatics analysis.
[0014] Furthermore, in step 1), use Genomic kit (Cat#13343) to extract gDNA; or use other long fragment DNA extraction kits to extract gDNA.
[0015] Further, in step 1), the extracted gDNA is obtained from abortion products or amniotic fluid or chorionic villus tissue.
[0016] Furthermore, in step 3), without amplification, the Ultra II End Repair / dA-tailing Kit (Cat# E7546) or other similar kits are directly used for end repair and dA addition, and then the official Oxford Nanopore LSK109, or LSK110 and other similar kits are used to complete the adapter connection.
[0017] Further, in step 4), the chip model may be R9.4 or R10.1.
[0018] Furthermore, in step 5), whole genome sequencing is performed, and the amount of sequencing data is not less than 5M reads.
[0019] Furthermore, in step 6), the bioinformatics analysis includes: identifying methylation information through the SUP pattern, detecting maternal contamination by identifying the methylation information, determining the proportion of maternal contamination, filtering out maternal contamination data using NanoFilt (v2.8.0), aligning sequences using Minimap2 (v2.22), and then analyzing CNV results using readscount.
[0020] Beneficial effects of the present invention: The present invention provides a prenatal diagnosis method based on nanopore methylation sequencing, comprising the following steps: extracting sample gDNA; repairing and adding A to the end of the sample gDNA to obtain the gDNA after adding A; connecting the gDNA after adding A to a connector combination to obtain a connection product; adding the sample sequencing library of the connection product to the Oxford nanopore sequencing chip for whole genome sequencing; bioinformatics analysis, due to the long read length of nanopore sequencing, which can reach more than 100 times the read length of the second-generation sequencing, various types of mutations can be easily detected, and a platform can achieve prenatal diagnosis of various mutations including CNV by sequencing once, and secondly, nanopore sequencing does not require enzyme treatment or sulfite treatment, and can directly detect the methylation modification information of nucleic acid molecules. Compared with enzyme or sulfite methods, nanopore sequencing detects methylation site information more comprehensively and faithfully, and has no effect on nucleic acids. The obtained methylation information can be used to identify maternal nucleic acid contamination, ensuring the accuracy of prenatal diagnosis results; and nanopore sequencing can be stopped at any time, the experimental operation is fast and convenient, the sample throughput is flexible, and the chip can be reused and mixed flexibly by cleaning. A prenatal diagnosis report can be issued within one day, leaving valuable time for clinicians. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 A schematic diagram of the methylation-assisted differentiation of maternal contamination of the present invention;
[0022] Figure 2 It is a schematic diagram of the process of the present invention;
[0023] Figure 3 Abnormal CNV results for the miscarriage products of the present invention.
[0024] Figure 4 This is the methylation information display of the present invention. DETAILED DESCRIPTION
[0025] Embodiment 1:
[0026] like Figure 2 As shown, a prenatal diagnosis method based on nanopore methylation sequencing comprises the following steps:
[0027] 1) Extract sample gDNA and use Genomic kit (Cat#13343) to extract gDNA; or use other long-fragment DNA extraction kits to extract gDNA, and the extracted gDNA is obtained in the flow product;
[0028] 2) Repair the ends of the sample gDNA and add A to obtain gDNA after adding A; directly use the Ultra II EndRepair / dA-tailing Kit (Cat#E7546) kit or other similar kits for end repair and adding A, the reagent usage system is shown in Table 1 below, and then put the mixed reagent into the PCR instrument, incubate at 20°C for 5 minutes and 65°C for 5 minutes. Then purify with AMPure XP magnetic beads;
[0029] Table 1 shows the end repair and A reagent usage system
[0030]
[0031]
[0032] Subsequently, an identity tag was added to each sample using the barcode (NB01-96) provided by ONT. The reagents used were shown in Table 2 below, and then incubated at room temperature for 20 minutes. After incubation, 1uL EDTA was added to stop the reaction and magnetic bead purification was performed.
[0033] Table 2
[0034]
[0035] 3) The gDNA after adding A and barcode is connected to the adapter combination to obtain the connection product without amplification, and then the adapter connection is completed using the official LSK109, LSK110 or other similar kits of Oxford Nanopore. The adapter reagents for connection sequencing are shown in Table 3 below, and the reaction is incubated at room temperature for 20 minutes, and purified using magnetic beads.
[0036] Table 3
[0037]
[0038] 4) Add the sample sequencing library of the ligation product to the Oxford Nanopore sequencing chip, the chip model can be R9.4 or R10.1; the following is an example of PromethION 2Solo model. First, the number of chip holes is quality checked. When the number of sequencing chip holes is not less than 5000, it can be used for sequencing; then open the chip inlet hole, use a pipette to remove the air in the chip flow channel, and then inject the priming mix provided by the official, prepare the machine library sample as shown in Table 4 below, add the configured machine library sample to the sequencing chip by pipetting, and then close the inlet hole. Click the button on the matching sequencing software interface to start sequencing.
[0039]
[0040]
[0041] 5) The Oxford Nanopore sequencing chip of step 4) is put into a machine for whole genome sequencing, and the sequencing data volume is not less than 5Mreads. The whole genome sequencing step also includes: the nanopore sequencing technology can perform basecalling while sequencing, and a fastq file containing sequence information can be obtained when the sequencing is completed.
