A method for constructing a library and sequencing of extrachromosomal circular DNA

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of gene sequencing. More specifically, the present invention relates to a method for constructing a library and sequencing extrachromosomal circular DNA. Background Art

[0002] Extrachromosomal circular DNA (eccDNA) is located outside the chromosome and has a single-stranded or double-stranded closed circular DNA structure. It widely exists in various eukaryotes. Most eccDNAs are less than 25 kb and are mainly distributed between 0.1 kb and 5 kb. Due to its circular structure characteristics, compared with free linear DNA, eccDNA has a stable structure and is not easily degraded. The generation of eccDNA is related to genomic instability. Some studies have 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, and can independently complete the replication process. It has high transcriptional activity itself and is a new amplification form of tumor oncogenes. Therefore, eccDNA is related to the occurrence and development of tumors and is a new type of tumor marker.

[0003] A literature published in PNAS this year (Identification and characterization of extrachromosomal circular DNA in maternal plasma (https: / / www.pnas.org / content / 117 / 3 / 1658)) detected eccDNA in the peripheral blood of pregnant women, confirming that eccDNA can be secreted into the blood circulation system and can be detected from plasma by high-throughput sequencing technology.

[0004] The existing methods for detecting eccDNA in plasma are based on the Illumina sequencing platform (https: / / www.pnas.org / content / 117 / 3 / 1658), including the detection method of eccDNA based on the digestion of restriction endonuclease MspI and the library construction method based on Tn5 tagmentation.

[0005] For the MspI-based detection method, 25 ng of circulating free DNA (cfDNA) is required as the starting amount. After digesting cfDNA with exonuclease V, it is purified and recovered by column. The circular DNA is digested into linear DNA by the restriction enzyme MspI and then a library is constructed for sequencing. This method can only detect circular DNA with one MspI cleavage site, while eccDNA without MspI cleavage site or with multiple MspI cleavage sites cannot be detected, resulting in a small and incomplete variety of detected eccDNA.

[0006] Another Tn5-based tagmentation library construction method, although it increases the number of detected circular DNA, requires as much as 30 ng of cfDNA as the starting amount, so it is not suitable for clinical samples of trace cfDNA.

[0007] In addition, there is no positive control in the experimental systems of these two detection methods, and the possible false negative problems during eccDNA detection cannot be solved. And currently, all eccDNA detection methods are based on the Illumina sequencing platform, while there is temporarily no detection technology based on the DNBseq sequencing platform.

[0008] Therefore, there is an urgent need in the art for a method for detecting eccDNA that can detect trace clinical samples and establish a positive control in the experimental system, so as to more accurately detect eccDNA. Summary of the Invention

[0009] As described above, the existing methods for detecting eccDNA are not comprehensive and accurate enough in detecting eccDNA, have a high requirement for the starting amount of sample cfDNA, and the obtained detection results are not accurate enough. Therefore, there is an urgent need in the art for a method for detecting eccDNA that can detect trace clinical samples, establish a positive control in the experimental system, so as to more accurately detect eccDNA.

[0010] Therefore, in a first aspect, the present invention provides a method for constructing a library of extrachromosomal circular DNA, comprising the following steps:

[0011] 1) Extract extrachromosomal circular DNA from the sample to be detected;

[0012] 2) Amplify the extrachromosomal circular DNA to obtain its amplification product;

[0013] 3) Fragment the amplification product and select DNA fragments;

[0014] 4) Repair the ends of the DNA fragments obtained by chip selection and add adenosine (A), and then ligate with adapters to obtain DNA fragments with adapters.

[0015] 5) Circularize the DNA fragments with adapters obtained in step 4), and digest with enzymes to remove uncircularized DNA fragments.

[0016] In a second aspect, the present invention provides a method for sequencing extrachromosomal circular DNA, the method comprising:

[0017] a) Performing the library construction method for extrachromosomal circular DNA of the first aspect of the present invention;

[0018] b) Sequencing the product obtained in step a).

[0019] In a third aspect, the present invention provides a double-stranded circular DNA used as a positive control in the sequencing of extrachromosomal circular DNA, and its sequence is shown in SEQ ID NO: 1.

