Preparation method of chromosome outer circular DNA library, sequencing method, kit and application thereof

Abstract

The invention provides a preparation method of a chromosome outer circular DNA library, a sequencing method, a kit and application thereof. The preparation method comprises the steps that total DNA in a sample is extracted, linear DNA in the total DNA is digested, a digestion product is obtained, and the digestion product comprises chromosome outer circular DNA; directly carrying out rolling circle amplification on the digestion product without purification to obtain an amplification product; an amplification product is directly subjected to library preparation without being purified, and the chromosome outer circular DNA library is obtained.

Description

Preparation method, sequencing method, kit and application of extrachromosomal circular DNA library Technical Field

[0001] The present invention relates to the field of gene sequencing, and in particular to a method for preparing an extrachromosomal circular DNA library, a sequencing method, a kit and applications thereof. Background Art

[0002] Extrachromosomal circular DNA (eccDNA) is a ubiquitous form of DNA in cells, existing independently of chromosomes. eccDNA ranges in length from a few hundred to several megabase pairs (bp) and can encode regulatory elements or entire genes. In 1965, researchers used electron microscopy to discover extrachromosomal circular DNA in pig sperm and wheat cells. Later, researchers also discovered eccDNA in yeast, human, mouse, and other cells. Related terms include eccDNA, spcDNA, double minutes, microDNA, and ecDNA. In recent years, using high-throughput sequencing technology, researchers have discovered free eccDNA in samples such as human plasma and urine. Researchers have found that fetal eccDNA is present in the plasma of pregnant women, and that the free eccDNA in the plasma of cancer patients also has certain characteristics. Therefore, free eccDNA is a potential liquid biopsy biomarker with important research and potential application value.

[0003] Advances in high-throughput sequencing technology and bioinformatics have enabled sequencing of extremely low levels of eccDNA. High-throughput sequencing-based eccDNA detection technologies primarily include ATAC-Seq using Tn5 transposase, linear DNA digestion followed by Tn5 transposase fragmentation and library construction, rolling circle amplification and sequencing using phi29 DNA polymerase after linear DNA digestion, and whole-genome sequencing combined with bioinformatics analysis. Sequencing platforms used include the Illumina platform for high-throughput short-read sequencing and the Nanopore and PacBio platforms for single-molecule sequencing.

[0004] However, eccDNA detection solutions based on Tn5 transposase and short-read sequencing technology have certain biases. Junction sites can only be detected in sequenced fragments and identified as eccDNA if the fragmentation site is near the circular junction site. Furthermore, because eccDNA is not amplified and enriched before fragmentation, a large amount of eccDNA information is easily lost during the experiment, affecting sequencing and analysis results.

[0005] The rolling circle amplification and sequencing method based on linear DNA digestion and the use of phi29 DNA polymerase involves digesting linear DNA and then amplifying eccDNA to the nanogram or microgram level using phi29 DNA polymerase. Conventional PCR amplification and library construction and sequencing methods can then be used for detection. However, this method was developed for eccDNA in tissue cells. The experimental process is long, the linear DNA digestion step takes several days, and there are multiple DNA purification steps. In addition, PCR amplification of the rolling circle amplification products is required during the preparation of the sequencing library. Since the replication errors generated during the PCR process accumulate exponentially, they will affect the final base sequence analysis, which is not conducive to base analysis with high accuracy requirements such as tumor mutations. The application scenarios of this technology are greatly limited.

[0006] In addition, the existing eccDNA detection technology has relatively high requirements for the starting amount of body fluid samples such as plasma or serum, requiring 1mL or more, which also limits the application scope of eccDNA detection.

[0007] Summary of the Invention

[0008] The main purpose of the present invention is to provide a method for preparing an extrachromosomal circular DNA library, a sequencing method, a kit and applications thereof, so as to solve the problem of high sample starting amount in the prior art.

[0009] To achieve the above-mentioned object, according to a first aspect of the present invention, a method for preparing an extrachromosomal circular DNA library is provided, the method comprising: extracting total DNA from a sample and digesting linear DNA in the total DNA to obtain digestion products, wherein the digestion products include extrachromosomal circular DNA; directly performing rolling circle amplification on the digestion products without purification to obtain amplification products; and directly performing library preparation on the amplification products without purification to obtain an extrachromosomal circular DNA library.

[0010] Furthermore, the sample is a body fluid sample, preferably, the extrachromosomal circular DNA is any one or more of the following: mitochondrial DNA, chloroplast DNA, microbial plasmid, circular DNA virus or extrachromosomal circular DNA produced by nuclear DNA; preferably, the body fluid sample includes plasma; preferably, the starting volume of the plasma sample is less than 500 μL; preferably, the starting volume of the plasma sample is 400-500 μL.

