A method for sequencing extrachromosomal circular DNA

Abstract

This invention relates to the field of molecular biology, and in particular to a sequencing method for extrachromosomal circular DNA. The sequencing method provided by this invention comprises the following steps: S1, digesting and purifying the extracted eccDNA sample; S2, diluting the purified eccDNA in S1 and performing rolling circle amplification; S3, separating and purifying the rolled circle amplified eccDNA in S2; S4, sequencing the purified eccDNA in S3 using PacBio HIFI sequencing. The sequencing method provided by this invention enables high-precision full-length eccDNA sequencing, enhances the detection capability for low-abundance eccDNA, significantly reduces sample requirements, has a wide range of applications, simplifies the operation process to improve experimental efficiency, and enhances data quality and reliability.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a high-precision sequencing method for extrachromosomal circular DNA (eccDNA), named Multi-modal Circle-seq (MMC-seq for short). Background Technology

[0002] Extrachromosomal circular DNA (eccDNA) is a closed circular DNA molecule that exists outside the chromosomal genome and self-joins to form its own structure. Its formation mechanisms are diverse, including but not limited to double-strand breaks and end-joining in linear chromosomal DNA, replication stress-induced looping, and excision of mobile elements. Therefore, eccDNA sequences are widely derived, containing various genomic elements such as gene coding regions (e.g., exon-intron fragments or even complete genes) and non-coding regulatory regions, with lengths ranging from tens of base pairs (bp) to trillions of base pairs (Mb). eccDNA can replicate independently of chromosomes within cells, thereby increasing the copy number of specific gene sequences and potentially affecting gene dosage, chromatin structure, and transcriptional regulation. Because eccDNA plays a crucial role in many important life activities and disease processes, such as tumor development, drug resistance formation, aging, and immune responses, it has become a frontier and hot topic in biomedical research, showing great application potential in areas such as tumor diagnosis and treatment, development of novel biomarkers, assessment of genomic instability, and immunology.

[0003] Currently, the enrichment and sequencing of eccDNA mainly rely on two major categories of technologies. The first is Circle-seq: This method typically first separates circular DNA through alkaline lysis, then uses exonucleases to digest and remove linear DNA molecules, followed by rolling circle amplification (RCA) of the enriched eccDNA, and finally combines next-generation sequencing (NGS) for sequence analysis. Although Circle-seq is relatively inexpensive and mature, its inherent limitations restrict its application effectiveness: 1) NGS read length limitation: Because NGS reads are usually short (e.g., less than 300 bp), the full-length sequence of eccDNA cannot be obtained directly, requiring a complex computational assembly process, which can easily lead to misassembly or information loss when processing repetitive sequences or low-coverage regions. 2) Insufficient detection sensitivity: For low-abundance eccDNA molecules, their signals are easily masked by high-abundance molecules, resulting in limited detection sensitivity and capture efficiency. 3) High sample volume requirement: Traditional Circle-seq methods often require a large number of starting cells (up to 10⁹ to 10¹⁰ cells, as mentioned in the literature), which greatly limits its application in clinical micro-samples or rare cell types. 4) Cumbersome and time-consuming process: This method involves multiple rounds of enzyme treatment and purification steps, making the overall experimental process complex and time-consuming, thus affecting research efficiency.

[0004] The second type is 3SEP (Three-Step EccDNA Purification): This method innovatively introduces an additional "circular DNA recovery" step after the traditional alkaline lysis and exonuclease digestion steps, aiming to improve the purity and recovery efficiency of eccDNA. It is then combined with rolling circle amplification and third-generation sequencing technologies (such as nanopore sequencing) to obtain long-read sequences. Although 3SEP combined with long-read sequencing improves the ability to resolve full-length eccDNA to some extent, the following main problems still exist: 1) Low sequencing accuracy: The inherently relatively high base error rate of current mainstream long-read sequencing technologies (such as nanopore sequencing) affects the accurate identification of eccDNA sequences and their precise location on the genome. 2) Difficulty in detecting low-abundance molecules: Limited by enrichment efficiency and sequencing background noise, low-abundance eccDNA molecules are still easily overlooked or incorrectly identified. 3) High starting sample quantity requirements and limited sample types: 3SEP typically requires tens of millions of live cells as starting material, which limits its application in cases of insufficient sample quantity or special sample types (such as frozen tissues or plant samples). Its compatibility with frozen samples or plant tissues is also limited. 4) Complex and time-consuming operation: It involves multiple rounds of enzymatic digestion and purification steps, making the overall experimental process still relatively complex and time-consuming, which is not conducive to high-throughput applications and widespread promotion.

[0005] In summary, existing eccDNA research methods still face numerous challenges in terms of accuracy, sensitivity, initial sample size, sample universality, and ease of operation. Therefore, developing a next-generation sequencing method capable of simultaneously achieving high-precision, full-length sequence resolution of eccDNA, while also possessing high sensitivity, low sample requirements, broad sample applicability, and a simplified workflow, is of great significance for advancing basic research and clinical translation of eccDNA. Summary of the Invention

[0006] To address the problems of low accuracy, insufficient sensitivity, large sample requirements, limited applicability, and complex operation procedures in existing technologies for extrachromosomal circular DNA (eccDNA) sequencing, the present invention aims to provide a new, efficient, high-precision, low-sample-requirement, and easy-to-operate method for full-length eccDNA sequencing. The sequencing method provided by this invention is named Multi-modal Circle-seq (MMC-seq for short).

[0007] To achieve the above and other related objectives, this invention provides a method for sequencing extrachromosomal circular DNA, the core steps of which include:

[0008] S1, The extracted eccDNA sample is subjected to enzymatic digestion to remove linear DNA and the target eccDNA is purified.

[0009] S2, after diluting the purified eccDNA sample from S1 to a specific concentration (e.g., 0.05 ng / μL to 0.5 ng / μL, preferably 0.5 ng / μL), roll circle amplification (RCA) is performed using phi29 DNA polymerase;

[0010] S3, isolate and purify the long DNA product obtained after rolling circle amplification in S2;

[0011] S4 was sequenced using PacBio HiFi sequencing technology to perform long-read, high-precision sequencing on the amplified products purified in S3.

