CN116064728A

CN116064728A - Method for constructing library and sequencing of extrachromosomal circular DNA

Info

Publication number: CN116064728A
Application number: CN202111288525.6A
Authority: CN
Inventors: 肖利云; 郭亭佑; 赵鑫; 吴逵
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2023-05-05

Abstract

The invention belongs to the field of gene detection, and more particularly provides a method for constructing and sequencing extrachromosomal circular DNA, double-stranded circular DNA for detecting extrachromosomal circular DNA detection and a kit comprising the double-stranded circular DNA. By the method for constructing the library and sequencing the extrachromosomal circular DNA, the extrachromosomal circular DNA with the content as low as 5pg in each milliliter of sample can be detected, and the true negative result and the false negative result can be accurately and effectively distinguished.

Description

Method for constructing library and sequencing of extrachromosomal circular DNA

Technical Field

The invention belongs to the field of gene sequencing, and in particular relates to a method for constructing a library of extrachromosomal circular DNA and sequencing.

Background

Extrachromosomal circular DNA (extrachromosomal circular DNA, eccna) is located extrachromosomally, in a closed circular DNA structure that is single-stranded or double-stranded, and is widely found in a variety of eukaryotic organisms. eccDNA is mostly smaller than 25kb, and is predominantly distributed between 0.1kb and 5 kb. Due to the characteristic of the ring structure, compared with free linear DNA, eccDNA has stable structure and is not easy to be degraded. The generation of eccna is associated with genomic instability. It has been shown that the chromatin of eccDNA in tumor cells is highly open, eccDNA can carry complete oncogenes, even promoter and enhancer elements upstream of the genes, can independently complete replication processes, has high transcriptional activity, and is a novel amplified form of tumor oncogenes. Therefore, eccDNA is related to tumorigenesis and development, and is a novel tumor marker.

The literature published on PNAS in this year (Identification and characterization of extrachromosomal circular DNA in maternal plasma (https:// www.pnas.org/content/117/3/1658)) detected eccNA in the peripheral blood of pregnant women, demonstrated that eccNA can be secreted into the blood circulation and can be detected from plasma by high throughput sequencing techniques.

The existing method for detecting the eccNA in the blood plasma is based on an Illumina sequencing platform (https:// www.pnas.org/content/117/3/1658), and comprises a method for detecting the eccNA based on restriction enzyme MspI digestion treatment and a method for constructing a taggant based on Tn 5.

For MspI-based detection methods, an initial amount of 25ng of circulating free DNA (cfDNA) is required, cfDNA is digested with exonuclease v and then purified and recovered by column purification, and circular DNA is digested into linear DNA by restriction enzyme MspI and then subjected to pool sequencing. The method can only detect circular DNA with one MspI restriction site, but eccDNA without MspI restriction site or with a plurality of MspI restriction sites cannot be detected, so that the detected eccDNA has few types and is not comprehensive.

While the other Tn 5-based fragment library construction method increases the number of circular DNA detection, the method has high requirement on the initial cfDNA amount to 30ng, so that the method is not suitable for a trace amount of cfDNA clinical samples.

In addition, the experimental systems of the two detection methods have no positive control, and the false negative problem possibly occurring during the eccDNA detection cannot be solved. And the current eccDNA detection methods are based on an Illumina sequencing platform, and detection technology based on a DNBseq sequencing platform is not available temporarily.

Therefore, there is a need in the art for a method for detecting eccna that can detect minute amounts of clinical samples and establish a positive control in an experimental system, thereby more accurately detecting eccna.

Disclosure of Invention

As described above, the existing eccDNA detection method is not comprehensive and accurate enough for eccDNA detection, and has high requirements on the initial quantity of the cfDNA of the sample, and the obtained detection result is not accurate enough. Therefore, there is a need in the art for a method for detecting eccna that can detect a trace amount of clinical samples, and establish a positive control in an experimental system, so as to more accurately detect eccna.

Accordingly, in a first aspect, the present invention provides a method of pooling extrachromosomal circular DNA comprising the steps of:

1) Extracting extrachromosomal circular DNA in a sample to be detected;

2) Amplifying the extrachromosomal circular DNA to obtain an amplified product thereof;

3) Breaking the amplified product and performing chip selection on the DNA fragments;

4) Performing terminal repair and addition of adenylate (A) on the DNA fragment obtained by chip selection, and connecting with a linker to obtain a DNA fragment with the linker;

5) Circularizing the ligated DNA fragment obtained in step 4), and removing the uncyclized DNA fragment by enzymatic digestion.

