FFPE sample library building method and application thereof

Abstract

The invention provides a library building method of FFPE samples and application of the library building method. The library building method comprises the following steps: S1) performing DNA extraction on an FFPE sample to obtain DNA of the FFPE sample; s2) pretreating the DNA of the FFPE sample, and then performing amplification to obtain a sequencing library of the FFPE sample; wherein before DNA extraction, protease is used for carrying out first incubation and second incubation on an FFPE sample; the pretreatment comprises the following steps: standing the DNA of the FFPE sample for 10 to 120 minutes under the condition of 15 to 45 DEG C; before carrying out the second incubation, carrying out third incubation on the product of the first incubation by using RNASE A; the amplification comprises multiple replacement amplification. The method can solve the problem of poor quality of the sequencing library of the FFPE sample in the prior art, and is suitable for the field of FFPE sample library building.

Description

Technical Field

[0001] The present invention relates to the field of FFPE sample library construction, and in particular to a FFPE sample library construction method and application thereof. Background Art

[0002] In the field of biomedical research, formalin fixed and paraffin embedded (FFPE) samples are widely used due to their ability to preserve tissue samples for a long time. This preservation method can maintain the integrity of tissue structure, which is crucial for pathological diagnosis and research. The preservation technology of FFPE samples allows researchers to retrospectively study disease samples decades later, which is invaluable for understanding disease development and validating new biomarkers. However, the chemicals used in the processing of FFPE samples, such as formalin, can chemically modify and break DNA, making it extremely difficult to extract high-quality, long-fragment DNA from these samples, which is a major technical obstacle for genomic research.

[0003] The formaldehyde in formalin can not only penetrate tissues and fix protein structures, but also react with DNA molecules, causing cross-linking and breakage of DNA chains. This chemical modification makes DNA molecules fragile and difficult to effectively amplify and sequence in subsequent experiments. In addition, the DNA in FFPE samples is usually highly fragmented, which limits its application in experiments that require long fragments of DNA, such as whole genome sequencing, structural variation analysis, and certain types of gene expression studies.

[0004] To overcome these challenges, there are currently a variety of FFPE sample DNA extraction and repair technologies to improve the quality and length of DNA in FFPE samples. These technologies include optimized DNA extraction processes, the use of special enzymes to repair DNA damage, and the use of advanced DNA amplification technologies, which can restore the integrity of DNA to a certain extent and improve its usability in subsequent molecular biology experiments.

[0005] Nevertheless, due to the particularity of FFPE samples, the above-mentioned extraction, repair or amplification techniques are still limited in restoring the integrity of their DNA, resulting in poor quality of FFPE sequencing libraries that are difficult to meet the growing research needs. Summary of the invention

[0006] The main purpose of the present invention is to provide a FFPE sample library construction method and application thereof, so as to solve the problem of poor quality of sequencing library of FFPE samples in the prior art.

[0007] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a method for constructing a library of FFPE samples is provided, and the method comprises: S1) extracting DNA from FFPE samples to obtain DNA of FFPE samples; S2) pre-treating the DNA of FFPE samples and then amplifying them to obtain a sequencing library of FFPE samples; wherein, before DNA extraction, the FFPE samples are subjected to a first incubation and a second incubation using a protease; the pretreatment comprises standing the DNA of the FFPE samples at 15 to 45° C. for 10 to 120 minutes; before the second incubation, the product of the first incubation is subjected to a third incubation using RNASE A; and the amplification comprises multiple displacement amplification.

[0008] Furthermore, the temperature of the first incubation is 50-60°C, and the first incubation time is 24-30 hours; the temperature of the second incubation is 60-70°C, and the second incubation time is 30-120 minutes; preferably, the protease includes proteinase K; preferably, in the first incubation, the concentration of the protease in the first incubation system is 45-60 mg / mL; preferably, in the second incubation, the concentration of the protease in the second incubation system is 15-30 mg / mL.