[0020] In a fourth aspect, the present invention provides a kit for detecting the sequencing of extrachromosomal circular DNA, comprising: plasmid pUC19 and / or the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) circular DNA shown in SEQ ID NO: 1, as a positive control for the experimental system.

[0021] The beneficial effects of the present invention include one or more of the following:

[0022] 1) Developed a method for detecting eccDNA based on the DNBseq sequencing platform;

[0023] 2) Reduced the starting amount of samples required for eccDNA detection, and even if each milliliter of the sample contains only 5 pg of eccDNA, effective detection can be carried out;

[0024] 3) Designed GAPDH circular double-stranded DNA suitable for use as a positive control in various eccDNA detections.

[0025] 4) By adding double-stranded circular DNA as a positive control to the experimental system, the true negative and false negative problems in eccDNA detection can be distinguished, so as to obtain more accurate detection results. Description of the Drawings

[0026] Figure 1 It is a detection result diagram obtained by an Agilent 2100 bioanalyzer before and after digestion of linear double-stranded DNA (dsDNA), plasmid pUC19, and a mixture of linear dsDNA and plasmid pUC19 with plasmid-safe ATP-Dependent DNase.

[0027] Figure 2 It is a detection diagram of the MDA amplification effect. Among them, the lanes from left to right in the figure are: Lane 1: 1kb DNA ladder; Lane 2: 50bp DNA ladder; Lanes 3 - 4: PCR products with the MDA amplification product of lung cancer cell line gDNA as the template; Lanes 5 - 6: PCR products with the MDA amplification product of deionized water as the template; Lanes 7 - 8: PCR products with deionized water as the template.

[0028] Figure 3 It is a composition ratio diagram of circular DNAs of different sizes (mitochondrial DNA, genomic DNA, pUC19 (positive control), and GAPDH circular DNA (positive control)).

[0029] Figure 4 It is a schematic diagram of the construction of GAPDH circular DNA. Detailed implementation manners

[0030] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments that those of ordinary skill in the art can obtain and they all fall within the scope of protection of the present invention.

[0031] As described above, the existing detection results of eccDNA based on the restriction endonuclease MspI are not comprehensive and accurate enough, and the required starting amount of samples for the library construction method based on Tn5 Tagmentation is too large. Therefore, the purpose of the present invention is to provide a new detection method for eccDNA, which can detect trace clinical samples and establish a positive control in the experimental system, so as to obtain more accurate and comprehensive detection results.

[0032] Therefore, in the first aspect, the present invention provides a method for constructing a library of extrachromosomal circular DNA, including the following steps:

[0033] 1) Extract extrachromosomal circular DNA from the sample to be detected;

[0034] 2) Amplify the extrachromosomal circular DNA to obtain its amplification product;

[0035] 3) Fragment the amplification product and perform fragment selection on the DNA fragments;

[0036] 4) End-repair and adenylation (A) addition are performed on the DNA fragments obtained by chip selection, and then they are ligated to adapters, thereby obtaining adapter-ligated DNA fragments;

[0037] 5) The adapter-ligated DNA fragments obtained in step 4) are circularized, and the uncircularized DNA fragments are removed by enzymatic digestion.

[0038] Since extrachromosomal circular DNA (eccDNA) has high heterogeneity in origin, its generation may involve different occurrence models and repair mechanisms in different backgrounds. EccDNA can affect cell life activities, promote tumor cell evolution and adaptive evolution, increase genomic plasticity and instability, and may have important application potential in tumor diagnosis and treatment, liquid biopsy, etc. Therefore, new eccDNA detection methods, especially high-throughput sequencing and other technologies, are very important for discovering and understanding more eccDNA.

[0039] In step 1), the circulating DNA (cfDNA) of the sample to be detected can be extracted first. The extraction of cfDNA can be carried out by using a conventional commercially available kit. It can be understood that the extracted cfDNA includes not only extrachromosomal circular DNA (eccDNA), but also linear DNA, and the latter is not the detection target. Therefore, before performing the subsequent steps, these linear DNAs need to be removed. Therefore, in some embodiments, step 1) includes: extracting cfDNA from the sample to be detected, and then digesting the linear DNA in the extracted cfDNA with exonuclease to obtain extrachromosomal circular DNA. In the present invention, Plasmid-Safe ATP-Dependent DNase or Exonuclease V (RecBCD) can be specifically used as the exonuclease for digesting linear DNA, but it is not limited thereto. Any exonuclease capable of digesting linear DNA can be used in the present invention.