[0011] Furthermore, the linear DNA is digested with any one or more of the following digestion enzymes: Plasmid-safe ATP-de pendent DNase, Exonuclease V (RecBCD) or Exonuclease III; preferably, the digestion enzyme is Exonuclease V (RecBCD); preferably, the digestion time is 0.5-1.5 h.

[0012] Furthermore, the rolling circle amplification further comprises: combining the amplification primer with the digestion product and then performing rolling circle amplification to obtain the amplified product.

[0013] Furthermore, the amplification primer is an oligonucleotide primer containing 4-8 random bases; wherein the random base is any one of A, T, C, and G; preferably, the number of random bases is 6; preferably, the random base is a modified random base; preferably, the modification is located 1-2 bases away from the 5' end or 3' end of the amplification primer; preferably, the modification includes a modification having any one of the following functions: 1) Function 1: preventing the amplification primer from being degraded by nucleases; 2) Function 2: preventing polymerase jumping during amplification to produce junk sequences; or 3) Function 3: preventing amplification primers from serving as templates for each other and extending to produce junk sequences; preferably, the modification having function 1 is a thiolation modification at the 3' end; preferably, the modifications having functions 2 and 3 are each independently selected from any one or more of the following 5' end modifications: 2', 3' ddT (5' reverse ddT) modification, dSpacer modification, spacer C3 modification, spacer C9 modification, spacer C18 modification; preferably, the sequence of the amplification primer is 5'-NpNpNpNp S Np S N-3'.

[0014] Furthermore, the amplification primers are combined with the digestion product under any of the following conditions: 1) at room temperature of 20-28°C; 2) under denaturing conditions of 50-95°C; 3) under an isothermal amplification reaction temperature of 25-37°C; preferably, the isothermal amplification reaction temperature is 30-35°C; preferably, the combination is performed under denaturing conditions of 75-95°C.

[0015] Furthermore, combining under denaturing conditions of 50-95°C includes: mixing the amplification primer and the digestion product and maintaining them at 50-95°C for a first period of time to obtain an incubated product; cooling the incubated product to below 4°C and maintaining it for a second period of time to obtain a cooled product; subjecting the cooled product to an isothermal amplification reaction under the catalysis of a rolling circle amplification enzyme to obtain an amplification product; preferably, mixing the amplification primer and the digestion product and placing them at 80-95°C; preferably, the first period of time is 1-10 minutes; preferably, the incubated product is cooled to 4-0°C; preferably, the second period of time is 1-15 minutes; preferably, the rolling circle amplification enzyme is selected from wild-type or mutant phi29 DNA polymerase; preferably, the temperature of the isothermal amplification reaction is 30-35°C; preferably, the time of the isothermal amplification reaction is 4-16 hours.

[0016] Furthermore, library preparation includes PCR-free library preparation, which includes: randomly shearing the amplified products while attaching library construction adapters to the sheared fragments to obtain a PCR-free library, wherein the random shearing is performed by a biological enzyme method or a physical shearing method; preferably, the PCR-free library is prepared using a PCR-free library preparation technology supporting the DNA nanoball-based DNB SEQ sequencing technology, preferably, the MGIEasy PCR-Free DNA Library Preparation Reagent Set or the MGIEasy Enzyme Digestion PCR-Free DNA Library Preparation Reagent Set is used to prepare the PCR-free library.

[0017] To achieve the above object, according to a second aspect of the present invention, a method for sequencing extrachromosomal circular DNA is provided, the method comprising: (1) preparing an extrachromosomal circular DNA library according to the above preparation method; (2) sequencing the extrachromosomal circular DNA library; preferably, sequencing is performed using any one of the following sequencing platforms: MGISEQ-2000, MGISEQ-200, DNBSEQ-E25, DNBSEQ-G99, DNBSEQ-T7, DNBSEQ-T10×4 or DNBSEQ-T20×2.

[0018] To achieve the above object, according to the third aspect of the present invention, a kit is provided, which comprises two or more of the following: a digestion enzyme for digesting linear DNA, a rolling circle amplification enzyme, an amplification primer, and a reaction buffer; wherein the digestion enzyme is selected from at least one of the following: Exonuclease V (RecBCD), Plasmid-safe ATP-dependent DNase, and Exonuclease III; and the rolling circle amplification enzyme is selected from any one of the following: wild-type or mutant phi29 DNA polymerase; the amplification primer is selected from an oligonucleotide primer containing multiple, preferably 4-8, more preferably 6 random bases; preferably, the random base is a modified random base; preferably, the modification is located 1-2 bases away from the 5' end or 3' end of the amplification primer; preferably, the modification includes a modification having any one of the following functions: 1) Function 1: preventing the amplification primer from being degraded by nucleases; 2) Function 2: preventing polymerase jumping during amplification to produce junk sequences; or 3) Function 3: preventing amplification primers from serving as templates for each other and extending to produce junk sequences; preferably, the modification having function 1 is a thiolation modification at the 3' end; preferably, the modifications having functions 2 and 3 are each independently selected from any one or more of the following 5' end modifications: 2', 3' ddT (5' reverse ddT) modification, dSpacer modification, spacer C3 modification, spacer C9 modification, spacer C18 modification; preferably, the sequence of the amplification primer is 5'-NpNpNpNp S Np S N-3'.