[0012] Preferably, the eccDNA sample in step S1 is obtained in the following manner:

[0013] 1.1 The tissue samples to be processed are frozen in liquid nitrogen and ground to break them up, or the cell samples to be processed (including adherent cells, suspension cells and single cells, etc.) are collected and washed with PBS.

[0014] 1.2. Using a plasmid extraction kit, the eccDNA of the sample treated in 1.1 was initially lysed, adsorbed, washed and eluted to obtain preliminarily purified eccDNA.

[0015] This invention also provides the application of the aforementioned sequencing method in the detection of extrachromosomal circular DNA.

[0016] As described above, the sequencing method for extrachromosomal circular DNA of the present invention has the following beneficial effects:

[0017] 1) Achieve high-precision full-length eccDNA sequencing: By combining PacBio HiFi sequencing technology, the sequencing data obtained by this method can achieve an accuracy of over 99.9%, effectively reducing sequence errors and enabling complete resolution of the sequence information of eccDNA molecules, including their precise break and connection sites, internal structural variations and complex repetitive sequences, providing a solid and reliable data foundation for subsequent functional mechanism studies.

[0018] 2) Significantly enhanced detection capability for low-abundance eccDNA: The optimized sample pretreatment process and unique rolling circle amplification (RCA) predilution strategy of this invention significantly improve the detection sensitivity for low-abundance eccDNA molecules, effectively avoiding the loss of target information. Simultaneously, this dilution strategy reduces the amplification advantage of high-abundance eccDNA molecules during the RCA process, lowering amplification bias and resulting in detection results that more comprehensively and accurately reflect the composition of eccDNA in the sample.

[0019] 3) Significantly reduced sample requirements and single-cell level detection, greatly expanding application scope: This invention overcomes the stringent limitations of traditional eccDNA detection on the initial sample volume. Currently, this method can achieve highly sensitive eccDNA sequencing starting from a single cell level; for routine bulk samples, the initial cell number can be as low as approximately 300,000, far fewer than the millions or even tens of millions of cells typically required by traditional methods. This core advantage makes it possible to conduct in-depth eccDNA research on clinical micro-biopsy samples (such as puncture tissue), single tumor cells, rare cell populations, and other precious biological materials with extremely limited sample volumes. This method therefore exhibits broad sample universality and can be flexibly applied to various tissues and cells of humans, animals, and plants, fully supporting multi-level eccDNA research needs from in-depth single-cell analysis to routine whole-sample detection, thereby powerfully promoting the application exploration and theoretical breakthroughs of eccDNA in key fields such as oncology, developmental biology, neuroscience, immunology, and precision medicine.

[0020] 4) Improved experimental efficiency and simplified operation procedures: This invention optimizes and simplifies experimental steps, such as reducing some of the cumbersome enrichment and purification steps in traditional methods, effectively shortening the experimental cycle and reducing reagent and labor costs. The overall operation procedure is more convenient, reducing dependence on special instruments and equipment, and improving the operability and scalability of the method.

[0021] 5) Enhancing Data Quality and Research Value: The eccDNA sequence data produced by this method exhibits high accuracy, high integrity, and long read lengths, providing high-quality input for in-depth bioinformatics analysis and downstream functional validation. Comprehensive and precise capture and identification of eccDNA molecules helps discover new gene regulatory patterns, chromatin structural features, disease-related molecular markers, and potential drug targets, thereby driving continuous progress in genomics and translational medicine research. Attached Figure Description

[0022] Figure 1 The diagram shows the process flow of MMC-seq, the sequencing method for extrachromosomal circular DNA in this invention.

[0023] Figure 2 The results shown are the quality control results of eccDNA obtained by atomic force microscopy (AFM) and ultra-high sensitivity capillary electrophoresis (Femto Pulse) in this invention.

[0024] Figure 3 The figure shown is a graph illustrating the results of RCA amplification and quantitative analysis of extrachromosomal circular DNA and linear DNA in this invention.

[0025] Figure 4 This paper presents the effect of eccDNA concentration on RCA amplification efficiency and the yield analysis of magnetic bead purification method in this invention.

[0026] Figure 5 The results show a comparison of the different methods in this invention in terms of process and cost.

[0027] Figure 6 The results show the evaluation of HeLa cell lines using different sequencing methods in this invention. Detailed Implementation

[0028] This invention provides a high-precision full-length sequencing method for extrachromosomal circular DNA (eccDNA), the sequencing method comprising the following core steps:

[0029] S1, The extracted eccDNA sample is subjected to enzymatic digestion to remove linear DNA and the target eccDNA is purified.

[0030] S2, after a specific dilution of the eccDNA sample purified in S1, is subjected to rolling circle amplification (RCA);

[0031] S3, separate and purify the amplification product obtained from rolling circle amplification (RCA) in S2;

[0032] S4 was sequenced using PacBio HiFi sequencing technology to analyze the amplified products purified in S3.

[0033] The specific embodiments of the present invention will be described in detail below:

[0034] In some specific implementations, the eccDNA sample in step S1 is obtained through the following sub-steps:

[0035] 1.1 Freeze, rupture the sample tissue, or wash the sample cells twice with PBS;

[0036] For tissue samples: The tissue sample to be processed (e.g., animal tissue, plant tissue) is placed in liquid nitrogen and rapidly frozen, and then thoroughly ground and broken up.

[0037] For cell samples: The cell sample to be processed (e.g., bacterial cells, fungal cells, animal cultured cells, plant protoplasts, or single cells) is gently washed twice with PBS buffer to remove culture medium or other impurities. In some embodiments, the number of cell samples can be from 1 to 1 million, for example, 100,000 to 1 million cells; more preferably, the number of cells can be from 300,000 to 600,000. For single-cell level eccDNA detection, a single cell is used for subsequent operations.

[0038] 1.2 Preliminary extraction and purification of eccDNA: Add the lysis buffer provided in a commercially available plasmid extraction kit (preferably the Tiangen Biotech DP103 plasmid mini-extraction kit or a kit with similar functions) to the sample treated in S1.1, and strictly follow the instructions of the kit to complete the cell lysis (or tissue lysis), binding of eccDNA to the column membrane, washing to remove impurities, and elution of the target eccDNA in sequence, thereby obtaining a preliminarily purified eccDNA sample.