[0042] 6) Bioinformatics analysis: methylation information was identified through the SUP pattern, maternal contamination was detected by identifying the methylation information, the maternal contamination ratio was determined, NanoFilt (v2.8.0) was used to filter out maternal contamination data, Minimap2 (v2.22) was used to align the sequences, and then CNV results were analyzed by readscount.
[0043] Specifically, maternal contamination detection is performed by identifying methylation information, and the steps of determining the maternal contamination ratio include: when identifying methylation sequencing, the methylation mode can be turned on in the computer interface, or after choosing to go offline, the pod5 data can be analyzed by dorado software to obtain a bam file with methylation information. The command line is such as: dorado basecaller sup,5mCG_5hmCG,6mA pod5s / >calls.bam. The options are 5-methylcytosine(5mC), 5-hydroxymethylcytosine(5hmC) and N6-methyladenosine(6mA). In the command, the methylation types to be identified are separated by ','. pod5s represents the pod folder path of the offline machine as the input file. The base recognition mode can be fast, hac or sup. Here we recommend using sup to obtain more accurate reads, which is conducive to more accurate downstream analysis.
[0044] For further analysis, you need to use dorado software to align the sequences of the unaligned bam files and sort them to get the aligned aligned.bam file. The command is as follows: dorado aligner reference calls.bam>aligned.bam, reference is the human reference genome file, hg19 or hg38 can be used. Then use Modkit software to get the bedMethyl file containing methylation statistics and summary information. The command line is as follows: modkit pileupaligned.bam pileup.bed--log-filepath pileup.log, calls.bam is the file containing methylation information generated in the previous step, and the generated pileup.bed file lists the base modification counts read on each read at each reference genome position. The overall methylation level of the sample is obtained by further statistics on the bedMethyl file, and then compared with the reference system sample to determine whether maternal contamination exists.
[0045] Among them, for the area of interest, the bam file of the corresponding sample can be opened through the IGV software to view the methylation details on the specific genome.
[0046] The specific steps for analyzing CNV results through readscount include: performing CNV copy number variation analysis on the aligned.bam file obtained from the previous analysis. First, filter the quality values of the bam file. Here, we remove reads with a quality lower than 9 and a MAPQ lower than 20. Subsequently, count the readcounts at the corresponding positions of the bam by dividing the genome into windows of a specified size bin. This statistical work can be completed using either samtools or bedtools software. Here, the analysis command of bedtools is provided (taking a 1000kb window as an example): First, divide the window, faidx reference / hg19.fasta -i chromsizes|grep -E -v'_|chrM'>hg19.genome.size && bedtools makewindows -g hg19.genome.size -w <1000000>hg19.1000kb.bed. Then, count the number of reads and GC content in the window. The command is bedtools coverage -a hg19.1000kb.bed -b aligned.bam>sample.count.
[0047] Then, further perform GC correction and reference system correction for CNV negative samples through loess regression, and obtain the final CNV result with the help of the CBS algorithm.
[0048] As Figure 3 shown, through bioinformatics analysis, it is found that this sample is trisomy 22.
[0049] As Figure 4 shown, by observing the methylation information, no abnormal methylation information is found, indicating that there is no maternal contamination in this sample.
[0050] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.
Claims
1. A prenatal diagnosis method based on nanopore methylation sequencing, characterized in that: The following steps are involved: 1) Extract sample gDNA; 2) repairing the ends of the sample gDNA and adding A to obtain A-added gDNA; 3) connecting the gDNA after A addition to the adapter combination to obtain a connection product; 4) Adding the sample sequencing library obtained by ligation products to the Oxford Nanopore sequencing chip; 5) placing the Oxford Nanopore sequencing chip from step 4) on a machine for whole genome sequencing; 6) Bioinformatics analysis.
2. The prenatal diagnosis method based on nanopore methylation sequencing according to claim 1, characterized in that: In step 1), use Genomic kit (Cat#13343) to extract gDNA; or use other long fragment DNA extraction kits to extract gDNA.
3. The prenatal diagnosis method based on nanopore methylation sequencing according to claim 1, characterized in that: In step 1), the extracted gDNA is obtained from abortion products or amniotic fluid or chorionic villus tissue.
4. The prenatal diagnosis method based on nanopore methylation sequencing according to claim 1, characterized in that: In step 3), no amplification is performed, and the Ultra II End Repair / dA-tailing Kit (Cat#E7546) or other similar kits are used directly for end repair and dA addition, and then the official Oxford Nanopore LSK109, or LSK110 and other similar kits are used to complete the adapter connection.
5. The prenatal diagnosis method based on nanopore methylation sequencing according to claim 1, characterized in that: In step 4), the chip model may be R9.4 or R10.
1.
6. The prenatal diagnosis method based on nanopore methylation sequencing according to claim 1, characterized in that: In step 5), whole genome sequencing is performed, and the amount of sequencing data is not less than 5M reads.
7. The prenatal diagnosis method based on nanopore methylation sequencing according to claim 1, characterized in that: In step 6), the bioinformatics analysis includes: identifying methylation information through the SUP mode, detecting maternal contamination by identifying the methylation information, determining the proportion of maternal contamination, filtering out maternal contamination data using NanoFilt (v2.8.0), aligning sequences using Minimap2 (v2.22), and then analyzing CNV results using readscount.