[0009] Furthermore, the temperature of the third incubation is 15-30° C., and the time is 1-5 min; the concentration of RNASE A in the third incubation system is 5-20 mg / mL.

[0010] In order to achieve the above-mentioned object, according to a second aspect of the present invention, a method for extracting DNA from a FFPE sample is provided, the extraction method comprising: extracting the DNA from the FFPE sample after a first incubation and a second incubation of the FFPE sample with a protease; and before the second incubation, performing a third incubation of the product of the first incubation with RNASE A.

[0011] Furthermore, the temperature of the first incubation is 50-60°C, and the first incubation time is 24-30 hours; the temperature of the second incubation is 60-70°C, and the second incubation time is 30-120 minutes; preferably, the protease includes proteinase K; preferably, in the first incubation, the concentration of the protease in the first incubation system is 45-60 mg / mL; preferably, in the second incubation, the concentration of the protease in the second incubation system is 15-30 mg / mL.

[0012] Furthermore, the temperature of the third incubation is 15-30° C., and the time is 1-5 min; preferably, the concentration of RNASE A in the third incubation system is 5-20 mg / mL.

[0013] In order to achieve the above-mentioned purpose, according to the third aspect of the present invention, a method for amplifying DNA of FFPE samples is provided, and the amplification method comprises: pretreating the DNA of the FFPE samples obtained by the above-mentioned DNA extraction method to obtain a pretreated product; performing multiple displacement amplification on the pretreated product; the pretreatment comprises leaving the DNA of the FFPE samples at 15 to 45° C. for 10 to 120 minutes.

[0014] In order to achieve the above object, according to the third aspect of the present invention, a sequencing library of a FFPE sample is provided, wherein the sequencing library is a sequencing library of a FFPE sample constructed using the above library construction method.

[0015] In order to achieve the above object, according to the fourth aspect of the present invention, a high-throughput sequencing method is provided, which comprises constructing a sequencing library of the FFPE sample obtained by the above library construction method; and sequencing the sequencing library of the FFPE sample on a machine.

[0016] By applying the technical solution of the present invention, the FFPE sample is subjected to the first and second incubations with protease before the DNA extraction, so that the DNA of the FFPE sample with high integrity can be obtained. Before the second incubation, the product of the first incubation is subjected to the third incubation with RNASE A, which is beneficial to improve the effect of protein removal in the second incubation, thereby improving the quality of DNA extraction. Then, the FFPE sample DNA is pretreated at 55 to 75°C for 10 to 30 minutes, and then multiple displacement amplification is performed to obtain a high-quality sequencing library of the FFPE sample, which can meet the requirements of the third generation sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 The figure shows the quality inspection result of the DNA library according to Example 3 of the present application specification.

[0019] Figure 2 The figure shows the quality inspection result of DNA extraction according to Test Example 1-Example 1 of the present application specification.

[0020] Figure 3 The figure shows the quality inspection result of DNA extraction according to Test Example 1-Comparative Example 1 in the specification of this application.

[0021] Figure 4 The graph shows the DNA extraction quality inspection results of Test Example 1-Comparative Example 2 according to the specification of this application.

[0022] Figure 5 The graph shows the DNA extraction quality inspection results of Test Examples 1 to 4 according to the specification of this application.

[0023] Figure 6 The graph shows the quality inspection results of DNA extraction according to Test Examples 1 to 5 of the present application specification.

[0024] Figure 7 The figure shows the MDA amplification quality inspection result according to Test Example 2-Example 2 of the present application specification.

[0025] Figure 8 The graph shows the MDA amplification quality inspection results according to Test Example 2 to Example 6 of the present application specification. DETAILED DESCRIPTION

[0026] 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 in conjunction with the embodiments.