In a second aspect, the invention provides a method of sequencing extrachromosomal circular DNA, the method comprising:

a) Executing the method for constructing the extrachromosomal circular DNA of the first aspect of the present invention;

b) Sequencing the product obtained in step a).

In a third aspect, the invention provides a double-stranded circular DNA for use as a positive control in extrachromosomal circular DNA sequencing, the sequence of which is shown in SEQ ID NO. 1.

In a fourth aspect, the present invention provides a kit for detecting extrachromosomal circular DNA sequencing comprising: the plasmid pUC19 and/or GAPDH (glyceraldehyde-3-phosphate dehydrogenase) circular DNA shown in SEQ ID NO. 1 was used as a positive control for the experimental system.

The beneficial effects of the invention include one or more of the following:

1) An eccDNA detection method based on DNBreq sequencing platform was developed;

2) The initial sample amount required for the eccDNA detection is reduced, and even if only 5pg of eccDNA is contained in each milliliter of sample, the effective detection can be performed;

3) GAPDH circular double-stranded DNA suitable for use as a positive control in a variety of eccna assays was designed.

4) By adding double-stranded circular DNA as positive control in the experimental system, the problems of true negative and false negative in the eccDNA detection can be distinguished, so that a more accurate detection result is obtained.

Drawings

FIG. 1 is a graph showing the results of the detection obtained by the Agilent 2100 bioanalyzer of the linear double-stranded DNA (dsDNA), the plasmid pUC19 and the mixture of the linear dsDNA and the plasmid pUC19 before and after the cleavage by the plasmid-safe ATP-Dependent DNase.

FIG. 2 is a graph showing the effect of MDA amplification, wherein lanes from left to right in the graph are lane 1:1kb DNA ladder; lane 2: a 50bp DNA ladder; lane 3-lane 4: PCR products taking lung cancer cell line gDNA MDA amplification products as templates; lane 5-lane 6: PCR products using MDA amplification products of deionized water as templates; lane 7-lane 8: PCR products using deionized water as templates.

FIG. 3 is a graph showing the composition ratio of circular DNA of different sizes (mitochondrial DNA, genomic DNA, pUC19 (positive control) and GAPDH circular DNA (positive control)).

FIG. 4 is a schematic diagram of the construction of GAPDH circular DNA.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The described embodiments are only some, but not all, embodiments of the invention. All other embodiments that are available to one of ordinary skill in the art based on the embodiments of the present invention and which fall within the scope of the present invention.

As described above, the detection result of eccDNA by the existing restriction endonuclease MspI is not comprehensive and accurate enough, and the sample initiation amount required by the Tn 5-based fragment library construction method is too large. Therefore, the present invention aims to provide a novel method for detecting eccDNA, which can detect a trace amount of clinical samples, and establish a positive control in an experimental system, so as to obtain more accurate and comprehensive detection results.

1) Extracting extrachromosomal circular DNA in a sample to be detected;

Due to the high heterogeneity in origin of extrachromosomal circular DNA (eccna), the generation may involve different occurrence models and repair mechanisms in different contexts. The eccDNA can influence the vital activity of cells, promote the evolution and adaptive evolution of tumor cells, increase the plasticity and the instability of genome, and have important application potential in the aspects of tumor diagnosis, liquid biopsy and the like. Thus, new methods of eccDNA detection, particularly high throughput sequencing and like techniques, are important for the discovery and understanding of more eccdnas.

In step 1), the circulating DNA (cfDNA) of the sample to be tested may be extracted first. The extraction of cfDNA can be performed by using conventional commercial kits. It will be appreciated that the cfDNA extracted includes not only extrachromosomal circular DNA (eccna) but also linear DNA, the latter not being the detection target. Therefore, these linear DNA needs to be removed before proceeding to the subsequent steps. Thus, in some embodiments, step 1) comprises: extracting cfDNA in the sample to be detected, and digesting linear DNA in the extracted cfDNA by exonuclease, thereby obtaining extrachromosomal circular DNA. In the present invention, plasmid-Safe ATP-Dependent DNase or Exoneuclease V (RecBCD) can be specifically used as Exonuclease for digesting linear DNA, but is not limited thereto. Any exonuclease capable of digesting linear DNA may be used in the present invention.

