Genome-wide r-loop detection method and use

Abstract

A genome-wide R-loop detection method, in particular, a genome-wide R-loop detection method independent of affinity enrichment and a use. According to the genome-wide R-loop detection method, nucleases are used to perform enzyme digestion on genomic DNA fragments, and R-loop detection can be achieved by performing one-step enzyme digestion simply using a system of Nuclease P1, T5 exonuclease, and Lambda exonuclease. The whole R-loop detection process does not require affinity enrichment or labeling treatment. The method is not only simple and easy to operate, but also can avoid the loss of samples and fragments, thereby reducing the cost and risk of detection, and improving the applicability.

Description

A whole-genome R-loop detection method and its application Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a whole-genome R-loop detection method, and more particularly to a whole-genome R-loop detection method and its application that does not depend on affinity enrichment. Background Technology

[0002] R-loops are a special nucleic acid structure, distinct from common double-stranded DNA and single-stranded RNA. An R-loop consists of three strands: unwinding occurs in a region of a double-stranded DNA strand, where one DNA strand binds to a complementary single-stranded RNA strand to form a DNA / RNA hybrid, while the other DNA strand remains single-stranded. This region is called the R-loop. R-loops are a triple-stranded nucleic acid structure composed of a DNA:RNA hybrid and associated non-template single-stranded DNA. They exist in various species and are widely involved in physiological and pathological processes.

[0003] Currently, various methods have been developed in this field to improve the understanding of R-loops at the molecular level, but each method has its limitations. DRIP-seq technology based on S9.6 antibodies is the earliest developed and most widely used method, followed by variants of DRIP-seq, including RDIP, DRIPC-seq, S1-DRIP-seq, ssDRIP-seq, and bisDRIP-seq. However, S9.6-based methods have limited resolution and are biased towards GC-rich sequences. Other RNase H-based alternatives, such as R-ChIP, MapR, and R-loop CUT&Tag, capture R-loops using the RNA:DNA hybrid binding domain (HBD) of RNase H. These methods effectively avoid the sequence bias of S9.6 methods, but their efficiency in R-loop capture is lower, and they often preferentially detect R-loops in promoter regions, also affecting the efficiency of the methods. Furthermore, the non-specific binding of S9.6 antibodies or HBDs may affect the specificity of R-loop detection. Other antibody-free methods, such as spKAS-seq, use N3-ketoaldehyde to label the ssDNA strand in the R-loop. However, the labeling of N3-ketoaldehyde may be affected by ssDNA binding proteins and other non-classical DNA structures containing ssDNA strands, which may affect the accuracy of spKAS-seq in capturing the R-loop.

[0004] In summary, traditional R-loop detection methods often suffer from complex workflows and excessively long operation times, which severely impact experimental success rates. Furthermore, inconsistencies in annotated R-loops can prevent most R-loops captured by one method from being captured by others. Therefore, there is a need in the art to develop a method that can effectively improve the resolution, specificity, and sensitivity of R-loop detection, thereby addressing the issue of inconsistent R-loop detection sites in existing technologies. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a whole-genome R-loop detection method that does not rely on affinity enrichment. The method does not rely on affinity enrichment treatment and has the advantages of high detection resolution, high specificity and high sensitivity.

[0006] The second technical problem to be solved by this invention is to provide the application of the above-mentioned whole-genome R-loop detection method in the field of gene detection.

[0007] To address the aforementioned technical problems, the present invention provides a whole-genome R-loop detection method, comprising the following steps:

[0008] (1) Extract genomic DNA from the sample to be tested;

[0009] (2) The extracted genomic DNA is fragmented;

[0010] (3) Use nucleases to digest the processed genomic DNA fragments;

[0011] (4) Use transposase to insert the library construction adapter into both ends of the RNA:DNA hybrid;

[0012] (5) Perform PCR amplification of the library and sequencing.

[0013] Specifically, in the whole genome R-loop detection method, in step (1), the extraction of genomic DNA can be carried out using methods disclosed in the prior art, such as extraction using phenol-chloroform-isoamyl alcohol, or various genomic DNA extraction kits can be used.