[0027] As mentioned in the background technology, due to the particularity of FFPE samples, the existing extraction, repair or amplification technologies still have limitations in restoring the integrity of the DNA of FFPE samples, resulting in poor quality of FFPE sequencing libraries, which is difficult to meet the growing research needs. Based on this, in this application, the inventors attempted to develop a library construction method for FFPE samples, which improved the integrity of FFPE sample DNA, and the amplified fragments met the requirements of subsequent library construction and sequencing, and thus proposed a series of protection schemes of the present application.

[0028] In a first typical embodiment of the present application, a method for constructing a library of FFPE samples is provided, and the method comprises: S1) extracting DNA from the FFPE samples to obtain DNA from the FFPE samples; S2) pre-treating the DNA from the FFPE samples and then amplifying them to obtain a sequencing library of the FFPE samples; wherein, before the DNA extraction, the FFPE samples are subjected to a first incubation and a second incubation using a protease; the pretreatment comprises allowing the DNA of the FFPE samples to stand for 10 to 120 minutes (including but not limited to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 minutes) at 15 to 45°C (including but not limited to 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C5); before the second incubation, the product of the first incubation is subjected to a third incubation using RNASE A; and the amplification comprises multiple displacement amplification (MDA, MμLtiple Displacement Amplification).

[0029] In biomedical research, processing FFPE samples for nucleic acid extraction and sequencing is a complex process. Formalin fixation causes cross-linking between nucleic acids, between proteins, and between nucleic acids and proteins, making purification more difficult. Changes in sample cell morphology and protein structure may affect the results and accuracy of pathological diagnosis and molecular biology research.

[0030] In terms of DNA extraction and quality, during the formalin fixation and paraffin embedding process of FFPE samples, the DNA will be chemically modified and broken, resulting in short and poor quality extracted DNA fragments, which directly affects the subsequent sequencing quality and the efficiency of library construction. It is particularly difficult to construct a library suitable for third-generation sequencing because third-generation sequencing technology requires longer DNA fragments to achieve high accuracy and long read length sequencing. However, although the third-generation sequencing technology has a long read length, its accuracy is relatively low and needs to be corrected by an algorithm. Damage to the DNA of FFPE samples may increase the error rate, thereby affecting the accuracy of the sequencing results.

[0031] Although relatively advanced DNA amplification technologies, such as MDA amplification, are used in the prior art to improve the DNA integrity of FFPE samples, the improvement effect is still relatively limited. For example, the nucleic acid of the FFPE samples has been partially degraded during the storage process, and the DNA is tightly cross-linked with histones, which results in the DNA extracted from the FFPE samples often being of poor quality and short fragments, which directly affects the effect of MDA amplification.

[0032] PCR reactions of DNA from FFPE samples may face inhibition and unbalanced amplification, resulting in poor sensitivity and accuracy of MDA amplification. Moreover, a long-standing problem in the MDA amplification process is template-independent amplification (TIA), especially when amplifying ultra-low concentrations of templates, which can lead to false positive amplification in negative controls, produce junk DNA, and undermine the credibility of the amplification results. In existing commercially available MDA amplification kits, operators are generally advised to increase the template amount (≥10ng) and shorten the incubation time during MDA amplification to avoid TIA, but it is often difficult for FFPE sample DNA to reach this template amount, so the use of MDA amplification to improve the DNA integrity of FFPE samples still has certain limitations.

[0033] Since the DNA in FFPE samples is often tightly cross-linked with proteins such as histones, this cross-link must be broken to release the DNA for subsequent purification and analysis. When FFPE samples are processed, they are usually incubated with proteases, which can effectively degrade these proteins, thereby helping to release DNA molecules cross-linked with proteins.

[0034] In the prior art, a single protease digestion is usually used to treat FFPE samples. However, since FFPE samples contain formaldehyde reagents, it is difficult to completely remove the impurity proteins in FFPE samples by using protease digestion. There are still proteins remaining in the samples, affecting subsequent experiments. However, if the amount of protease is increased to digest FFPE samples, although more proteins can be removed, it will affect the length of the nucleic acid fragments, and thus affect the extraction effect and the effect of subsequent library construction. Therefore, in the pre-treatment of FFPE samples, there is a bottleneck that needs to be broken through for the removal of proteins.