[0019] In order to achieve the above-mentioned object, according to the fourth aspect of the present invention, there is provided a use of the above-mentioned kit in the preparation and / or sequencing of an extrachromosomal circular DNA library.

[0020] Applying the technical solution of the present invention, in the preparation of eccDNA libraries, the present invention achieves amplification of the test sample through rolling circle amplification after linear DNA digestion. This process omits the DNA purification steps after linear DNA digestion and rolling circle amplification reactions. While ensuring amplification efficiency, it also reduces sample loss and the required initial sample amount for testing. Therefore, it can be applied to the preparation of libraries containing low-input free eccDNA in body fluids, broadening the application range of eccDNA library preparation.

[0021] In a preferred embodiment, the digestion and amplification reaction conditions, including the digestion reagents, are further optimized, greatly shortening the library preparation time. A PCR-free approach is used for library construction and sequencing, avoiding mutations introduced by human manipulation. This, in turn, avoids the difficulty in accurately distinguishing replication errors caused by conventional PCR amplification library construction from true mutations such as tumor mutations, thereby improving the accuracy of eccDNA sequencing base analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0023] FIG1 shows a schematic diagram of the process of preparing and detecting an eccDNA library in Example 1 of the present invention;

[0024] FIG2 shows the eccDNA length distribution diagram in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0026] As mentioned in the background art, there are some shortcomings in the detection method based on the principle of linear DNA digestion followed by phi29 DNA polymerase amplification in the existing art of eccDNA sequencing. The library construction experimental process based on this principle is relatively long, in which the linear DNA digestion step takes several days and there are multiple DNA purification steps. During sequencing, the replication errors generated by the sequencing platform based on PCR amplification will accumulate exponentially, affecting the final base sequence analysis. In addition, the samples sequenced by this method have certain limitations, the sample starting amount is required to be high, and it is mostly used for tissue samples. Therefore, the inventors of this application provide a method for preparing an eccDNA library. By optimizing the experimental steps of library construction, multiple purification steps are omitted to achieve the purpose of low sample starting amount library construction, while shortening the library construction time and greatly improving the efficiency of eccDNA sequencing. In addition, the accuracy of eccDNA sequencing is further improved by PCR-Free library construction.

[0027] In a first typical embodiment of the present invention, a method for preparing an extrachromosomal circular DNA library is provided, which comprises: extracting total DNA from a sample and digesting linear DNA in the total DNA to obtain a digestion product, wherein the digestion product comprises extrachromosomal circular DNA; directly performing rolling circle amplification on the digestion product without purification to obtain an amplification product; and directly performing library preparation on the amplification product without purification to obtain an extrachromosomal circular DNA library.

[0028] The optimized library construction method of the present invention omits the DNA purification steps after linear DNA depletion and rolling circle amplification, making the experimental procedure simpler and faster. Omitting the DNA purification step reduces sample loss, making it applicable to library preparation of free eccDNA in low-input body fluids, thereby broadening the application range of eccDNA library preparation.

[0029] The above-mentioned methods for extracting DNA from samples include genomic DNA extraction methods, plasmid DNA extraction methods, and free DNA extraction methods.

[0030] The samples for the above-mentioned preparation of the extrachromosomal circular DNA library can be body fluid samples or tissue samples, and are particularly suitable for body fluid samples. The inventors of this application have found through experimental testing that the existing extrachromosomal circular DNA (eccDNA) library construction method is developed for tissue samples and cell samples. The starting amount of DNA in tissue and cell samples is relatively high and relatively easy to detect. Since the DNA content in body fluid samples is relatively small, there are certain difficulties in detecting body fluid samples using existing technologies developed based on tissue and cell samples with a higher DNA content. Therefore, it is difficult to achieve library construction and detection of free eccDNA in body fluid samples.