In addition, as described in the background section, there is no positive control in the existing eccna detection methods, and thus the false negative problem that may occur in the eccna detection cannot be solved. For this reason, the present inventors have found and/or designed a double-stranded circular DNA suitable for use as a positive control in various eccna assays, and added the double-stranded circular DNA to a sample to be tested at the time of the eccna assay, whereby the problem of discriminating between true and false negatives in the eccna assay can be solved.

Thus, in some embodiments, in step 1), double stranded circular DNA is added to the sample to be tested as a positive control prior to extracting the extrachromosomal circular DNA from the sample to be tested. In the present invention, the double-stranded circular DNA as a positive control may include pUC19 plasmid shown in SEQ ID NO. 20 and/or GAPDH circular DNA shown in SEQ ID NO. 1, but is not limited thereto. One skilled in the art can select the most appropriate size of double-stranded circular DNA according to the type of sample.

Plasmid refers to double-stranded closed circular DNA that exists outside the bacterial chromosome and is capable of independent replication. As a genetically engineered vector, a plasmid usually contains a replication origin, a single recognition site for a plurality of restriction enzymes, and a selectable marker gene such as ampicillin gene for screening recombinants. Plasmids such as pBR322, pUC, pGEM and pGEX series, which are commercially available at present, have a replication initiation site region containing 2 MspI cleavage sites and 5 MspI cleavage sites on the ampicillin gene, and thus cannot be used as a positive control in the existing MspI cleavage-based eccna assay. However, the principle of the eccDNA detection in the present invention is different from that of the MspI cleavage method, and therefore the eccDNA detection is not limited by whether or not restriction sites are contained and the number of restriction sites contained. The pUC19 plasmid was selected as a positive control of the present invention because of its ease of obtaining high copy number, high quality samples and its small length (only 2686 base pairs).

GAPDH is widely distributed in various tissue cells and is highly expressed in almost all tissues, with the expression of proteins being substantially constant in the same cell or tissue. Selected for positive quality control due to the nature of their housekeeping genes. The GAPDH circular double-stranded DNA designed by the inventor has three characteristics: firstly, the gene sequence is of human origin, and the eccDNA in the detected human body fluid is used as an experimental positive control, so that a reference genome does not need to be additionally selected in bioinformatic analysis; secondly, the designed GAPDH circular double-stranded DNA size is 406bp, which is slightly larger than the main eccNA in the reported pregnant woman plasma, and the peak value of the pregnant woman plasma eccNA is distributed at 338bp. The GAPDH circular double-stranded DNA is used as a positive control for eccNA detection, so that the positive quality control of the experimental process can be performed by simulating a detection target, and the eccNA detection in blood plasma can not be influenced; finally, the GAPDH circular double-stranded DNA loop does not contain an MspI cleavage site and thus can be used as a positive control for detecting eccna based on MspI cleavage.

According to the invention, double-stranded circular DNA is added as a positive control in the eccDNA detection, verification can be performed from a sequencing result, the problem of setting experimental positive control in the same tube sample detection is realized, the quality control of an experimental process is not required by other additional experimental designs, the experimental operation is simple and convenient, and the problems of true negative and false negative in the eccDNA detection can be solved.

In some embodiments, in step 2), the extrachromosomal circular DNA is amplified using multiplex displacement amplification (Multiple Displacement Amplification, MDA). MDA is an isothermal amplification method that relies on the principle of strand displacement amplification and is widely used for amplification of genomes or transcriptomes. MDA typically uses phi29 DNA polymerase, which exhibits excellent strand displacement and exonuclease activity. Therefore, MDA is superior to PCR in both template coverage and amplification fidelity, and can obtain a large amount of high-quality DNA from a very small amount of DNA sample. In addition, strand displacement amplification brings about a DNA product of high molecular weight (. Gtoreq.10 kb), and the amplified DNA is suitable for construction of libraries.

In some embodiments, in step 3), ultrasound may be used to break up the amplification product. The conditions of the ultrasound may be specifically selected depending on the specific conditions, as long as a DNA fragment of a desired length can be obtained.