[0040] In addition, as described in the background art section, there is no positive control in the existing eccDNA detection methods, so the problem of false negatives that may occur during eccDNA 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 eccDNA detections, and added this double-stranded circular DNA to the sample to be detected during eccDNA detection, thereby solving the problem of distinguishing true negatives and false negatives in eccDNA detection.

[0041] Thus, in some embodiments, in step 1), before extracting extrachromosomal circular DNA from the sample to be detected, double-stranded circular DNA is added to the sample to be detected as a positive control. In the present invention, the double-stranded circular DNA serving as the positive control may include the pUC19 plasmid shown in SEQ ID NO: 20 and / or the GAPDH circular DNA with the sequence shown in SEQ ID NO: 1, but is not limited thereto. Those skilled in the art can select double-stranded circular DNA of the most suitable size according to the sample type.

[0042] A plasmid refers to double-stranded closed circular DNA that exists outside the bacterial chromosome and can replicate independently. As a gene engineering vector, a plasmid usually contains an origin of replication, single recognition sites for a variety of restriction endonucleases, and a selectable marker gene such as an ampicillin gene for screening recombinants. Currently commercially available plasmids, such as vectors of the pBR322, pUC, pGEM, and pGEX series, contain 2 MspI restriction sites in the origin of replication region and 5 MspI restriction sites on the ampicillin gene, so they cannot be used as positive controls in existing MspI-based eccDNA detection. However, the principle of eccDNA detection in the present invention is different from the MspI digestion method, so the eccDNA detection is not limited by the presence or absence of restriction sites and the number of restriction sites contained. The pUC19 plasmid is selected as the positive control of the present invention because of its easy access to high-copy-number and high-quality samples and its small length (only 2686 base pairs).

[0043] GAPDH is widely distributed in various tissue cells and is highly expressed in almost all tissues, and the protein expression is basically constant in the same type of cells or tissues. It is selected as a positive control because of its housekeeping gene characteristics. The GAPDH circular double-stranded DNA designed by the present inventors has the following three characteristics: First, the gene sequence is human-derived, and there is no need to additionally select a reference genome during bioinformatics analysis when detecting eccDNA in human body fluids as an experimental positive control; second, the designed GAPDH circular double-stranded DNA is 406 bp in size, which is slightly larger than the main eccDNA in pregnant women's plasma. The peak distribution of eccDNA in pregnant women's plasma is at 338 bp. Using this GAPDH circular double-stranded DNA as a positive control for eccDNA detection can not only simulate the experimental positive control for the detection target during the experiment process, but also not affect the detection of eccDNA in plasma; finally, this GAPDH circular double-stranded DNA loop does not contain MspI restriction sites, so it can be used as a positive control for detecting eccDNA based on MspI digestion.

[0044] In the present invention, double-stranded circular DNA is added as a positive control in eccDNA detection. It can be verified from the sequencing results, which realizes the problem of setting an experimental positive control in the detection of the same tube of samples. There is no need for other additional experimental designs to quality control the experimental process. The experimental operation is simple, and it can solve the problems of true negative and false negative in eccDNA detection.

[0045] In some embodiments, in step 2), multiple displacement amplification (MDA) is used to amplify extrachromosomal circular DNA. MDA is an isothermal amplification method based on the principle of strand displacement amplification and is widely used for the amplification of genomes or transcriptomes. MDA usually uses phi29 DNA polymerase, which exhibits excellent strand displacement and exonuclease activities. Therefore, MDA is superior to PCR in terms of template coverage and amplification fidelity, and a large amount of high-quality DNA can be obtained from a very small amount of DNA sample. In addition, strand displacement amplification results in high molecular weight (≥10 kb) DNA products, and the amplified DNA is suitable for library construction.

[0046] In some embodiments, in step 3), the amplified product can be fragmented by ultrasound. The conditions of ultrasound can be specifically selected according to specific conditions as long as DNA fragments of the desired length can be obtained.