[0035] In the DNA extraction process of the present application, the release efficiency of DNA is significantly improved by adding two protease digestions (first incubation and second incubation). Before the second incubation, the present application uses RNASE A to perform a third incubation on the product of the first incubation. In addition to reducing the RNA in the FFPE sample, the RNA in the higher-order structure of RNA-DNA-protein binding can also be digested, so that this part of the residual protein and DNA can be released and completely digested by protease in the second incubation.

[0036] In view of the characteristics of FFPE samples, the present application uses protease to perform a first incubation and a second incubation before extracting DNA from them, and adds a third incubation between the first incubation and the second incubation. The FFPE samples are processed by an enzymatic hydrolysis method of protease-RNASE A-protease. A large amount of protein is removed without destroying the length of nucleic acid fragments, so that the protein cross-linked with RNA can also be digested, and the DNA cross-linked with protein can be fully released, which significantly improves the extraction efficiency and quality of DNA from FFPE samples.

[0037] However, during the amplification stage, due to the special sample type of FFPE samples, it is difficult to completely remove the protein and reagents such as EDTA used in the early lysis process even if the incubation time is increased during the DNA extraction process. Therefore, before the MDA amplification is performed in this application, the DNA of the FFPE sample is pretreated, and the random primers are first effectively combined with the template DNA at a lower denaturation temperature, and then the isothermal amplification step in the MDA amplification is performed. Controlling the pretreatment temperature within the above-mentioned temperature range can denature the protein while the binding of the primer to the template DNA is not affected. This is beneficial to improving the quality of amplification and avoiding the effects of proteins and complex reagents on amplification and TIA on amplification.

[0038] The library construction method of the present application solves the problem of difficulty in extracting complete DNA from FFPE samples, and further obtains DNA fragments of FFPE samples with a length of more than 7000bp through the method of multiple displacement amplification, constructs a library suitable for third-generation Nanopore (Oxford Nanopore Technologies, referred to as ONT) sequencing, and improves the yield and quality of long-fragment DNA of FFPE samples. The library construction method of the present application is simple, low-cost and reproducible, and has wide applicability and promotion value. Moreover, through third-generation sequencing, the genome structure and variation information of FFPE samples can be analyzed more accurately, providing strong support for fields such as disease diagnosis, drug development and personalized medicine. In a preferred embodiment, the temperature of the first incubation is 50-60°C (including but not limited to 50°C, 55°C or 60°C), and the first incubation time is 24-30 hours (including but not limited to 24, 25, 26, 27, 28, 29 or 30 hours); the temperature of the second incubation is 60-70°C (including but not limited to 60°C, 65°C or 70°C), and the second incubation time is 30-120min (including but not limited to 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120min); preferably, the protease comprises proteinase K; preferably, in the first incubation, the protein The concentration of the enzyme in the first incubation system is 45-60 mg / mL (including but not limited to 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mg / mL), more preferably 50-55 mg / mL; preferably, in the second incubation, the concentration of the protease in the second incubation system is 15-30 mg / mL (including but not limited to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mg / mL), more preferably 20-25 mg / mL.

[0039] In a preferred embodiment, before the second incubation, the product of the first incubation is subjected to a third incubation using RNASE A; preferably, the temperature of the third incubation is 15-30°C (including but not limited to 15°C, 20°C, 25°C or 30°C), and the time is 1-5 min (including but not limited to 1, 2, 3, 4 or 5 min).

[0040] The present application controls the temperature and time of the third incubation within the above range, which can be beneficial to the subsequent second incubation and to improving the overall DNA extraction effect and library construction effect.

[0041] In a preferred embodiment, after the DNA of the FFPE sample is amplified, a DNA fragment with a length greater than 7000 bp is obtained.