[0031] In a preferred embodiment, the extrachromosomal circular DNA includes any one or more of the following: mitochondrial DNA, chloroplast DNA, microbial plasmids, circular DNA viruses or extrachromosomal circular DNA produced by nuclear DNA; in a preferred embodiment, the body fluid sample includes plasma; when the body fluid sample is plasma, the extrachromosomal circular DNA includes extrachromosomal circular DNA produced by mitochondrial DNA and nuclear DNA. In a preferred embodiment, the starting amount of the plasma sample is less than 500 μL, and more preferably, the starting amount of the plasma sample is 400-500 μL. The existing technology has high requirements for the starting amount of body fluid samples, requiring at least 1 mL of starting amount. However, by using the method of omitting the two-step purification step mentioned above in the present application, the purpose of library construction and detection of body fluid samples with a lower starting amount can be achieved.

[0032] In the above-mentioned linear DNA digestion reaction, any enzyme that can digest linear DNA can be used in the present invention. Among them, any one or more of the following digestive enzymes can be used to digest linear DNA: Plasmid-safe ATP-dependent DNase, Exonuclease V (RecBCD), Exonuclease III; in a preferred embodiment, the digestive enzyme is Exonuclease V (RecBCD); the digestion time is 0.5-1.5h. Digesting linear DNA can increase the relative content or purity of eccDNA in the library sample. The existing technology is mostly for tissue or cell samples. Since the content of genomic long fragment DNA extracted from tissue or cell samples is high, in order to eliminate the influence of genomic long fragment DNA, the linear DNA digestion step takes several days, and there are multiple DNA purification steps, so the experimental process of library construction is longer. The present application optimizes the digestive enzyme used and since the purification step is omitted, the digestion method of the present invention can greatly shorten the digestion time to about 1 hour, thereby improving the efficiency of library construction.

[0033] The aforementioned rolling circle amplification also includes: combining amplification primers with digestion products and then performing rolling circle amplification to obtain an amplified product; directly performing rolling circle amplification on the digestion products, which can capture and amplify extremely small amounts of eccDNA. This application reduces the sample input requirement by optimizing the primers, amplification enzyme, and amplification reaction system conditions used in the rolling circle amplification reaction, thereby improving the efficiency of rolling circle amplification.

[0034] The above-mentioned amplification primer is an oligonucleotide primer containing 4-8 random bases; wherein the random base is any one of A, T, C, and G. In a preferred embodiment, the number of random bases is 6; the random base is a modified random base. The modification is located 1-2 bases away from the 5' end or 3' end of the amplification primer; the modification includes a modification having any one of the following functions: 1) Function 1: preventing the amplification primer from being degraded by nucleases; 2) Function 2: preventing polymerase jumping during amplification to produce junk sequences; or 3) Function 3: preventing amplification primers from serving as templates for each other and extending to produce junk sequences; preferably, the modification having function 1 is a thiolation modification at the 3' end; preferably, the modifications having functions 2 and 3 are each independently selected from any one or more of the following 5' end modifications: 2', 3' ddT (5' reverse ddT) modification, dSpacer modification, spacer C3 modification, spacer C9 modification, and spacer C18 modification. Among them, 2', 3'ddT (5' reverse ddT) is 2', 3'dideoxy-dT base (5'inverted ddT), also known as 2', 3'dideoxythymine (5' reversed dideoxythymine); dSpacer, spacer C3, spacer C9, and spacer C18 are all base-free spacers. In the above modifications, the thio modification at the 3' end is to prevent the amplification primer from being degraded by nucleases; the modification at the 5' end is a modification with a steric hindrance effect, which can prevent the generation of junk sequences during the amplification process due to mutual template extension and polymerase jumping between primers. This application uses special groups to modify the primers to achieve better amplification effects.

[0035] In a preferred embodiment, the sequence of the amplification primer is 5'-NpNpNpNp S Np S N-3', wherein the N base is base A, base T, base C or base G, and the two bases at the 3' end of the primer are thiolated to prevent the primer from being degraded by nucleases.

[0036] In the aforementioned rolling circle amplification, the amplification primers and digestion products bind under any of the following conditions: 1) at room temperature between 20 and 28°C; 2) under denaturing conditions between 50 and 95°C; or 3) at an isothermal amplification reaction temperature between 25 and 37°C. In a preferred embodiment, the isothermal amplification reaction temperature is 30-35°C. In a preferred embodiment, the amplification primers and digestion products are denatured at 75-95°C, then rapidly cooled to below 4°C, and finally, at the isothermal reaction temperature, the single-stranded primers bind to the single strands. At high temperatures within this range, the double strands melt, and when the temperature returns to the annealing temperature, the single-stranded primers can bind to the single strands with high specificity.

[0037] The above-mentioned amplification primers and digestion products are combined under denaturing conditions of 50-95°C, including: mixing the amplification primers and digestion products and maintaining them at 50-95°C for a first period of time to obtain an incubated product; cooling the incubated product to below 4°C and maintaining it for a second period of time to obtain a cooled product; and performing an isothermal amplification reaction on the cooled product under the catalysis of a rolling circle amplification enzyme to obtain an amplified product.