[0047] After fragmenting the amplified product, it is also necessary to further select DNA fragments of the desired length. The selection can be carried out by magnetic beads, but is not limited thereto. Any method that can achieve the selection is within the scope of the present invention. In the present invention, preferably, DNA fragments with a length of 200 bp - 400 bp are obtained through selection.

[0048] It can be understood that in subsequent on-machine sequencing, more DNA fragments are more suitable. For this reason, in some embodiments, after step 4) and before step 5), there is also a step of PCR amplifying the DNA fragments with adapters. The systems and conditions for PCR amplification are within the capabilities of those skilled in the art.

[0049] As described above, in step 5), it includes removing uncircularized DNA fragments by enzymatic digestion. In the present invention, any enzyme that can be used to digest uncircularized DNA fragments can be used. The enzyme can be, for example, an exonuclease, such as exonuclease I and exonuclease III, but is not limited thereto.

[0050] In addition, as described above, the present invention may require fewer samples compared to existing methods, partly because it involves the amplification of eccDNA. Therefore, to ensure proper amplification of eccDNA, the amplification effect can be detected after the amplification of eccDNA. Thus, in step 2) of some embodiments, after the amplification of the extrachromosomal circular DNA, the amplification effect can be verified by PCR, such as multiplex PCR, through housekeeping genes. It can be understood that multiplex PCR is also within the capabilities of those skilled in the art.

[0051] In some embodiments, the sample may be a body fluid such as blood, plasma, urine, saliva, pleural effusion, peritoneal effusion, mucus, or cerebrospinal fluid, but is not limited thereto. In a preferred embodiment, the sample is plasma.

[0052] In some embodiments, the lower limit of detection of the method can be as low as 5 pg of extrachromosomal circular DNA / mL of the sample to be detected. In this case, 1 mL of plasma or 10 ng of cfDNA can generally meet the requirements for detection.

[0053] Through the eccDNA library construction method of the present invention, an eccDNA library can be obtained. This library not only includes the eccDNA to be detected, but also includes double-stranded circular DNA as a positive control, so that the detection result can be ensured to be a true negative, excluding false negative results.

[0054] In addition, in the early stage of this method, it involves the amplification of eccDNA, such as MDA amplification. Therefore, it does not require as much as 30 ng of cfDNA as the starting amount like the existing Tn5-based tagmentation library construction method, but only requires as low as 5 pg of extrachromosomal circular DNA to perform the detection, with higher sensitivity.

[0055] In a second aspect, the present invention provides a method for sequencing extrachromosomal circular DNA, the method comprising:

[0056] a) Performing the method for constructing a library of extrachromosomal circular DNA according to the first aspect of the present invention;

[0057] b) Sequencing the product obtained in step a).

[0058] Before the present invention, the sequencing of extrachromosomal circular DNA was basically carried out based on the Illumina sequencing platform, and only circular DNA with MspI restriction sites could be detected, while eccDNA without MspI restriction sites could not be detected. However, the sequencing method of the present invention can be completed on the DNBseq sequencing platform. The DNBseq sequencing platform is a sequencing technology based on DNA nanoballs. For the same template, rolling circle replication is performed. Even if a single incorrect base is introduced during the replication process, this incorrect base will not amplify the error signal due to PCR, thus ensuring more accurate sequencing results, which is particularly important for low-copy DNA. Therefore, in some embodiments, step b) is carried out on the DNBseq sequencing platform. The method of the present invention provides more options for the sequencing of eccDNA.

[0059] In a third aspect, the present invention provides a double-stranded circular DNA used as a positive control in the sequencing of extrachromosomal circular DNA, and its sequence is shown in SEQ ID NO:1.

[0060] In a fourth aspect, the present invention provides a kit for the sequencing of extrachromosomal circular DNA, including: plasmid pUC19 shown in SEQ ID NO:20 and / or the double-stranded circular DNA shown in SEQ ID NO:1, as a positive control for the experimental system.

[0061] There is no positive control in the existing eccDNA detection system, and the content of eccDNA in the sample is relatively low originally. Therefore, false negative results are likely to occur during the detection process. By adding double-stranded circular DNA to the detection system, such as the double-stranded circular DNA shown in SEQ ID NO:1 designed by the present inventor or pUC19 shown in SEQ ID NO:20, the occurrence of false negative problems can be very effectively avoided.