[0042] In a second typical embodiment of the present application, a method for extracting DNA from a FFPE sample is provided. The method comprises: extracting DNA from the FFPE sample after first incubating and second incubating the FFPE sample with a protease.

[0043] In the present application, by incubating the FFPE sample twice with protease (i.e., the first incubation and the second incubation mentioned above), the protein and nucleic acid in the FFEP sample are separated, the DNA molecules cross-linked with the protein are released, the protein that affects the extraction effect is fully digested, and the protein that affects the DNA extraction effect in the FFPE sample is reduced, which is beneficial to improving the effect of DNA extraction from the FFPE sample, improving the integrity of the FFPE sample DNA, and facilitating subsequent amplification and library construction to obtain DNA fragments that meet the sequencing requirements.

[0044] In a preferred embodiment, the temperature of the first incubation is 50-60°C, and the first incubation time is 24-30 hours; the temperature of the second incubation is 60-70°C, and the second incubation time is 30-120 minutes; preferably, the protease includes proteinase K. Preferably, in the first incubation, the concentration of the protease in the first incubation system is 45-60 mg / mL; preferably, in the second incubation, the concentration of the protease in the second incubation system is 15-30 mg / mL.

[0045] In a preferred embodiment, before the second incubation, the product of the first incubation is subjected to a third incubation using RNASE A; preferably, the temperature of the third incubation is 15-30°C, and the time is 1-5 min. Preferably, the concentration of RNASE A in the third incubation system is 5-20 mg / mL.

[0046] In a third typical embodiment of the present application, a method for amplifying DNA of an FFPE sample is provided, the method comprising: pretreating the DNA of the FFPE sample to obtain a pretreatment product; and performing multiple displacement amplification on the pretreatment product; the pretreatment comprises allowing the DNA of the FFPE sample to stand at 55 to 75° C. for 10 to 30 minutes.

[0047] Due to the particularity of FFPE samples, it is difficult to completely remove proteins and reagents such as EDTA used in the early lysis and cross-linking during the DNA extraction process. Therefore, before the MDA amplification in this application, the DNA of the FFPE sample is pretreated, and the random primers are first effectively combined with the template DNA at a lower denaturation temperature, and then the isothermal amplification link in the MDA amplification is performed. Controlling the pretreatment temperature within the above-mentioned temperature range can denature the protein while the binding of the primer to the template DNA is not affected. This is beneficial to improving the quality of amplification and avoiding the effects of proteins and complex reagents on amplification and TIA on amplification.

[0048] In a preferred embodiment, after the DNA of the FFPE sample is amplified, a DNA fragment with a length greater than 7000 bp is obtained.

[0049] In a fourth typical embodiment of the present application, a sequencing library of a FFPE sample is provided. The sequencing library is a sequencing library of a FFPE sample constructed using the above-mentioned library construction method.

[0050] In a fifth typical embodiment of the present application, a high-throughput sequencing method is provided, which comprises constructing a sequencing library of the FFPE sample obtained by the above-mentioned library construction method; and sequencing the sequencing library of the FFPE sample on a machine.

[0051] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0052] Unless otherwise specified, the reagents in the examples of this application are conventional commercially available products.

[0053] Example 1

[0054] Taking mouse tissue section samples as an example, DNA extraction is performed on them. The specific steps are as follows:

[0055] 1. Sample dewaxing: Place FFPE sections (1 slice) in a 1.5 mL centrifuge tube, add 1 mL of xylene, cover the sections and shake thoroughly, incubate at 56 °C for 3 min, and then cool to room temperature.

[0056] 2. 20000g for 2 min, remove the supernatant and keep the precipitate;

[0057] 3. Add 1 mL of anhydrous ethanol to the precipitate, shake thoroughly, centrifuge at 20,000 g for 2 min at room temperature, and remove the supernatant;

[0058] 4. Repeat step 3 to completely remove the supernatant, then open the lid of the centrifuge tube at room temperature and let it stand until all residual alcohol evaporates.