[0038] In a preferred embodiment, the amplification primers and digestion product are mixed and incubated at 80-95°C; the first time duration is 1-10 minutes; the incubated product is cooled to 4-0°C; the second time duration is 1-15 minutes; the rolling circle amplification enzyme is selected from wild-type or mutant phi29 DNA polymerase; the isothermal amplification reaction temperature is 30-35°C; and the isothermal amplification reaction time is 4-16 hours. The amplified product of the present invention does not require purification, shortening the library preparation time to 2 days, significantly reducing the time period and improving experimental efficiency.

[0039] The above-mentioned library preparation includes PCR-free library preparation. The preparation of a PCR-free library involves randomly fragmenting the amplified product while attaching library construction adapters to the fragmented fragments, thereby obtaining a PCR-free library. Random fragmentation is performed using a bio-enzymatic method or a physical fragmentation method. In a preferred embodiment, the PCR-free library is prepared using a PCR-free library preparation technology that is compatible with DNA nanoball-based DNB SEQ sequencing technology. The preparation of a PCR-free library directly adds sequencing adapters, eliminating the need for PCR amplification during library construction. This reduces sample label hopping and sample cross-contamination caused by the PCR process, as well as the bias and base error accumulation caused by PCR amplification. This improves the accuracy of library sequencing and is beneficial for applications requiring high-accuracy base analysis, such as tumor mutations.

[0040] In a preferred embodiment, the MGIEasy PCR-Free DNA Library Preparation Reagent Set or the MGIEasy Enzyme Digestion PCR-Free DNA Library Preparation Reagent Set is used to prepare the PCR-free library.

[0041] In a second typical embodiment of the present invention, a method for sequencing extrachromosomal circular DNA is provided, comprising: (1) preparing an extrachromosomal circular DNA library according to the above-mentioned preparation method; and (2) sequencing the extrachromosomal circular DNA library. The sequencing of the present invention can be performed on any sequencing platform that can be used for library detection. In a preferred embodiment, sequencing is performed using any of the following sequencing platforms: MGISEQ-2000, MGISEQ-200, DNBSEQ-E25, DNBSEQ-G99, DNBSEQ-T7, DNBSEQ-T10×4, or DNBSEQ-T20×2.

[0042] In a third exemplary embodiment of the present invention, a kit is provided, comprising two or more of the following: a digestion enzyme for digesting linear DNA, a rolling circle amplification enzyme, amplification primers, and a reaction buffer; wherein the digestion enzyme is selected from at least one of the following: Exonuclease V (RecBCD), Plasmid-safe ATP-dependent DNase, and Exonuclease III; and the rolling circle amplification enzyme is selected from any one of the following: wild-type or mutant phi29 DNA polymerase. In a preferred embodiment, the rolling circle amplification enzyme is selected from a mutant phi29 DNA polymerase. The amplification primers are selected from a plurality of oligonucleotide primers, preferably 4-8, and more preferably 6, random bases. In a preferred embodiment, the random base is a modified random base; the modification is located 1-2 bases away from the 5' end or 3' end of the amplification primer; the modification includes a modification having any of the following functions: 1) Function 1: preventing the amplification primer from being degraded by nucleases; 2) Function 2: preventing polymerase jumping during amplification to produce junk sequences; or 3) Function 3: preventing amplification primers from serving as templates for each other and extending to produce junk sequences; preferably, the modification having function 1 is a thiolation modification at the 3' end; preferably, the modifications having functions 2 and 3 are each independently selected from any one or more of the following 5' end modifications: 2', 3' ddT (5' reverse ddT) modification, dSpacer modification, spacer C3 modification, spacer C9 modification, spacer C18 modification. The sequence of the amplification primer is 5'-NpNpNpNp S Np S N-3'.

[0043] In a fourth exemplary embodiment of the present invention, a kit is provided for use in the preparation and / or sequencing of an extrachromosomal circular DNA library. The use of the kit for the construction and sequencing of an eccDNA library optimizes the experimental process, reduces experimental time, improves the efficiency of DNA amplification and library sequencing, and reduces experimental costs compared to kits used in the prior art. The kit can also be used for detecting free eccDNA in the field of liquid biopsy, broadening the application range of eccDNA sequencing.

[0044] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0045] Example 1: Enrichment of eccDNA, library preparation, and sequencing analysis (step flow shown in Figure 1)

[0046] 1. Extraction of plasma free DNA

[0047] Plasma samples from three healthy individuals were collected, each containing 400 μL of plasma. Samples were designated as Sample 1, Sample 2, and Sample 3. DNA was extracted using the MGIEasy Cell-Free DNA Extraction Kit (MGI, 1000017017) in strict accordance with the manufacturer's instructions. DNA samples were then resolubilized in 30 μL of Elution Buffer.