[0062] The present invention will be described more specifically and in detail below with reference to the examples. The test methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from regular reagent stores unless otherwise specified. It should be noted that the above-mentioned invention content part and the following detailed description are only for the purpose of specifically explaining the present invention and are not intended to limit the present invention in any way.

[0063] Examples

[0064] 1. eccDNA Extraction and Enrichment

[0065] Add 5 pg of the experimental positive plasmid pUC19 (SEQ ID NO: 20, 2686 bp) or 5 pg of GAPDH circular DNA (SEQ ID NO: 1, 406 bp) to 1 ml of plasma. The GAPDH circular DNA was obtained by PCR using the primers shown in SEQ ID NOs: 18 - 19 with the genomic DNA of a lung cancer cell line as the template. Subsequently, use the QIAamp Circulating Nucleic Acid Kit (Qiagen) to extract cfDNA from the plasma according to the instructions.

[0066] Then digest the extracted cfDNA with Plasmid-Safe ATP-Dependent Dnase (Epicentre, catalog number E3110K) to remove linear DNA. The reaction system is shown in Table 1.

[0067] Table 1. Reaction system for digesting linear DNA in cfDNA

[0068]

[0069] Mix the above reaction system, then incubate it at 37 °C for 30 minutes, and after the reaction, incubate it at 70 °C for 30 minutes.

[0070] Figure 1 The detection result graphs obtained by the Agilent 2100 Bioanalyzer before and after digestion with plasmid-safe ATP-Dependent DNase for linear dsDNA, plasmid pUC19, and a mixture of linear dsDNA and plasmid Puc19 are shown. From Figure 1 the results, it can be seen that linear DNA was completely digested after reacting at 37 °C for 30 minutes under the action of plasmid-safe ATP-Dependent DNase, while circular DNA was retained during the digestion process.

[0071] 2. eccDNA Amplification and Amplification Effect Verification

[0072] Take 5 μL of the reaction product obtained in step 1 for multiple displacement amplification (MDA). The MDA is performed using the MGIEasy Single Cell Whole Genome Amplification Kit according to the instructions therein. In this experiment, genomic DNA (gDNA) of a lung cancer cell line was used as a positive control, and deionized water was used as a negative control.

[0073] After performing MDA on the reaction product obtained in Step 1, multiplex PCR was used to verify the amplification effect through housekeeping genes. The PCR amplification system is shown in Table 2, where the primer mixture was prepared by diluting each primer to 10 μM and mixing them in the volumes shown in Table 3. The reaction conditions for PCR are shown in Table 4.

[0074] Table 2. PCR Amplification System

[0075]

[0076] Table 3. Composition of PCR Primer Mixture

[0077]

[0078]

[0079] Table 4. PCR Reaction Conditions

[0080]

[0081] After the multiplex PCR amplification was completed, 1 μL of the PCR product was subjected to 2% agarose gel electrophoresis at 170 V for 25 minutes.

[0082] Figure 2 The detection graph showing the MDA amplification effect is presented. The lanes from left to right in the graph are as follows: Lane 1: 1 kb DNA ladder; Lane 2: 50 bp DNA ladder; Lanes 3 and 4: PCR products with the gDNA MDA amplification product of the lung cancer cell line as the template; Lanes 5 and 6: PCR products with the MDA amplification product of deionized water as the template; Lanes 7 and 8: PCR products with deionized water as the template. Generally, in the agarose gel electrophoresis results, six or more electrophoresis bands indicate a good amplification effect. In this graph, the electrophoresis bands of the positive control (Lanes 3 and 4) are six bands or more, indicating that MDA can effectively amplify and the amplification product can be used for the next step of fragmentation and fragment selection.

[0083] 3. Fragmentation and Size Selection of Amplification Products

[0084] 1 μg of the MDA amplification product was taken and sonicated using a Covaris instrument with the following fragmentation parameters: Duty cycle value of 21, PIP / Intensity value of 500, CPB value of 500, Treatment Times value of 20 s * 14. The fragmented product was purified with 0.8× + 0.2× AMPure beads and redissolved in 40 μL of deionized water.