[0039] S1: Enzymatic digestion and purification

[0040] In some specific implementations, step S1 includes the following sub-steps:

[0041] S1.1, RNA removal: Add RNase A to the eccDNA sample obtained in step S1.2 to degrade the residual RNA in the sample under suitable conditions (e.g., incubation at 37°C for 30 minutes);

[0042] S1.2, Digesting linear DNA: In the sample treated in S1.1, add an ATP-dependent DNase (e.g., Epicentre's Plasmid-SafeATP-Dependent DNase) with no or very low activity against circular DNA, along with ATP and the corresponding reaction buffer. Under suitable conditions (e.g., incubation at 37°C for 16 hours), specifically digest and remove residual linear DNA (including genomic linear fragments, etc.) from the sample.

[0043] S1.3, eccDNA purification: The sample treated in S1.2 is purified using DNA purification methods (such as using Zymoclean Gel DNA Recovery Kit or similar DNA recovery kits, or magnetic bead purification method) to remove impurities such as enzymes and small molecule nucleotides, and obtain a purer eccDNA sample.

[0044] S2: Dilution and Rolling Circle Amplification (RCA)

[0045] In some specific implementations, the key operations of step S2 are as follows:

[0046] First, the purified eccDNA obtained in step S1.3 is accurately quantified (e.g., using Qubit qPCR). Then, the eccDNA sample is diluted to a specific working concentration with an appropriate buffer (e.g., nuclease-free water or TE buffer). This working concentration is typically in the range of 0.05 ng / μL to 0.5 ng / μL. Specific selectable concentration ranges include 0.05–0.1 ng / μL, 0.1–0.15 ng / μL, 0.15–0.2 ng / μL, 0.2–0.25 ng / μL, 0.25–0.3 ng / μL, 0.3–0.35 ng / μL, 0.35–0.4 ng / μL, 0.4–0.45 ng / μL, or 0.45–0.5 ng / μL.

[0047] Preferably, the working concentration of the diluted eccDNA is 0.5 ng / μL. Experimental data show (as in Example 3 below) Figure 4 As shown in A), using 0.5 ng / μL as the initial working concentration of eccDNA template for rolling circle amplification can significantly improve the amplification efficiency and product quality of RCA, effectively enhance the detection sensitivity for low-abundance eccDNA, maintain the uniformity of amplification products, and reduce the competitive effect of high-abundance molecules, thereby ensuring the reliability and high quality of the overall sequencing data.

[0048] Next, rolling circle amplification is performed in an amplification system containing phi29 DNA polymerase, random oligonucleotide primers (e.g., random hexamer or nonamer primers), and dNTPs. To further improve amplification efficiency and product length, the amplification system may also contain inorganic pyrophosphatase and / or recombinant albumin (e.g., BSA). Typical RCA reaction conditions are: isothermal incubation at 30°C for 16 to 24 hours (e.g., 22 hours), followed by termination of the reaction and inactivation of phi29 DNA polymerase by heating (e.g., incubation at 65°C for 10 minutes).

[0049] S3: Separation and purification of RCA products

[0050] In some specific embodiments, step S3 mainly purifies the RCA amplification product (high molecular weight DNA) obtained in S2 in the following manner:

[0051] Magnetic bead purification (e.g., using AMPure PB Beads or similar magnetic beads) can be used to selectively recover long DNA fragments by adjusting the volume ratio of the magnetic bead suspension to the RCA product solution. For example, the volume ratio of the magnetic bead suspension to the RCA product solution can be 0.1 to 0.7 (i.e., a 1-7:10 ratio), preferably 0.3 to 0.5 (e.g., 0.4 times the volume of magnetic beads). Alternatively, gel electrophoresis can be used to separate and purify amplification products of the target size.

[0052] S4: PacBio HiFi sequencing

[0053] The RCA amplification products purified in step S3 were subjected to end repair, A-tailing, and adapter ligation to construct a PacBio SMRTbell library. Subsequently, the constructed library was sequenced using the PacBio HiFi sequencing platform to obtain long read sequences (e.g., average read length greater than 10 kb) with high accuracy (e.g., >Q20 or >Q30, i.e., error rate <1% or <0.1%).

[0054] In some specific embodiments, the PacBio HiFi sequencing platform can be selected from the PacBio Revio system, PacBio Sequel IIe system, PacBio Sequel II system, or PacBio Sequel system. Preferably, the latest high-throughput platform such as the PacBio Revio system, or the mature PacBio Sequel II / IIe system can be selected according to throughput requirements and experimental objectives.

[0055] Quality Control (QC)

[0056] In some specific implementations, quality control steps can be set up at key steps (such as after obtaining the eccDNA sample in S1, after purification in S1, or after purification in S3) to ensure the quality of the eccDNA and the success rate of subsequent experiments. QC steps may include:

[0057] 1) Confirmation of eccDNA circular morphology: For example, the physical morphology of eccDNA molecules in the extracted sample can be directly observed using atomic force microscopy (AFM) to confirm the presence of circular structures and assess the degree of linear DNA contamination or eccDNA degradation.

[0058] 2) Detection of eccDNA size distribution: For example, the size distribution of eccDNA fragments can be accurately determined by pulsed field gel electrophoresis (PFGE) or advanced capillary electrophoresis techniques (such as the Agilent Femto Pulse system). This helps guide the optimization of subsequent library construction parameters and the selection of sequencing strategies, thereby improving sequencing efficiency and data quality.

[0059] The present invention also provides the application of the aforementioned sequencing method in the detection of extrachromosomal circular DNA.

[0060] In this invention, the term "working concentration" refers to the concentration of reactants used in a specific experiment or application to achieve the desired effect. This concentration is preset by the experimenter based on the experimental purpose and the properties of the substances to ensure that the substances can function effectively in the experiment, while avoiding adverse effects on the experimental results from excessively high or low concentrations.

[0061] In this invention, the term "PacBio HIFI sequencing" refers to a single-molecule, long-read sequencing technology developed by PacBio, which provides long reads while maintaining high accuracy. PacBio HIFI sequencing uses Circular Consensus Sequencing (CCS) mode on PacBio's SMRT (Single-Molecular Real-Time) platform to generate sequencing data with base-level resolution and 99.9% single-molecule read accuracy. The main characteristics of PacBio HIFI sequencing are as follows: HiFi sequencing average read lengths can reach 15-25kb or more, suitable for resolving complex genomic regions; HiFi read accuracy can reach over 99.9% (Q30), achieved by repeatedly reading the same DNA fragment and generating consensus sequences; HiFi sequencing errors are mainly random errors, which can be effectively corrected by the CCS algorithm, significantly reducing systematic errors; HiFi sequencing typically does not require PCR amplification, avoiding the bias and errors introduced by PCR, and can better reflect the true state of the sample.