[0048] 2. Linear DNA Digestion

[0049] Digest the linearized DNA using Exonuclease V (RecBCD) (NEB, M0345L) and NEB Buffer 4. In a PCR reaction tube, prepare the linear DNA digestion reaction according to Table 1. Vortex the reaction tube to mix thoroughly and centrifuge briefly to collect the reaction mixture at the bottom of the tube. Place the reaction tube in a PCR instrument and run the linear DNA digestion reaction according to Table 2.

[0050] Table 1 Linear DNA digestion reaction system

[0051] Table 2 Linear DNA digestion reaction conditions

[0052] 3. Rolling Circle Amplification of eccDNA

[0053] 3.1 Preparation of amplification reaction solution

[0054] Prepare the rolling circle amplification reaction solution according to Table 3, mix thoroughly, and place on ice until ready for use.

[0055] Table 3 Rolling circle amplification reaction solution

[0056] 3.2 Primer annealing

[0057] Add 4 μL of DNA amplification primer (Sangon Biotechnology, 500 μM) to the PCR reaction tube of the linear DNA digestion product, whose sequence is 5'-NpNpNpNp S Np S N-3', where N is A, T, C, or G, and p represents the phosphate backbone. The two bases at the 3' end of the primer are phosphothioate-modified (denoted by S) to prevent degradation by nucleases. Vortex the reaction tube, centrifuge briefly, and place in a PCR instrument. Run the high-temperature denaturation reaction conditions in Table 4.

[0058] Table 4 High temperature denaturation reaction conditions

[0059] After the reaction program is completed, take out the PCR reaction tube immediately and proceed to the next step quickly.

[0060] 3.3 Isothermal amplification reaction

[0061] Use a pipette to draw 9 μL of the rolling circle amplification reaction solution prepared in step 3.1 and add it to the PCR reaction tube in the previous step. Vortex to mix thoroughly. Centrifuge briefly to collect the reaction solution at the bottom of the tube. Immediately run the isothermal amplification reaction in Table 5.

[0062] Table 5 Isothermal amplification reaction conditions

[0063] After the reaction, centrifuge briefly and place on ice until ready to use. The concentration of the amplified product was determined using the Qubit dsDNA BR Assay Kit. The concentrations of the amplified product for Sample 1, Sample 2, and Sample 3 were 1040 ng / μL, 950 ng / μL, and 1100 ng / μL, respectively.

[0064] 4. PCR-free library preparation

[0065] Based on the concentration of the DNA amplification product detected in step 3.3, take a total of 1500 ng of the amplification product into a new PCR tube and add nuclease-free HO to 80 μL.

[0066] Sequencing libraries were prepared from the amplified products using the MGI Easy PCR-Free DNA Library Preparation Kit (MGI, 1000013452) according to the manufacturer's instructions. DNA fragmentation was performed using a Covaris ultrasonic disruptor to fragment the amplified DNA products to between 150 and 1000 bp, with a major band length of 300 to 500 bp. The fragments were screened according to the manufacturer's instructions, using magnetic bead double selection conditions with a major band of 400 bp.

[0067] The remaining steps, including DNA end repair, adapter ligation, purification of ligation products, denaturation, single-strand circularization, enzyme digestion, purification of digestion products, and quality control of digestion products, were performed strictly in accordance with the instructions of the MGIEasy PCR-Free DNA Library Preparation Reagent Set (MGI, 1000013452).

[0068] 5. High-throughput sequencing

[0069] Sequencing was performed using the MGISEQ-2000RS High-Throughput Sequencing Kit (FCL PE100) (MGI, 1000012554) in strict accordance with the kit instructions. Sequencing was performed on the MGISEQ-2000 gene sequencer using the PE100+10 (Paired End 100+10) sequencing method to obtain base sequence information.

[0070] 6. eccDNA Sequencing Data Analysis and Results Presentation

[0071] The raw sequencing data (raw reads) were filtered for low-quality bases, adapters were removed, and unpaired reads were removed to generate clean reads. All clean reads were aligned to the human genome (GRCh38) as a reference. Statistics were calculated for the number of raw reads, the number of filtered reads, the number of reads aligned to the human genome, the number of reads aligned to nuclear DNA, the number of reads aligned to mitochondrial DNA, and the human genome coverage. The results are shown in Table 6.