[0085] 4. End Repair and Adenylation (A) Addition

[0086] Add the reagents listed in Table 5, and react successively at 37°C for 30 minutes, at 65°C for 15 minutes in a PCR instrument, and then maintain at 4°C.

[0087] Table 5. Reaction system for end repair and adenylation addition

[0088]

[0089] *: PNK represents polynucleotide kinase

[0090] 5. Adapter Ligation

[0091] Add the reagents listed in Table 6 to the reaction solution in Step 4, mix well and react at 23°C for one hour. The reaction product is purified and recovered with 0.5×AMPure beads and redissolved with 50 μL of deionized water.

[0092] Table 6. Reaction system for adapter ligation

[0093]

[0094]

[0095] 6. PCR Amplification

[0096] Take 25 μL of the DNA obtained in Step 5 and mix it with PCR amplification reagents to form a PCR amplification system as shown in Table 7:

[0097] Table 7. PCR reaction system

[0098]

[0099] After mixing the above substances evenly, perform PCR according to the reaction conditions shown in Table 8 below. The PCR product is purified and recovered with 1×AMPure beads, redissolved with 22 μL of deionized water, and the concentration is detected using Qubit (Invitrogen).

[0100] Table 8. PCR reaction conditions

[0101]

[0102] 7. Circularization

[0103] Take 330 ng of the product obtained in Step 6, add water to make up a 48.2 μL system, mix well and react at 95°C for 3 minutes, and then immediately place on ice. Successively add the reagents listed in Table 9 below, mix well, and react at 37°C for 30 minutes.

[0104] Table 9. Reaction system for cyclization

[0105]

[0106] 8. Enzymatic Digestion

[0107] After the cyclization reaction is completed, the reagents listed in Table 10 below are added sequentially. After mixing evenly, the reaction is carried out at 37 °C for 30 minutes. Then, purification and recovery are carried out using 2.5×XP magnetic beads, and then it is dissolved in 42 μL of deionized water.

[0108] Table 10. Reaction system for enzymatic digestion

[0109]

[0110] 9. Sequencing on Machine

[0111] The single-stranded ring obtained in step 8 is subjected to paired-end sequencing using a DIPSEQ-T1 sequencer, and the sequencing method is PE100 + 10. The sequencing data is used to analyze eccDNA in plasma.

[0112] 10. Data Analysis Results

[0113] Table 11 shows the detection results of eccDNA in plasma. It can be found from the sequencing analysis results that by using the method of the present invention, circular DNA as low as 5 pg in 1 mL of plasma can be detected, and the sample control pUC19 is close to 10,000, the GAPDH circular DNA is more than 20,000, and the size of the ring is consistent with the expectation (as Figure 3 shown), and at the same time, more than 280,000 eccDNA in plasma itself are also detected (as shown in Table 11).

[0114] Table 11. Statistical results of the detection of eccDNA in plasma

[0115]

[0116] Sequence information:

[0117]

[0118]