After disruption of the amplified product, further selection of DNA fragments of the desired length is required. The chip selection may be performed by magnetic beads, but is not limited thereto. Any method by which chip selection may be accomplished is within the scope of the present invention. In the present invention, it is preferable that a DNA fragment of 200bp to 400bp in length is obtained by chip selection.

It will be appreciated that in subsequent on-machine sequencing, more DNA fragments are more suitable. To this end, in some embodiments, after step 4) and before step 5), further comprising the step of PCR amplifying the adaptor-ligated DNA fragments. The systems and conditions for PCR amplification are within the ability of those skilled in the art.

As described above, in step 5), the removal of the uncyclized DNA fragments by enzymatic digestion is included. In the present invention, any enzyme that can be used to digest the uncyclized DNA fragment can be used. The enzyme may be, for example, an exonuclease such as exonuclease I and exonuclease III, but is not limited thereto.

In addition, as noted above, the present invention may require fewer samples than prior methods, in part because of the amplification of eccDNA involved therein. Therefore, to ensure that eccDNA is properly amplified, it is considered to detect the amplification effect after eccDNA amplification. Thus, in step 2) of some embodiments, the amplification effect may be verified by PCR, e.g., multiplex PCR, by housekeeping genes after amplification of the extrachromosomal circular DNA. It will be appreciated that multiplex PCR is also within the ability of those skilled in the art.

In some embodiments, the sample may be, but is not limited to, blood, plasma, urine, saliva, pleural effusion, peritoneal effusion, mucous, or cerebrospinal fluid. In a preferred embodiment, the sample is plasma.

In some embodiments, the lower limit of detection of the method may be as low as 5pg extrachromosomal circular DNA/mL of the sample to be detected. In this case, 1mL of plasma or 10ng of cfDNA would normally be sufficient for detection.

By the eccna library construction method of the present invention, an eccna library can be obtained. The library includes not only the eccna to be detected but also double-stranded circular DNA as a positive control, and thus can ensure that the detection result is true negative, excluding false negative results.

In addition, the amplification of eccDNA, such as MDA amplification, is involved earlier in the method, so that cfDNA up to 30ng in initial amount is not required as in the existing Tn 5-based fragment library method, but detection can be performed with only 5pg of extrachromosomal circular DNA, and the sensitivity is higher.

b) Sequencing the product obtained in step a).

Prior to the present invention, sequencing of extrachromosomal circular DNA was performed based on the Illumina sequencing platform, only circular DNA with MspI cleavage site could be detected, while eccNA without MspI cleavage site could not be detected. However, the sequencing method of the present invention may be accomplished on a DNBseq sequencing platform. The DNBseq sequencing platform is a DNA nanosphere-based sequencing technology. The rolling circle replication is carried out on the same template, even if a single error base is introduced in the replication process, the error base can not amplify error signals due to PCR, so that the sequencing result is more accurate, and the rolling circle replication is particularly important for low-copy DNA. Thus, in some embodiments, step b) is performed on a DNBseq sequencing platform. By the method of the invention, more selectivity is provided for the sequencing of eccDNA.

In a fourth aspect, the present invention provides a kit for extrachromosomal circular DNA sequencing comprising: the plasmid pUC19 shown in SEQ ID NO. 20 and/or the double-stranded circular DNA shown in SEQ ID NO. 1 are used as positive control of the experimental system.

The existing eccna detection system has no positive control, and the content of the eccna in the sample is relatively low originally, so that false negative results are easy to occur in the detection process. The double-stranded circular DNA, such as the double-stranded circular DNA shown in SEQ ID NO. 1 or pUC19 shown in SEQ ID NO. 20 designed by the inventor, is added into the detection system, so that the occurrence of false negative problems can be effectively avoided.

The present invention will be described in more detail with reference to examples. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below were purchased from a conventional reagent store unless otherwise specified. It should be noted that the summary of the invention above and the detailed description below are for purposes of specific illustration only and are not intended to limit the invention in any way.

Examples

eccDNA extraction and enrichment

To 1ml of plasma was added 5pg of the experimentally positive plasmid pUC19 (SEQ ID NO:20, 2686 bp) or 5pg of GAPDH circular DNA (SEQ ID NO:1,406 bp) obtained by PCR using the lung cancer cell line genome as a template, using the primers shown in SEQ ID NO: 18-19. cfDNA was then extracted from plasma using QIAamp Circulating Nucleic Acid Kit (Qiagen) and following instructions for use.