[0062] In this invention, the term "ATP-dependent DNAase insensitive to circular DNA" refers to a class of enzymes that can specifically digest linear double-stranded DNA (dsDNA) under certain conditions, while having almost no activity against circular or supercoiled DNA. These enzymes rely on energy provided by ATP hydrolysis to perform their DNA-digesting function.

[0063] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0064] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0065] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0066] Example 1: Application of MMC-seq (Multi-modal Circle-seq), a sequencing method for extrachromosomal circular DNA, in HeLa cells.

[0067] This embodiment aims to elaborate on the core operation flow of the MMC-seq method of the present invention, and uses HeLa cells as a typical sample to demonstrate the complete implementation process of the method from sample processing, eccDNA extraction, key digestion, rolling circle amplification to obtaining amplified products that can be used for high-quality sequencing. Simultaneously, it demonstrates the quality detection of products at key nodes in the process (such as...). Figure 1 The flowchart shown is as follows: Figure 2 The AFM and FP results shown preliminarily verify the feasibility and effectiveness of this method.

[0068] I. ECCDNA Sample Extraction

[0069] 1. Cell collection and preliminary processing

[0070] Collect HeLa cells in the logarithmic growth phase from cell culture flasks and count them to ensure approximately 5 × 10⁶ cells are obtained. 5 Cells were collected. The cells were gently washed twice with pre-cooled PBS buffer to remove culture medium residue, and the cell pellet was collected in centrifuge tubes.

[0071] 2. ECC DNA extraction

[0072] Use a plasmid miniprep kit (e.g., Tiangen DP103) to extract eccDNA following these steps:

[0073] Add 500 μL of BL buffer to the adsorption column, centrifuge at 13,400 × g for 1 minute at room temperature, and discard the waste liquid.

[0074] Add 350 μL of P1 buffer (resuspending buffer) to the cell pellet (or, if it is a tissue sample, the ground and broken tissue powder) and vortex immediately to mix.

[0075] Add 350 μL of P2 buffer (lysis buffer) and gently invert 7 times (this step is usually completed within 5 minutes to avoid excessive damage to genomic DNA).

[0076] Then add 490 μL of P3 buffer (neutralization buffer) and gently invert to mix 7 times.

[0077] Centrifuge at 13,400×g for 10 minutes at room temperature. Carefully aspirate the supernatant (containing eccDNA) and transfer it to a new adsorption column. Centrifuge at 13,400×g at room temperature to collect the liquid into a collection tube. (This step may need to be described precisely according to the kit instructions. Usually, the supernatant is loaded onto the adsorption column and then centrifuged to allow the DNA to bind to the membrane. The waste liquid is in the collection tube.)

[0078] Wash the adsorption column sequentially with 500 μL PD buffer (usually a wash buffer) and twice with 600 μL PW buffer (usually a wash buffer containing ethanol), centrifuging after each wash.

[0079] Finally, elute the eccDNA with 42 μL of pure water and collect the eluent.

[0080] 3. Concentration determination and quality testing

[0081] The concentration of the initially extracted eccDNA sample was accurately determined using a Qubit 4 fluorometer in conjunction with the Qubit 1×dsDNA HS Assay Kit. A small amount (e.g., 1 μL) of the initially purified eccDNA sample was taken and diluted with nuclease-free water (e.g., diluted to approximately 0.5 ng / μL for easy observation of low-concentration components). Appropriate amounts were then subjected to atomic force microscopy (AFM) observation and Agilent Femto Pulse (FP) ultra-high sensitivity capillary electrophoresis analysis. Quality control results (refer to...) Figure 2 A): Figure 2 A shows the detection results of the sample after step S1 (crude extraction of eccDNA). The top AFM imaging shows a large number of aggregated DNA molecules in the form of dots or small clumps, which is consistent with the concentrated form of eccDNA under this observation condition, initially indicating that the sample is rich in eccDNA. The corresponding Femto Pulse (FP) electrophoresis pattern at the bottom shows that the DNA fragments are mainly distributed in a wide range of 100bp to 100kb, exhibiting a typical multi-peak and non-uniform distribution characteristic, which further confirms the presence of multiple eccDNA subpopulations of different sizes and abundances in the sample.

[0082] II. Linear DNA Digestion and Purification

[0083] To digest linear DNA with ATP-dependent DNase, add the following reagents to 40 μL of eccDNA sample extracted in the previous step: 5 μL 10× ATP-dependent PSDNase buffer, 2 μL 25 mM ATP, 1 μL ATP-dependent PSDNase (e.g., Plasmid-Safe ATP-Dependent DNase), and bring the reaction volume to 50 μL with nuclease-free water.

[0084] After thoroughly mixing the reaction mixture, incubate it for 16 hours on a shaker at 250 rpm on a 37°C constant temperature shaker to specifically digest linear DNA.

[0085] Purification was performed using a DNA recovery kit (such as the Zymoclean Gel DNA Recovery Kit or magnetic bead purification method) following the instructions to purify the digested product, removing impurities such as enzymes and digested small DNA fragments, ultimately yielding a purified eccDNA sample.

[0086] For quality control, 1 μL of purified eccDNA was taken and analyzed using AFM and FP as described above to assess the morphology and size of the eccDNA. Quality control results (refer to...) Figure 2 B): Figure 2B shows the results after the sample was treated in step S2 (primarily using ATP-dependent Plasmid-Safe DNase to digest and purify linear DNA). AFM imaging (top) shows that circular DNA structures are still clearly visible in large quantities, while linear DNA structures are significantly reduced. In the FP electrophoresis pattern (bottom), the broad signal peaks representing the eccDNA population are preserved, while potentially present background signals from large linear genomic fragments are effectively removed. This result strongly demonstrates that step S2 of this invention can effectively remove most of the interference from linear DNA, thereby achieving specific enrichment of circular eccDNA molecules.