[0119] Sequence Listing <110> BGI Shenzhen Institute of Life Sciences <120> Method for constructing library and sequencing of extrachromosomal circular DNA <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 406 <212> DNA / RNA <213> Artificial Sequence <400> 1 aattcccttc ttgcctcttg tctcttagat ttggtcgtat tgggcgcctg gtcaccaggg 60 ctgcttttaa ctctggtaaa gtggatattg ttgccatcaa tgaccccttc attgacctca 120 actacatggt gagtgctaca tggtgagccc caaagctggt gtgggaggag ccacctggct 180 gatgggcagc cccttcatac cctcacgtat tcccccaggt ttacatgttc caatatgatt 240 ccacccatgg caaattccat ggcaccgtca aggctgagaa cgggaagctt gtcatcaatg 300 gaaatcccat caccatcttc caggagtgag tggaagacag aatggaagaa atgtgctttg 360 gggaggcaac taggatggtg tggctccctt gggtatatgg taaccg 406 <210> 2 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 2 ggcaacgctt agactctgtg tg 22 <210> 3 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 3 ctgcccttgg cctaactaac ct 22 <210> 4 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 4 gttcctcaag aagctgcacg ag 22 <210> 5 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 5 cgttagactc tggatctggc gt 22 <210> 6 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 6 ccagccaatt catgagtcgg tg 22 <210> 7 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 7 cctgacaact cgcaagtagc ac 22 <210> 8 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 8 gctcaatggg gtacttcagg gt 22 <210> 9 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 9 gtggacgtta cgtaaaggcc c 21 <210> 10 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 10 tgctctggat gtgaagatgc ca 22 <210> 11 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 11 ttccaggtaa atccagccca gg 22 <210> 12 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 12 cagccagtaa gcatcatcca ac 22 <210> 13 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 13 gaaagccgga ttgcggtaac at 22 <210> 14 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 14 ggatagctct gcaaggggag ag 22 <210> 15 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 15 tcgtcgcagt agaaatacgg ct 22 <210> 16 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 16 agaagtcagg cacgtagctc ag 22 <210> 17 <211> 22 <212> DNA / RNA <213> Artificial Sequence <400> 17 ggcacgttgg tgtttacgat ga 22 <210> 18 <211> 33 <212> DNA / RNA <213> Artificial Sequence <400> 18 tattgaattc ccttcttgcc tcttgtctct tag 33 <210> 19 <211> 33 <212> DNA / RNA <213> Artificial Sequence <400> 19 tattgaattc ggttaccata tacccaaggg agc 33 <210> 20 <211> 2686 <212> DNA / RNA <213> Artificial Sequence <400> 20 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct 480 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct 480 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 1320 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500 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 constructing a library of extrachromosomal circular DNA, comprising the following steps: 1) Extract extrachromosomal circular DNA from a sample to be detected; 2) Amplify the extrachromosomal circular DNA to obtain an amplification product thereof; 3) Fragment the amplification product and select DNA fragments; 4) Repair the ends of the selected DNA fragments and add adenosine (A), and then ligate with adapters to obtain DNA fragments with adapters; 5) Circularize the DNA fragments with adapters obtained in step 4), and digest with enzymes to remove uncircularized DNA fragments; Wherein, in step 1), before extracting extrachromosomal circular DNA from the sample to be detected, double-stranded circular DNA is added to the sample to be detected as a positive control; Wherein, the double-stranded circular DNA is the pUC19 plasmid with the sequence shown in SEQ ID NO: 20 and / or the GAPDH circular DNA with the sequence shown in SEQ ID NO: 1; Wherein, step 1) includes: extracting cfDNA from the sample to be detected, and then digesting the linear DNA in the extracted cfDNA with exonuclease to obtain the extrachromosomal circular DNA; Wherein, in step 2), the amplification method is multiple displacement amplification; Wherein, in step 5), the enzyme is exonuclease; Wherein, after step 4) and before step 5), there is also a step of performing PCR amplification on the DNA fragments with adapters.

2. The method according to claim 1, wherein The exonuclease in step 1) is Plasmid-Safe ATP-Dependent DNase or Exonuclease V.

3. The method according to claim 1 or 2, characterized in that, In step 3), the amplification product is fragmented by ultrasound.

4. The method according to claim 1 or 2, characterized in that, In step 3), DNA fragments with a length of 200bp - 400bp are obtained by size selection.

5. The method according to claim 1 or 2, characterized in that, The exonuclease in step 5) is Exonuclease I and Exonuclease III.

6. The method according to claim 1 or 2, characterized in that, In step 2), it also includes: after amplifying the extrachromosomal circular DNA, verifying the amplification effect by PCR with a housekeeping gene.

7. The method according to claim 6, wherein The PCR is multiplex PCR.

8. The method according to claim 1 or 2, characterized in that The sample is blood, plasma, urine, saliva, pleural effusion, ascites, mucus or cerebrospinal fluid.

9. The method according to claim 1 or 2, characterized in that, The detection limit of the method is as low as 5 pg of extrachromosomal circular DNA / mL of the sample to be detected.

10. A method for sequencing extrachromosomal circular DNA, the method comprising: a) Performing the method for constructing a library of extrachromosomal circular DNA according to any one of claims 1 - 9; b) Sequencing the product obtained in step a); Wherein, the method is for non-diagnostic use.

11. The method according to claim 10, wherein Step b) is performed on a DNBseq sequencing platform.

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