[0014] Specifically, in the whole-genome R-loop detection method, step (2) includes at least one of restriction endonuclease treatment, micrococcal nuclease treatment, DNA fragmentation enzyme treatment, or ultrasonic fragmentation treatment.

[0015] Specifically, in the whole-genome R-loop detection method, the restriction endonuclease includes at least one of Alu I, Dde I, Mbo I, Mse I, Hind III, EcoR I, BsrGI, Xba I, or Ssp I; and / or,

[0016] The ultrasonic fragmentation step controls the size of the fragment after fragmentation to be 250-300bp.

[0017] Specifically, in the whole-genome R-loop detection method, in step (3), the nuclease includes at least one of nuclease P1, T5 exonuclease or Lambda exonuclease;

[0018] Preferably, the nuclease comprises a mixture of nuclease P1, T5 exonuclease, and Lambda exonuclease;

[0019] Preferably, the volume ratio of nuclease P1, T5 exonuclease or Lambda exonuclease is 1:3-5:3-5, more preferably 1:4:4.

[0020] Specifically, in the whole genome R-loop detection method, step (3) includes a digestion step of incubating at 35-40°C for 1-3 hours.

[0021] Specifically, in the whole-genome R-loop detection method, step (3) of the digestion step further includes the step of adding Proteinase K for treatment;

[0022] Preferably, the Proteinase K digestion process includes incubation at 50-60°C for 20-40 min and incubation at 65-75°C for 20-40 min.

[0023] Specifically, in the whole-genome R-loop detection method, in step (4), the transposase includes Tn5.

[0024] Specifically, in the whole genome R-loop detection method, step (4) includes the transposase treatment step of incubating at 50-60℃ for 5-15 minutes.

[0025] Specifically, in the whole genome R-loop detection method, in step (5), the PCR system of the PCR amplification step includes: template, primers, polymerase and premixed solution;

[0026] Preferably, the PCR program for the PCR amplification step includes gap completion and PCR amplification procedures.

[0027] As an example, the gap-filling procedure can be conventionally selected according to different PCR systems.

[0028] As an example, the PCR amplification program can select the appropriate PCR amplification program according to different PCR systems, and adjust the parameters adaptively according to the amplification situation.

[0029] The whole-genome R-loop detection method of this invention allows for the use of a conventional PCR system when amplifying the PCR library. For example, the polymerase may include a hot-start DNA polymerase or a reverse transcription polymerase. Those skilled in the art can select a suitable system based on conventional theory.

[0030] Specifically, in the whole-genome R-loop detection method, in step (5), the library construction can also be performed using a DNA or RNA library construction kit.

[0031] This invention also discloses the application of the whole-genome R-loop detection method in the field of gene detection and analysis.

[0032] The whole-genome R-loop detection method of this invention uses nucleases to digest genomic DNA fragments. R-loop detection can be achieved in one step using a system of Nuclease P1, T5 exonuclease and Lambda exonuclease. The entire R-loop detection process does not require affinity enrichment or labeling. The method is not only simple and easy to operate, but also avoids sample and fragment loss, reduces detection costs and risks, and improves applicability.

[0033] The genome-wide R-loop detection method described in this invention requires only a one-step digestion process using Nuclease P1, T5 exonuclease, and Lambda exonuclease. This method not only boasts high sensitivity, yielding an order of magnitude more R-loops, but can also be applied to the detection of R-loops in as few as 10 cells. In particular, the method achieves base-level resolution with a high signal-to-noise ratio and no bias. Compared to traditional detection methods, the genome-wide R-loop detection method of this invention effectively improves detection resolution, specificity, and sensitivity, and solves the problem of site inconsistency in existing R-loop detection technologies. Attached Figure Description

[0034] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0035] Figure 1 is a technical flowchart of the RIAN-seq of the present invention;

[0036] Figure 2 shows the detection results of the scheme in Embodiment 1 of the present invention;

[0037] Figure 3 shows the detection results of the scheme in Embodiment 2 of the present invention;

[0038] Figure 4 shows the detection results of the schemes in Embodiment 1 and Embodiment 3 of the present invention;