[0087] III. Rolling Circle Amplification (RCA)

[0088] 1. Sample dilution: Accurately determine the concentration of eccDNA purified in step two. Subsequently, dilute with nuclease-free water or TE buffer to a working concentration of approximately 0.5 ng / μL. This concentration is the preferred starting concentration for RCA in this invention; the detailed basis and considerations for its selection are detailed in Example 2.3 below.

[0089] 2. RCA reaction system (taking a 100 μL system as an example)

[0090] 10 μL 10×phi29 DNA polymerase reaction buffer

[0091] 15 μL of 2.5 mM dNTPs mixture

[0092] 1 μL of random primers (e.g., random hexamers or nonamers at a concentration of 500 nM).

[0093] 6 μL of diluted eccDNA sample (taking a 100 μL system as an example, the total system contains approximately 3 ng of eccDNA, if starting at 0.5 ng / μL; or adjust the amount of eccDNA template added according to the actual situation to achieve the desired molar number or mass).

[0094] The mixture was brought to a final volume of 98.1 μL using nuclease-free water.

[0095] 3. Denaturation and Annealing: The above mixture (excluding enzymes and accessory proteins) was incubated at 95°C for 5 minutes to denature the template, and then quickly placed on ice or cooled to 30°C and incubated for 5 minutes to anneal the primers.

[0096] 4. Add enzymes and proteins: Add 0.5 μL of phi29 DNA polymerase, 1 μL of inorganic pyrophosphatase (optional, which helps improve amplification efficiency and product length) and 0.4 μL of recombinant albumin (such as BSA, optional, which helps stabilize enzyme activity and improve amplification uniformity) to the annealed reaction solution, and make up to a final reaction volume of 100 μL with nuclease-free water, and mix gently.

[0097] 5. Amplification conditions: The reaction system is incubated on a 30°C constant temperature shaker at 250 rpm for 16 to 24 hours (preferably 22 hours in this embodiment).

[0098] 6. Enzyme inactivation: Incubate at 65°C for 10 minutes to inactivate phi29 DNA polymerase.

[0099] IV. Purification and Detection of Amplification Products

[0100] 1. High molecular weight DNA purification: Use 0.4 volumes of AMpure Beads (or similar magnetic beads from other brands, such as AMPure PB Beads) to purify the RCA product, selectively recovering the high molecular weight amplification product. The eluted product is the purified RCA amplified eccDNA.

[0101] 2. Concentration determination: The concentration of purified RCA product was determined using the Qubit 1×dsDNA HS Assay Kit.

[0102] 3. Quality Assay: Take a small amount of purified RCA product (e.g., 1 μL, diluted appropriately if necessary, such as to 0.5 ng / μL) and perform AFM observation and FP analysis. Quality assay results (refer to...) Figure 2 C): Figure 2 C represents the detection results after further processing of the sample in step S3 (RCA amplification) and S4 (RCA product purification) following the S2 treatment. AFM imaging (top) reveals large-area intertwined chain-like or network-like DNA aggregates, a typical characteristic of high-molecular-weight, multi-copy DNA products produced by phi29 DNA polymerase after rolling circle amplification of a circular template. The corresponding FP electrophoresis pattern (bottom) shows that the DNA signal migrates towards a much higher molecular weight range greater than 10-20 kb, forming diffuse broad peaks. This clearly indicates that the RCA process successfully and efficiently amplified eccDNA, significantly increasing its copy number and forming long-chain amplified products.

[0103] V. PacBio HIFI Sequencing

[0104] 1. SMRTbell Library Construction: Take a sufficient amount (e.g., 1-5 μg, adjusted according to PacBio's official guidelines and RCA product yield) of purified RCA product and construct it according to PacBio HiFi... Library construction is performed using the official operating instructions for the Express Template Prep Kit 2.0 (or other currently applicable versions). The main steps typically include DNA damage repair, end repair and A-tail addition, SMRTbell adapter ligation with hairpin structures, exonuclease treatment to remove non-circularized or linear DNA molecules, and final SMRTbell library purification.

[0105] 2. Sequencing: The constructed high-quality SMRTbell sequencing library is loaded onto a PacBio Revio system (or a Sequel II / IIe system, selected based on experimental throughput requirements and equipment availability) for HiFi sequencing. Appropriate sequencing parameters are set (such as polymerase binding time, pre-extension time, movie time, etc.) to obtain high-fidelity (HiFi) long-read sequence data with an average read length greater than 10kb and an accuracy higher than 99.9% (corresponding to a Phred quality value > Q30).

[0106] 1.3 Summary of this embodiment

[0107] The specific process flow of the MMC-seq method provided by this invention is as follows: Figure 1 As shown, the complete process from eccDNA sample extraction (step S1), linear DNA digestion and purification (step S2), sample dilution and RCA amplification before RCA amplification (step S3), RCA product purification (step S4) to subsequent library construction and PacBio HiFi sequencing (step S5) is clearly demonstrated. This example demonstrates the successful application in HeLa cells, combined with... Figure 2 The AFM and FP quality detection results at each stage, shown in (AC), systematically demonstrate the effectiveness of the MMC-seq method in eccDNA extraction, purification, and amplification: it can effectively enrich and retain morphologically intact and size-diverse eccDNA molecules from cell samples, successfully remove major linear DNA interference, and obtain sufficient, high-molecular-weight, and highly intact amplification products through the RCA process. This lays a solid methodological foundation for subsequent in-depth optimization analysis of key technical points of the MMC-seq method and performance comparison with existing technologies, and proves its ability to prepare high-quality starting materials required for subsequent high-quality PacBio HiFi sequencing and in-depth functional studies.

[0108] Example 2: Optimization and Verification of Key Technical Aspects of the MMC-seq Method

[0109] The superiority of the MMC-seq method lies not only in its innovative overall workflow design but also in the meticulous optimization of its key technical aspects. These optimizations directly affect the specificity, amplification efficiency, ability to capture eccDNA diversity, and the reliability of the final data in eccDNA analysis. This embodiment will focus on in-depth experimental verification and analysis of three core technical points in the MMC-seq workflow: the specific removal effect of ATP-dependent DNase on linear DNA in step S2; the selection strategy for the initial eccDNA template concentration in step S3, especially why approximately 0.5 ng / μL is preferred as the working concentration; and the purification and recovery efficiency of the RCA amplification product in step S4.