[0072] Table 6 Basic information of sample sequencing and eccDNA detection

[0073] Split reads and junction sites were identified based on the Circle_finder analysis process (https: / / github.com / pk7zuva / Circle_finder). Split reads are a special type of sequencing reads. The front and back parts can be aligned to different positions on the genome, and both are uniquely aligned. They are the main source of information for detecting eccDNA loop junctions (Junction sites). In this embodiment, Circle_finder was used to preliminarily identify potential eccDNAs, and then the potential eccDNAs were filtered by limiting the number of Split reads and the sequencing depth and coverage of the loop region. For the detection of each eccDNA, the minimum number of Split reads for the Junction site is required to be 2; the two alignment positions of the Split read on the genome are required to be on the same chromosome, and the length distance is less than 1Mb; the average sequencing depth of the bases aligned to the loop region on the genome (from the starting position to the ending position on the genome) is required to be more than 4.5 times the average sequencing depth of the bases in the two flanking regions (extending 1 / 2 of the length of the loop region to both sides). Based on the above analysis conditions, eccDNA was filtered and detected, and basic information such as the number, average GC content, and length distribution of eccDNA was statistically analyzed. The number and average GC content of eccDNA in each sample are shown in Table 6. The length distribution of eccDNA in each sample is shown in Figure 2.

[0074] As shown in Table 6, abundant eccDNA was detected in all three samples, with specific numbers of 28517, 31077, and 38541. The genome coverage was all below 5%, indicating that the digestion of linear DNA was relatively complete and there was little linear DNA residue. Mitochondrial DNA was detected in the sequencing results, but the proportion was not high, and most of the detection signals came from eccDNA generated on nuclear DNA. As shown in Figure 2, the detected circular DNA had obvious periodicity, and its periodicity of about 160 to 200 bp may be related to the periodicity of nucleosomes, and its periodicity of about 10 bp may be related to the structure of double-stranded DNA. The above periodic characteristics are consistent with the known periodicity of eccDNA, indicating that the experimental method of the present invention is suitable for the detection of eccDNA and can capture the relevant characteristics of eccDNA.

[0075] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the eccDNA library preparation method of the present invention can be used to prepare and detect free eccDNA in body fluid samples. By optimizing the preparation steps, the DNA purification steps after linear DNA digestion and rolling circle amplification reaction are omitted, making the experimental operation simpler and faster, reducing sample loss, and lowering the requirement for the starting amount of detection sample. Therefore, it can be applied to the preparation of libraries of free eccDNA in low starting amounts of body fluids, broadening the application range of eccDNA library preparation.

[0076] In addition, the digestion and amplification reaction conditions, including digestion reagents, were further optimized, greatly shortening the library preparation time. The PCR-free method was used for library construction and sequencing, avoiding mutations introduced by human manipulation. This, in turn, avoided the problem of difficulty in accurately distinguishing between replication errors caused by conventional PCR amplification library construction and real mutations such as tumor mutations, which is conducive to improving the accuracy of eccDNA sequencing base analysis.

[0077] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing an extrachromosomal circular DNA library, characterized in that: The preparation method comprises: Extracting total DNA from a sample and digesting linear DNA in the total DNA to obtain a digestion product, wherein the digestion product includes extrachromosomal circular DNA; The digestion product is directly subjected to rolling circle amplification without purification to obtain an amplified product; The amplified product is directly used for library preparation without purification to obtain the extrachromosomal circular DNA library.

2. The preparation method according to claim 1, characterized in that: The sample is a body fluid sample, Preferably, the extrachromosomal circular DNA includes any one or more of the following: mitochondrial DNA, chloroplast DNA, plasmid of microorganisms, circular DNA virus or extrachromosomal circular DNA produced by nuclear DNA; Preferably, the body fluid sample comprises plasma; Preferably, the initial volume of the plasma sample is less than 500 μL; Preferably, the initial volume of the plasma sample is 400-500 μL.

3. The preparation method according to claim 1, characterized in that: Digest the linear DNA with any one or more of the following digestive enzymes: Plasmid-safe ATP-dependent DNase, Exonuclease V (RecBCD) or Exonuclease III; Preferably, the digestive enzyme is Exonuclease V (RecBCD); Preferably, the digestion time is 0.5-1.5h.

4. The preparation method according to claim 1, characterized in that: The rolling circle amplification further comprises: The amplification primer is combined with the digestion product and then the rolling circle amplification is performed to obtain the amplification product.