[0039] Figure 5 shows the cell detection results in Example 4;

[0040] Figure 6 shows the cell detection results in Example 5;

[0041] Figure 7 shows the cell detection results in Example 6;

[0042] Figure 8 shows the experimental results of the control group in Comparative Example 1;

[0043] Figure 9 shows the experimental results of the scheme in Comparative Example 2. Detailed Implementation

[0044] As shown in the flowchart in Figure 1, in the following embodiments of the present invention, nuclease is used to detect R-loop, and the overall method is referred to as RIAN-seq, which specifically includes the following steps:

[0045] (1) Extract genomic DNA;

[0046] (2) Select restriction endonucleases to fragment genomic DNA;

[0047] (3) Use Nuclease P1, T5 exonuclease and Lambda exonuclease to digest dsDNA, ssDNA and ssRNA.

[0048] (4) Use transposase to insert the library construction adapter into both ends of the RNA:DNA hybrid;

[0049] (5) Perform PCR amplification of the library and sequencing.

[0050] In the following embodiments of the present invention, different cell systems are used to perform RIAN-seq library construction and R-loop detection based on the aforementioned steps.

[0051] Example 1

[0052] This embodiment uses HEK293T for RIAN-seq library construction, specifically including the following steps:

[0053] (1) Collect HEK293T cells, extract genomic DNA using phenol-chloroform-isoamyl alcohol, dissolve in 20 μL ddH2O, and take 1 μg of genomic DNA for subsequent steps;

[0054] (2) Continue to add 3 μL of rCutsmart buffer and 2 μL of a mixture of restriction endonucleases Alu I, Dde I, Mbo I and Mse I (volume ratio 1:1:1:1) for fragmentation;

[0055] (3) Continue to add 0.5 μL nuclease P1, 2 μL T5 exonuclease and 2 μL Lambda exonuclease, mix and incubate at 37℃ for 2 hours; continue to add Proteinase K, mix and incubate at 55℃ for 30 min and 70℃ for 30 min respectively.

[0056] (4) Add 1 μL of Tn5 and mix, then incubate at 55°C for 10 min; add 5 μL of 0.25% SDS and mix, then incubate at room temperature for 5 min;

[0057] (5) Add 10 μL ddH2O, 30 μL NEB Q5 Mastermix, and 2 μL Bst3.0 Polymease; and perform gap filling and PCR amplification according to the following program: 72℃, 10 min; 80℃, 10 min; 98℃, 45 s; (98℃, 15 s; 65℃, 1 min 45 s; (10 cycles)); 72℃, 5 min; 16℃. Collect the amplification product and purify it using 0.85× Backman Ampure beads, then add 20 μL ddH2O for elution; collect the product for PE150 sequencing.

[0058] Meanwhile, the same system was detected using existing methods such as S9.6, RNase H, and spKAS-seq, and the results are shown in Figure 2(a)-(d).

[0059] As can be seen, the R-loop detection method described in this embodiment, RIAN-seq, can capture nearly 500,000 R-loop sites in HEK293T cells, while existing methods can only capture 10,000 to 80,000 sites (as shown in Figure 2a).

[0060] Meanwhile, this invention exhibits the highest signal-to-noise ratio and sensitivity (as shown in Figure 2bc). Therefore, the method described in this invention can present whole-genome R-loop information and obtain the distribution pattern of R-loops even with only 5M sequencing reads, while also demonstrating high signal-to-noise ratio and sensitivity (as shown in Figure 2d).

[0061] In summary, the R-loop detection method described in this embodiment, compared with existing methods such as S9.6, RNase H, and spKAS-seq, not only has an order of magnitude more R-loops, but also exhibits the highest signal-to-noise ratio and sensitivity.