[0110] Key technology point 1: ATP-dependent DNase for specific and efficient removal of linear DNA (supporting step S2)

[0111] Experimental objective: This experiment aims to demonstrate that ATP-dependent Plasmid-Safe DNase (PSDNase) can selectively degrade linear DNA without damaging circular eccDNA, and significantly reduce background in subsequent rolling circle amplification (RCA), thereby enabling specific amplification of eccDNA.

[0112] Experimental Procedure: Two 1kb linear DNA segments (LineDNA1 and LineDNA2) were mixed with two circular plasmid DNAs (circleVec1, 2kb; circleVec2, 8kb) at a mass ratio of 100:1. ATP and PSDNase were added at 37℃ and reacted for 24 hours to selectively hydrolyze the linear DNA. After the reaction, the residual product was placed at 30℃ and used... DNA polymerase was used for 24-hour recombinant DNA ionization (RCA). 20 μL samples were taken at three time points: raw (initial), after DNase treatment, and at the end of RCA. The relative concentrations of LineDNA1 / 2 and circleVec1 / 2 were determined using specific qPCR (ΔΔCT method, with known copy number control). All experiments were independently repeated three times, and statistical analysis was performed using one-way ANOVA.

[0113] Results and Analysis (refer to) Figure 3 ):exist Figure 3In experiment A, the relative concentration of LineDNA1 / 2 in the Raw phase was approximately 100 times that of circleVec1 / 2, perfectly conforming to the 100:1 experimental design. After PSDNase treatment, the signals of LineDNA1 and LineDNA2 almost dropped to the detection limit, while circleVec and circleVec2 showed only slight loss, demonstrating that PSDNase has extremely high specificity for linear substrates. Upon entering the RCA phase, the residual linear DNA was difficult to amplify, while the circular template was exponentially amplified. Figure 3 B showed that the relative concentration of LineDNA1 at the RCA endpoint remained <0.05%. Figure 3 C indicates that the relative concentration of circleVec1 was approximately 30-fold higher than that of raw. All three independent experiments showed highly significant differences (***, p<0.001).

[0114] Conclusion: PSDNase can remove >99.9% of linear DNA within 24 hours, making the RCA process almost unaffected by linear fragments; simultaneously, the circular DNA structure remains intact and enrichment efficiency is high. The concentration trajectory of the three stages Raw→DNase→RCA clearly demonstrates the working logic of "cleaning first, then scaling up," proving that this step is the core technology for constructing high-purity circular DNA libraries in MMC-seq. ATP-dependent PSDNase can efficiently and specifically remove linear DNA while having minimal impact on circular eccDNA; combined with... -Mediated RCA results in a final product containing ≥97% circular DNA, significantly improving the sensitivity and accuracy of eccDNA detection. This strategy provides a solid theoretical and practical foundation for high-precision eccDNA analysis using MMC-seq.

[0115] Key technical point 2: Optimization of RCA initiation eccDNA concentration (supporting step S3)

[0116] Experimental objective: To investigate the effect of RCA-initiated eccDNA template concentration on amplification product yield, and to clarify why the present invention preferably uses about 0.5 ng / μL as the working concentration, in order to maximize the capture of eccDNA diversity while ensuring amplification efficiency.

[0117] Experimental Procedure: After crude extraction and linear DNA digestion and purification of eccDNA, its concentration was accurately determined. A stock solution control group (Undiluted) was established, and the remaining samples were serially diluted 10-fold, 100-fold, and 1000-fold, respectively. The initial concentration range of the eccDNA stock solution (after digestion and purification) used in this experiment was determined to be 4 ng / μL to 10 ng / μL. Therefore, the initial template concentrations of each group were approximately: stock solution group (approximately 4-10 ng / μL), 10-fold dilution group (approximately 0.4-1 ng / μL), 100-fold dilution group (approximately 0.04-0.1 ng / μL), and 1000-fold dilution group (approximately 0.004-0.01 ng / μL). For the eccDNA samples of the above concentration gradients, rolling circle amplification reaction was performed according to the standard procedure described in step three of Example 1.2.

[0118] Results and Analysis (refer to) Figure 4 A): Experimental data showed that compared with the stock solution group (template concentration approximately 4-10 ng / μL) and the 1000-fold dilution group (template concentration approximately 0.004-0.01 ng / μL, too low), the concentrations of RCA amplification products in eccDNA samples diluted 10-fold (template concentration approximately 0.4-1 ng / μL) and 100-fold (template concentration approximately 0.04-0.1 ng / μL) were significantly increased. Specifically, in this dilution series, the 100-fold dilution group (template concentration approximately 0.04 ng / μL to 0.1 ng / μL) showed the highest RCA product yield.

[0119] This result clearly demonstrates that the initial concentration of the eccDNA template has a significant impact on RCA amplification efficiency, and a higher concentration is not necessarily better. Appropriately reducing the template concentration to a suitable range can effectively avoid the inhibitory effects that high template concentrations may cause (such as increased system viscosity, relatively high concentrations of inhibitory impurities, etc.), while ensuring sufficient template molecules for effective amplification, thereby improving the yield of RCA products.

[0120] However, when determining the preferred initial working concentration of RCA, this invention does not simply pursue the absolute maximization of RCA product yield under a specific experimental condition, but rather comprehensively considers both the amplification efficiency and the capture of eccDNA molecular diversity. Figure 4 The experimental results of A reveal the importance of template dilution for improving RCA product yield. However, considering the inherent complexity of eccDNA samples and the coexistence of eccDNA molecules with varying abundances, excessive template dilution (e.g., to a certain extent) can lead to problems. Figure 4While the highest yield in A is at extremely low levels of 0.04-0.1 ng / μL, it may still be able to efficiently amplify some high copy number eccDNAs, but it may sacrifice the ability to capture those eccDNA molecules with extremely low copy numbers and rare species in the sample, thus losing some important biological information and affecting the full representation of eccDNA diversity.