5. The preparation method according to claim 4, characterized in that: The amplification primer is an oligonucleotide primer containing 4-8 random bases, wherein the random base is any one of A, T, C, and G; Preferably, the number of random bases is 6; Preferably, the random base is a modified random base; Preferably, the modification is located 1-2 bases away from the 5' end or 3' end of the amplification primer; Preferably, the modification includes a modification having any of the following functions: 1) Function 1: Prevent the amplification primer from being degraded by nuclease; 2) Function 2: Preventing junk sequences from being generated by polymerase jumping during amplification; or 3) Function 3: Prevent the amplification primers from serving as templates for each other and extending to generate junk sequences; Preferably, the modification having function 1 is a thio modification at the 3' end; Preferably, the modifications having the function 2 and the function 3 are each independently selected from any one or more of the following 5' end modifications: 2', 3' ddT (5' reverse ddT) modification, dSpacer modification, spacer C3 modification, spacer C9 modification, spacer C18 modification; Preferably, the sequence of the amplification primer is 5'-NpNpNpNp S Np S N-3'.

6. The preparation method according to claim 4, characterized in that: The amplification primer is combined with the digestion product under any of the following conditions: 1) At room temperature of 20-28°C; 2) Under denaturation conditions of 50-95°C; 3) at an isothermal amplification reaction temperature of 25-37°C; Preferably, the isothermal amplification reaction temperature is 30-35°C; Preferably, the binding is performed under denaturing conditions of 75-95°C.

7. The preparation method according to claim 6, characterized in that: Carrying out the binding under denaturing conditions of 50 to 95° C. comprises: The amplification primer and the digestion product are mixed and kept at 50-95° C. for a first period of time to obtain an incubation product; Cooling the heat-insulated product to below 4° C. and maintaining it for a second period of time to obtain a cooled product; The cooling product is subjected to the isothermal amplification reaction under the catalysis of the rolling circle amplification enzyme to obtain the amplification product; Preferably, the amplification primers and the digestion product are mixed and placed at 80-95°C; Preferably, the first duration is 1-10 minutes; Preferably, the temperature of the insulated product is lowered to 4-0°C; Preferably, the second duration is 1-15 minutes; Preferably, the rolling circle amplification enzyme is selected from wild-type or mutant phi29 DNA polymerase; Preferably, the temperature of the isothermal amplification reaction is 30-35°C; Preferably, the isothermal amplification reaction time is 4-16 hours.

8. The preparation method according to claim 1, characterized in that: The library preparation includes PCR-free library preparation, and the PCR-free library preparation includes: randomly shearing the amplified product and attaching library construction adapters to the sheared fragments, thereby obtaining the PCR-free library, wherein the random shearing is performed by a biological enzyme method or a physical shearing method; Preferably, the PCR-free library is prepared using a PCR-free library preparation technology that is compatible with the DNA nanoball-based DNB SEQ sequencing technology. Preferably, the PCR-free library is prepared using MGIEasy PCR-Free DNA Library Preparation Reagent Kit or MGIEasy Enzyme Digestion PCR-Free DNA Library Preparation Reagent Kit.

9. A method for sequencing extrachromosomal circular DNA, characterized in that: The method comprises: (1) Preparing an extrachromosomal circular DNA library according to the preparation method according to any one of claims 1 to 8; (2) sequencing the extrachromosomal circular DNA library; Preferably, sequencing is performed using any one of the following sequencing platforms: MGISEQ-2000, MGISEQ-200, DNBSEQ-E25, DNBSEQ-G99, DNBSEQ-T7, DNBSEQ-T10×4 or DNBSEQ-T20×2.

10. A kit, characterized in that: The kit comprises two or more of the following: a digestion enzyme for digesting linear DNA, a rolling circle amplification enzyme, an amplification primer and a reaction buffer; Wherein, the digestive enzyme is selected from at least one of the following: Exonuclease V (RecBCD), Plasmid-safe ATP-dependent DNase, Exonuclease III; The rolling circle amplification enzyme is selected from any one of the following: wild-type or mutant phi29 DNA polymerase; The amplification primer is selected from an oligonucleotide primer comprising a plurality of, preferably 4-8, more preferably 6, random bases; Preferably, the random base is a modified random base; Preferably, the modification is located 1-2 bases away from the 5' end or 3' end of the amplification primer; Preferably, the modification includes a modification having any of the following functions: 1) Function 1: Prevent the amplification primer from being degraded by nuclease; 2) Function 2: Preventing junk sequences from being generated by polymerase jumping during amplification; or 3) Function 3: Prevent the amplification primers from serving as templates for each other and extending to generate junk sequences; Preferably, the modification having function 1 is a thio modification at the 3' end; Preferably, the modifications having the function 2 and the function 3 are each independently selected from any one or more of the following 5' end modifications: 2', 3' ddT (5' reverse ddT) modification, dSpacer modification, spacer C3 modification, spacer C9 modification, spacer C18 modification; Preferably, the sequence of the amplification primer is 5'-NpNpNpNp S Np S N-3'.

11. Use of the kit according to claim 10 in the preparation and / or sequencing of an extrachromosomal circular DNA library.

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