[0062] Example 2

[0063] This embodiment utilizes a small number of HEK293T cells for RIAN-seq library construction, specifically including the following steps:

[0064] (1) Collect 1000, 100 and 10 HEK293T cells respectively, and extract genomic DNA using phenol-chloroform-isoamyl alcohol method, then add 20 μL ddH2O to dissolve;

[0065] (2) Continue to add 3 μL rCutsmart buffer and 2 μL of a mixture of restriction endonucleases Alu I, Dde I, Mbo I and Mse I (volume ratio 1:1:1:1) for fragmentation;

[0066] (3) Continue to add 0.5 μL nuclease P1, 2 μL T5 exonuclease and 2 μL Lambda exonuclease, mix and incubate at 37℃ for 2 hours; continue to add Proteinase K, mix and incubate at 55℃ for 30 min and 70℃ for 30 min respectively.

[0067] (4) Add 1 μL of Tn5 and mix, and incubate at 55°C for 10 min; add 5 μL of 0.25% SDS and mix, and incubate at room temperature for 5 min; add 1 μL of Triton X-100 and mix, and incubate at 37°C for 10 min;

[0068] (5) Continue to add 10 μL ddH2O, 3 μL rCutsmart buffer, 1 μL 10mM dNTP, 10 μL 5×Hotstart buffer, and 2 μL Bst3.0 Polymease; and perform gap filling and PCR amplification according to the following program: 72℃, 10 min; 80℃, 10 min; (98℃, 45 s; 98℃, 15 s; 58℃, 30 s; 72℃, 30 s; (16 / 17 / 18 cycles)); 72℃, 3 min; 16℃ for the duration; in the above cycle, the PCR program for 1000 cells is performed for 16 cycles, the PCR program for 100 cells is performed for 17 cycles, and the PCR program for 10 cells is performed for 18 cycles.

[0069] The amplified product was collected and purified using 0.85× Backman Ampure beads, followed by elution with 20 μL ddH2O. The product was then collected for PE150 sequencing. The results are shown in Figure 3 (ac).

[0070] As can be seen, the sequencing libraries obtained from a small number of cells (1000, 100, and 10 HEK293T cells) in this embodiment showed a very high correlation with those obtained from a large number of cells (R>0.8) (Figure 3a), and the signal distribution of the R-loop was consistent with that of the large number of cells (Figure 3b). Most importantly, the signal-to-noise ratio of the small number of cells was similar to that of the large number of cells (Figure 3c).

[0071] In summary, the R-loop detection method described in this embodiment can detect R-loops using as few as 10 cells, thereby capturing the R-loop distribution pattern of the entire genome. Moreover, the R-loop map of 10 cells is consistent with the R-loop information of a large number of cells.

[0072] Example 3

[0073] The R-loop detection method described in this embodiment is the same as that in embodiment 1, except that in step (2), the genomic DNA fragmentation step uses a mixture of restriction endonucleases HindIII, EcoRI, BsrGI, XbaI and SspI (volume ratio 1:1:1:1:1).

[0074] The detection results of the restriction endonuclease mixture system in Example 1 (denoted as Combination 1) and the restriction endonuclease mixture system in Example 3 (denoted as Combination 2) are shown in Figure 4. It can be seen that the method described in this invention can obtain a high signal-to-noise ratio and highly reproducible R-loop signal distribution landscape using different endonuclease combinations.

[0075] Example 4

[0076] The R-loop detection method described in this embodiment is the same as that in Embodiment 1, except that HeLa cells are used. The detection results are shown in Figure 5.

[0077] As can be seen, the method in this embodiment can capture 241,532 high-confidence R-loop sites in two biological replicates. These signals can all be specifically eliminated by RNase H, proving the authenticity of the signals.

[0078] Example 5

[0079] The R-loop detection method described in this embodiment is the same as that in Embodiment 1, except that the cells used are mES cells, and the detection results are shown in Figure 6.

[0080] As can be seen, the method in this embodiment can capture 109,978 high-confidence R-loop sites in two biological replicates. These signals can all be specifically eliminated by RNase H, proving the authenticity of the signals.

[0081] Example 6

[0082] The R-loop detection method described in this embodiment is the same as that in Embodiment 1, except that yeast cells are used. The detection results are shown in Figure 7.

[0083] As can be seen, the method in this embodiment can capture 6,176 high-confidence R-loop sites in two biological replicates. These signals were all specifically dissolved by RNase H, proving their authenticity. However, other methods capture between 1,000 and 2,000 R-loops.