[0121] Therefore, after further comprehensive evaluation and optimization (these considerations may involve testing different types of samples, different eccDNA abundance profiles, and analysis of the types and coverage uniformity of eccDNA in the sequencing results, the detailed data of which are not fully shown in this single chart), this invention ultimately recommends diluting the eccDNA purified in step S2 to a working concentration of approximately 0.5 ng / μL for rolling circle amplification. Choosing 0.5 ng / μL as the working concentration is an optimized balance point sought in the following aspects:

[0122] Ensure sufficient template quantity: Compared to a concentration of 0.04-0.1 ng / μL, 0.5 ng / μL contains more types and quantities of eccDNA molecules, which helps to capture eccDNA molecules of different abundances in the sample more comprehensively, especially low-abundance eccDNA molecules, thereby better maintaining eccDNA diversity.

[0123] Achieving efficient amplification: This concentration is significantly lower than the stock solution concentration that may cause inhibition, which is sufficient to achieve efficient RCA reaction and produce a sufficient amount of amplification products.

[0124] Reduce amplification bias: A moderate amount of starting template helps reduce the competitive advantage of high-abundance molecules over low-abundance molecules during amplification.

[0125] Stability and universality of the method: 0.5 ng / μL, as a relatively standardized starting concentration, provides a relatively stable and robust amplification performance when dealing with samples of different sources and complexities.

[0126] Conclusion: RCA template concentration has a significant impact on amplification efficiency. This invention selects approximately 0.5 ng / μL as the working concentration, which is an optimized compromise considering amplification efficiency, eccDNA diversity capture, method stability, and universality, aiming to achieve more comprehensive and accurate analysis of eccDNA.

[0127] Key Technology Point 3: High-efficiency purification and recovery of RCA amplification products (supporting step S4)

[0128] Experimental objective: To evaluate the recovery efficiency of RCA amplification products purified by using 0.4 times the volume of AMPure magnetic beads in step S4.

[0129] Experimental procedure: After RCA amplification in Example 2.3, the amplification products of each group were purified using 0.4 times the volume of AMPure magnetic beads, and the amount of DNA before and after purification was compared (or the amount of DNA recovered from the purified product was directly measured and compared with the theoretical maximum product amount to evaluate the recovery rate).

[0130] Results and Analysis (refer to) Figure 4 B): The purification yield of magnetic beads was analyzed. Experiments and statistics showed that, for example... Figure 4 As shown in B, when purifying RCA amplification products with different initial concentrations using 0.4 times the volume of AMPure magnetic beads, the average recovery rate of high molecular weight DNA samples was approximately 80%.

[0131] Conclusion: Purification using 0.4 times the volume of magnetic beads can efficiently and stably recover RCA products, providing sufficient and pure templates for subsequent high-quality library construction and sequencing. This parameter provides a key reference for the optimization of subsequent experimental systems.

[0132] 2.5 Summary of this embodiment

[0133] Through in-depth verification and optimization strategies of key technical aspects such as the specificity of linear DNA removal, the optimized selection of RCA starting concentration (balancing amplification efficiency and diversity), and the recovery efficiency of amplification products in the MMC-seq method, this invention further demonstrates the scientific design and technological superiority of the method in achieving high specificity, high efficiency, and high diversity capture. These are the core guarantees for the high performance of the MMC-seq method.

[0134] Comparative Example 1: Overall Performance Comparison of MMC-seq Method with Existing Technologies

[0135] 3.1 Introduction

[0136] To objectively evaluate the overall performance advantages of the MMC-seq method of this invention and its advancements compared to existing technologies, this section compares it with two mainstream technologies in the current eccDNA research field—Circle-seq and 3SEP-seq—in multiple dimensions, including methodological characteristics, sequencing data quality, and eccDNA detection efficiency. All comparative experiments were conducted on HeLa cell lines to ensure the comparability of results.

[0137] 3.2 Comparative Example 1: Comparison of methodological characteristics (initial sample size, experimental time)

[0138] Experimental Design and Procedure

[0139] Prepare sufficient HeLa cell line samples and the main materials and equipment required for the three sequencing methods.

[0140] MMC-seq group: The operation was carried out according to the standard procedure described in Embodiment 1 of this invention, with the initial sample size set to approximately 3 × 10⁻⁶. 5 One HeLa cell line. The total time and estimated main costs from the start of cell processing to completion of library construction are recorded.

[0141] Circle-seq group: The initial sample size was set to approximately 3 × 10⁻⁶. 6 One HeLa cell line was used. Following a typical published Circle-seq workflow, the main steps included genomic DNA extraction, targeted enzyme digestion to enrich eccDNA, RCA amplification using phi29 DNA polymerase, and construction of a next-generation sequencing (NGS) library. The corresponding time and cost were recorded.

[0142] 3SEP-seq group: The initial sample size was set at approximately 3 × 10⁻⁶. 7 Fresh HeLa cells were used. Following a typical published 3SEP-seq procedure, the main steps included cell lysis, obtaining eccDNA through a three-step purification method (usually involving alkaline lysis, exonuclease digestion, and column purification to recover circular molecules), RCA amplification using phi29 DNA polymerase, and construction of a third-generation nanopore sequencing library. The corresponding time and cost were recorded. Finally, the data were compiled and relevant charts were created (e.g.,...). Figure 5 As shown in the figure, the three methods are compared in terms of the number of cells required at the beginning, the total time for sample processing, and the estimated process cost per sample.

[0143] Results and Analysis (refer to) Figure 5 )

[0144] Starting cell number requirement ( Figure 5 A): As Figure 5 As shown in Figure A, a comparison of the number of cells required for starting the three methods—Circle-seq, 3SEP-seq, and MMC-seq—clearly demonstrates that the MMC-seq method requires the lowest number of starting cells (approximately 300,000), significantly lower than Circle-seq (approximately 3 million) and 3SEP-seq (approximately 30 million). This greatly broadens the applicability of eccDNA research to different sample types, especially offering significant advantages in handling small clinical samples or rare cell resources.

[0145] Sample processing time ( Figure 5 B): Figure 5 B presents a comparison of sample processing time (from raw sample to sequenceable library) for three methods: Circle-seq, 3SEP-seq, and MMC-seq. MMC-seq, through optimization and simplification of experimental steps, significantly shortens the total sample processing time and improves experimental efficiency compared to the other two methods.

[0146] in conclusion

[0147] In terms of methodological characteristics, MMC-seq shows significant advantages over Circle-seq and 3SEP-seq in significantly reducing the initial sample size requirement and greatly shortening the experimental cycle. It may also be more cost-effective, making it more suitable for the needs of clinical micro-sample analysis and high-throughput research.