The extracted cfDNA was then digested with Plasmid-Safe ATP-Dependent Dnase (Epicentre, cat No. E3110K) to remove linear DNA. The reaction system is shown in Table 1.

TABLE 1 reaction System for digestion of Linear DNA in cfDNA

The reaction system was mixed uniformly, then allowed to react at 37℃for 30 minutes, and after the completion of the reaction, allowed to react at 70℃for another 30 minutes.

FIG. 1 shows graphs of the results obtained by the Agilent 2100 bioanalyzer before and after cleavage of the linear dsDNA, plasmid pUC19 and a mixture of linear dsDNA and plasmid Puc 19. As can be seen from the results of FIG. 1, the linear DNA was completely digested off after 30 minutes of reaction at 37℃under the action of the plasma-safe ATP-Dependent DNase, and the circular DNA was retained during the digestion.

eCDNA amplification and verification of amplification Effect

mu.L of the reaction product obtained in step 1 was subjected to Multiple Displacement Amplification (MDA) using MGIEasy single cell whole genome amplification kit and operated according to the instructions therein. In this experiment, lung cancer cell line genomic DNA (gDNA) was used as positive control and deionized water was used as negative control.

After MDA was performed on the reaction product obtained in step 1, the amplification effect was verified by housekeeping genes using multiplex PCR. The PCR amplification system is shown in Table 2, in which the primer mixture was prepared by diluting each primer to 10. Mu.M and mixing them in the volumes shown in Table 3. The reaction conditions of PCR are shown in Table 4.

TABLE 2 PCR amplification System

TABLE 3 PCR primer mix composition

TABLE 4 PCR reaction conditions

After the multiplex PCR amplification was completed, 1. Mu.L of the PCR product was subjected to electrophoresis on a 2% agarose gel at 170 volts for 25 minutes.

FIG. 2 shows a graph of the effect of MDA amplification, with lanes from left to right in the graph being lane 1:1kb DNA ladder; lane 2: a 50bp DNA ladder; lane 3 and lane 4: PCR products with the lung cancer cell line gDNA MDA amplification products as templates; lanes 5 and 6: a PCR product taking MDA amplification product of deionized water as a template; lanes 7 and 8: PCR products using deionized water as templates. Generally, the number of electrophoresis bands of 6 or more in agarose gel electrophoresis results indicates that the amplification effect is good. In this figure, the electrophoresis bands of the positive control (lanes 3 and 4) were 6 and above, which indicates that MDA was amplified efficiently and that the amplified product was available for further disruption and chip selection.

3. Disruption and chip selection of amplified products

Taking 1 mug of MDA amplification product, carrying out ultrasonic disruption by using a Covaris instrument, and setting the disruption parameters as follows: the Duty cycle value was 21, the pip/integrity value was 500, the cpb value was 500,Treatment Times and the value was 20 s.14. The broken product was purified with 0.8x+0.2xAMPure beads and redissolved with 40. Mu.L deionized water.

4. End repair and adenylate (A) addition

The reagents listed in Table 5 were added and reacted sequentially in a PCR apparatus at 37℃for 30 minutes, at 65℃for 15 minutes, and then maintained at 4 ℃.

TABLE 5 reaction System for end repair and adenylate addition

* : PNK represents a Polynucleotide kinase

5. Joint connection

The reagents listed in Table 6 were added to the reaction solution of step 4, and the mixture was reacted at 23℃for one hour after being mixed. The reaction product was recovered by purification with 0.5 XAMPure beads and reconstituted with 50. Mu.L deionized water.

TABLE 6 reaction System for Joint connection

PCR amplification

Mixing 25. Mu.L of the DNA obtained in step 5 with a PCR amplification reagent, thereby forming a PCR amplification system as shown in Table 7:

TABLE 7 PCR reaction System

After the above materials were uniformly mixed, PCR was performed under the reaction conditions shown in Table 8 below. The PCR product was recovered using 1 XAMPure beads, reconstituted with 22. Mu.L deionized water and concentration checked using Qubit (Invitrogen).