[0084] Comparative Example 1

[0085] The operation described in this comparative example is the same as that in Example 1, except that in step (3), 25URNase H is added and mixed with 0.5μL nuclease P1, 2μL T5 exonuclease and Lambda exonuclease, and then incubated at 37°C for 2 hours.

[0086] The gold standard for R-loop detection is that the signal can be eliminated by RNase H, as shown in Figure 8ab. It is evident that in this comparative scheme, RNase H leads to a significant reduction in the signal detected by this method.

[0087] It is evident that the signal captured by the method of this invention is a real R-loop signal.

[0088] Comparative Example 2

[0089] According to existing literature, knockdown of the helicase SETX or treatment with the cleavage inhibitor Pladienolide B (PlaB) and the topoisomerase inhibitor Camptothecin (CPT) did not lead to an abnormal increase in R-loop levels.

[0090] In this comparative example, HEK293T cells treated with the above three enzymes were collected. The specific operation procedure was as described in Example 1, and the R-loop changes at the whole genome level were detected using the method of this invention.

[0091] As shown in Figure 9, the results indicate that knocking down SETX, PlaB, and CPT all lead to varying degrees of increase in R-loop levels, consistent with previous reports.

[0092] In summary, the whole-genome R-loop detection method of the present invention is not only highly sensitive, capable of obtaining an order of magnitude more R-loops, but also applicable to the detection of R-loops in as few as 10 cells. In particular, the detection resolution of the method can reach the base level, and it has a high signal-to-noise ratio and no bias, effectively solving the problem of site inconsistency in existing R-loop detection technologies.

[0093] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting whole-genome R-loop, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) The extracted genomic DNA is fragmented; (3) Use nucleases to digest the processed genomic DNA fragments; (4) Use transposase to insert the library construction adapter into both ends of the RNA:DNA hybrid; (5) Perform PCR amplification of the library and sequencing.

2. The whole-genome R-loop detection method according to claim 1, characterized in that, In step (2), the fragmentation process includes at least one of restriction endonuclease treatment, micrococcal nuclease treatment, DNA fragmentation enzyme treatment, or ultrasonic fragmentation treatment.

3. The whole-genome R-loop detection method according to claim 2, characterized in that: The restriction endonuclease includes at least one of Alu I, Dde I, Mbo I, Mse I, Hind III, EcoRI, BsrGI, Xba I, or Ssp I; and / or, The ultrasonic fragmentation step controls the size of the fragment after fragmentation to be 250-300bp.

4. The whole-genome R-loop detection method according to any one of claims 1-3, characterized in that, In step (3), the nuclease includes at least one of nuclease P1, T5 exonuclease or λ-exonuclease; Preferably, the nuclease comprises a mixture of nuclease P1, T5 exonuclease and λ-exonuclease; Preferably, the volume ratio of the nuclease P1, T5 exonuclease and λ-exonuclease is 1:3-5:3-5, more preferably 1:4:

4.

5. The whole-genome R-loop detection method according to any one of claims 1-4, characterized in that, In step (3), the digestion step includes a constant temperature incubation at 35-40°C for 1-3 hours.

6. The whole-genome R-loop detection method according to any one of claims 1-5, characterized in that, In step (3), the digestion step further includes the step of adding proteinase K for treatment; Preferably, the proteinase K digestion process includes incubation at 50-60°C for 20-40 min and incubation at 65-75°C for 20-40 min.

7. The whole-genome R-loop detection method according to any one of claims 1-6, characterized in that, In step (4), the transposase includes Tn5.

8. The whole-genome R-loop detection method according to any one of claims 1-7, characterized in that, In step (4), the transposase treatment step includes incubation at 50-60°C for 5-15 minutes.

9. The whole-genome R-loop detection method according to any one of claims 1-8, characterized in that, In step (5), the PCR system for the PCR amplification step includes: template, primers, polymerase and premix; Preferably, the PCR program for the PCR amplification step includes gap completion and PCR amplification procedures.

10. The application of the whole-genome R-loop detection method according to any one of claims 1-9 in the field of gene detection and analysis.

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