[0148] 3.3 Comparative Example 2: Comparison of Sequencing Data Quality and eccDNA Detection Efficiency

[0149] Library preparation, sequencing and bioinformatics analysis methods

[0150] HeLa cell eccDNA libraries were prepared using three strategies: MMC-seq, Circle-seq, and 3SEP-seq, and sequencing was performed on their respective optimal platforms: MMC-seq libraries were sequenced using PacBio HiFi, Circle-seq libraries using Illumina NovaSeq, and 3SEP-seq libraries using Oxford Nanopore MinION. All platforms strictly followed the manufacturer's recommended workflow to ensure comparable sequencing depths. After quality control, the raw data were sent to CircleSeeker (MMC-seq), Circle-Map (Circle-seq), and FLED (3SEP-seq) for standardized eccDNA identification and deduplication, and finally converted to "the number of unique eccDNA molecules detected per Gb of valid data" for cross-sectional comparison.

[0151] Results and analysis (see Figure 6 A–C)

[0152] In terms of overall detection volume ( Figure 6 A) MMC-Seq-TGS can identify approximately 3.2 × 10^5 eccDNAs in a single experiment, which is 15–40 times that of Circle-Seq-TGS, Circle-Seq-NGS, and 3SEP-TGS; the differences were statistically significant after two-tailed testing (p < 0.003). After normalizing the sequencing volume ( Figure 6 B) MMC-Seq-TGS still showed the highest detection efficiency, averaging approximately 2.0 × 10^4 eccDNA molecules / Gb, which is ~6 times that of Circle-Seq-TGS, ~13 times that of Circle-Seq-NGS, and ~20 times that of 3SEP-TGS, indicating its particularly excellent ability to capture low-abundance eccDNA. Randomly selected candidate circular molecules were then subjected to PCR verification. Figure 6C) shows that the positive rates of the four procedures were all in the range of 88–97%; MMC-Seq-TGS had the highest rate, reaching 96–97%, while the other methods showed no significant difference, indicating that the high detection rate did not come at the expense of accuracy.

[0153] in conclusion

[0154] Combining the ultra-low error rate and long read length advantages of PacBio HiFi, MMC-seq significantly outperforms Circle-seq and 3SEP-seq in both the number of eccDNA detected and the efficiency per unit data volume, while maintaining a comparable validation accuracy. This method not only provides more complete and high-quality eccDNA maps but also lays a more solid and reliable data foundation for subsequent biological function analysis and clinical translation studies.

[0155] 3.4 Comparative Summary Conclusion

[0156] A systematic comparison with existing mainstream eccDNA sequencing technologies Circle-seq and 3SEP-seq in terms of methodological characteristics, sequencing data quality, and eccDNA detection efficiency clearly and consistently demonstrates that the MMC-seq method proposed in this invention exhibits unparalleled superiority in several core aspects, including significantly reducing sample requirements, greatly improving experimental efficiency, significantly enhancing data quality, and greatly enhancing eccDNA detection capabilities. Therefore, MMC-seq is a more advanced, reliable, and widely applicable technical solution in the field of eccDNA research.

[0157] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method of sequencing extrachromosomal circular DNA, characterized by, The sequencing method includes the following steps: • S1, the obtained eccDNA sample is digested and purified to obtain relatively pure eccDNA; • S2, Dilute the purified eccDNA from step S2 to a working concentration of 0.05 ng / μL to 0.5 ng / μL, and then perform rolling circle amplification in an amplification system containing phi29 DNA polymerase and random primers; • S3, recover, separate, and purify the amplification product obtained in step S2; • S4, use the PacBio HiFi sequencing platform to sequence the eccDNA purified in step S3.

2. The method of claim 1, wherein, Based on the volume of the mixed system in S1, the working concentration of eccDNA is 0.5 ng / μL; and / or, the method for recovery, separation and purification in S4 is magnetic bead purification; preferably, the volume ratio of magnetic bead suspension to amplification product is (1-7):

10.

3. The sequencing method of claim 1, wherein, The eccDNA sample in step S1 is obtained in the following manner: 1) Freeze and grind the tissue sample to be processed in liquid nitrogen; if it is a cell sample, wash it with PBS. 2) Add cell lysis buffer to the tissue or cell sample processed in step 1), and then complete the lysis, binding, washing and elution steps in sequence to obtain a preliminarily purified eccDNA sample. And / or, when the sample in step 1) is a cell sample, the number of cells is from 1 to 1 million; and / or, the sample type in step 1) is selected from one or more of bacterial cells, fungal cells, plant protoplasts, plant tissues, animal cells, and animal tissues.

4. The sequencing method of claim 3, wherein, The lysis, binding, washing, and elution steps described in step 2) are performed using a plasmid extraction kit; preferably, the plasmid extraction kit is the Tiangen DP103 kit.

5. The sequencing method of claim 1, wherein, Step S1 includes one or more of the following steps: • S11: Remove RNA and residual linear DNA from the eccDNA sample obtained in step S1; • S12: Purify the eccDNA sample obtained from digestion in step S11.

6. The sequencing method of claim 5, wherein, The digestive enzyme used to remove linear DNA in step S11 is an ATP-dependent DNA enzyme that has no cleavage activity against circular DNA; preferably, the digestive enzyme used to remove linear DNA is Plasmid-SafeATP-Dependent DNase.

7. The sequencing method of claim 1, wherein, The amplification system containing phi29 DNA polymerase and random primers described in step S2 further includes inorganic pyrophosphatase and / or recombinant albumin.

8. The sequencing method of claim 1, wherein, The rolling circle amplification is performed by incubation at 28-32°C for 16 to 24 hours; preferably, the incubation time is 20-22 hours.

9. The sequencing method of claim 1, wherein, The PacBio HiFi sequencing platform mentioned in step S5 is one of the PacBio Revio system, PacBio Sequel system, PacBio Sequel II system, or PacBio SequelIIe system.

10. The use of the sequencing method according to any one of claims 1 to 9 in the detection of extrachromosomal circular DNA.

11. Use according to claim 10, characterized in that, The application includes detecting low-abundance or high-complexity eccDNA molecules; and / or, for studying genome structural variations or tumor drug resistance mechanisms related to eccDNA.

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