TABLE 8 PCR reaction conditions

7. Cyclization

330ng of the product obtained in step 6 was taken, water was added to make up 48.2. Mu.L of the system, and after mixing well, it was reacted at 95℃for 3 minutes, and then immediately placed on ice. The reagents listed in Table 9 below were added in this order, and after mixing well, the mixture was reacted at 37℃for 30 minutes.

TABLE 9 reaction System for cyclization

8. Digestion by enzyme digestion

After the cyclization reaction was completed, the reagents shown in Table 10 were added in this order, and after mixing uniformly, they were reacted at 37℃for 30 minutes. Then, the mixture was recovered by purification using 2.5 XP beads and then dissolved in 42. Mu.L of deionized water.

TABLE 10 reaction System for cleavage

9. Sequencing on machine

The single-stranded loop obtained in step 8 was subjected to double-ended sequencing in a DIPSEQ-T1 sequencer in a manner of PE100+10. Sequencing data were used to analyze eccna in plasma.

10. Data analysis results

Table 11 shows the results of the eccna detection in plasma. From the sequencing analysis results, it was found that by using the method of the present invention, circular DNA as low as 5pg in 1mL of plasma could be detected, and the sample control pUC19 was nearly ten thousand, GAPDH circular DNA was over twenty thousand, and the loop size was consistent with the expectations (as shown in FIG. 3), and eccDNA as high as 28 ten thousand or more in the plasma itself was also detected (as shown in Table 11).

TABLE 11 statistics of the results of the detection of eccna in plasma

Sequence information:

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gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560

cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620

gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680

tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740

ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800

ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860

atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920

cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980

tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040

aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100

tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160

ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220

agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280

gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340

agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400

accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460

gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520

cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580

ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 2640

atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 2686

Claims

1. A method for the construction of an extrachromosomal circular DNA comprising the steps of:

1) Extracting extrachromosomal circular DNA in a sample to be detected;

2) Amplifying the extrachromosomal circular DNA, e.g., multiple Displacement Amplification (MDA), to obtain an amplification product thereof;

3) Breaking up the amplification products (e.g., using ultrasound) and performing chip selection on the DNA fragments;

5) Circularizing the ligated DNA fragment obtained in step 4), and digesting with enzymes such as exonucleases such as exonuclease I and exonuclease III to remove uncyclized DNA fragments.

2. Method according to claim 1, characterized in that in step 1) a double stranded circular DNA, such as pUC19 plasmid with the sequence of SEQ ID No. 20 and/or GAPDH circular DNA with the sequence of SEQ ID No. 1, is added to the sample to be tested as positive control before extracting the extrachromosomal circular DNA in the sample to be tested.

3. The method according to claim 1 or 2, wherein step 1) comprises: extracting cfDNA in the sample to be detected, and digesting linear DNA in the extracted cfDNA by Exonuclease such as Plasmid-Safe ATP-Dependent DNase or Exoneuclease V (RecBCD), thereby obtaining the extrachromosomal circular DNA.

4. A method according to any one of claims 1-3, characterized in that in step 3) DNA fragments of 200bp-400bp in length are obtained by chip selection.

5. The method according to any one of claims 1 to 4, further comprising the step of PCR amplifying the ligated DNA fragments after step 4) and before step 5).

6. The method according to any one of claims 1-5, characterized in that in step 2), further comprising: after the extrachromosomal circular DNA is amplified, the amplification effect is verified by PCR, for example, multiplex PCR, by housekeeping genes.

7. The method of any one of claims 1-6, wherein the sample is blood, plasma, urine, saliva, pleural effusion, peritoneal effusion, mucous or cerebrospinal fluid.

8. The method of any one of claims 1-7, wherein the method has a lower limit of detection of up to 5pg extrachromosomal circular DNA/mL of the sample to be detected.

9. A method of sequencing extrachromosomal circular DNA, the method comprising:

a) Performing a pooling method of the extrachromosomal circular DNA of any of claims 1-8;

b) Sequencing the product obtained in step a).

10. The method of claim 9, wherein step b) is performed on a DNBseq sequencing platform.

11. A double-stranded circular DNA used as a positive control in extrachromosomal circular DNA sequencing has the sequence shown in SEQ ID NO. 1.

12. A kit for extrachromosomal circular DNA sequencing comprising: the plasmid pUC19 shown in SEQ ID NO. 20 and/or GAPDH circular DNA shown in SEQ ID NO. 1 are used as positive control of the experimental system.