[0059] 5. First incubation: Digest the above precipitate with protease, add 25 μL protein digestion buffer, the components of which are shown in Table 1, 55 μL RNase-free water and 50 μL proteinase K (Qiagen, 19133), and incubate at 55° C. overnight (15 h).

[0060] Table 1

[0061] Reagent name Final concentration Tris-HCl 10mM NaCl 150mM Nonidet P-40 0.5％

[0062] 6. Third incubation: RNASE A (Qiagen, 19101) treatment: add 10 μL RNASE A, mix well, and incubate at room temperature for 2 min.

[0063] 7. Second incubation: add 20 μL proteinase K, incubate at 65°C for 120 min, and centrifuge briefly.

[0064] 8. DNA purification: Add 450 μL FFPE lysis buffer (the components are shown in Table 2), mix well by pipetting with a wide-mouth pipette tip, and mix well for 2 minutes at room temperature using a four-dimensional mixer.

[0065] Table 2

[0066] Reagent name Final concentration Tris-HCl 10mM NaCl 150mM EDTA 100mM SDS 1％ PEG8000 5％

[0067] 9. Centrifuge at 12,000 x g for 15 min at room temperature.

[0068] 10. Use a wide-mouth pipette tip to transfer the supernatant to a new 1.5 mL centrifuge tube.

[0069] 11. Take Dynabeads TM MyOne TM Add 50 μL of Silane (Thermo, 37002D) into a 1.5 ml centrifuge tube and flick to mix.

[0070] 12. Add 500 μL of isopropanol, mix thoroughly by pipetting, and place on a four-dimensional mixer for 10 min at room temperature;

[0071] 13. After instant centrifugation, place the centrifuge tube on the magnetic rack, let it stand for 10 minutes, and carefully discard the supernatant;

[0072] 14. Keep the centrifuge tube on the magnetic rack, add 1 mL of freshly prepared 75% ethanol, and carefully aspirate the liquid after 30 seconds; repeat once;

[0073] 15. Centrifuge to completely remove residual ethanol, open the lid and let the magnetic beads dry until matte (about 2 minutes);

[0074] 16. Pipette 50 μL EB, flick the beads to mix, and mix at room temperature for 15 min using a four-dimensional mixer.

[0075] 17. Place the centrifuge tube on the magnetic rack, let it stand for 10 minutes, and carefully pipette the supernatant into a new centrifuge tube.

[0076] 18. DNA band detection: using AATI (Advanced Analytical Technological, equipment: Agilent FEMTO Pulse TM ) detection method was used to detect the quality of DNA extraction.

[0077] Example 2

[0078] MDA amplification:

[0079] The DNA extracted in Example 1 was used as a template and added to the MDA amplification reaction system. Under appropriate temperature and time conditions, the MDA amplification reaction was performed until a sufficient amount of DNA fragments longer than 10K were obtained. The specific operation steps are as follows:

[0080] 1. Transfer 20 ng of DNA to a 0.2 mL PCR tube according to the DNA concentration, dilute to 5 μL with nuclease-free water, and mix gently by pipetting 5 times.

[0081] 2. Prepare a new 1.5 mL centrifuge tube and prepare the repair buffer. The reagent formula is shown in Table 3 ( FFPE DNA Repair Mix, M6630S).

[0082] Table 3

[0083] Reagents Usage FFPE DNA Repair Buffer 2μL NEBNext μLtra II End Prep Buffer 2μL NEBNext FFPE DNA Repair Mix 1μL

[0084] 3. Mix the prepared repair buffer thoroughly by pipetting.

[0085] 4. Add 1 μL of repair buffer to the prepared 20 ng of DNA, pipette gently to mix 5 times, and centrifuge for 1 second.

[0086] 5. Pretreatment: Place in PCR instrument, react at 20℃ for 30min. After the reaction is completed, centrifuge for 1s.

[0087] 6. Prepare a new 1.5 mL centrifuge tube and place it on ice. Add the reagents listed in Table 4 (phi29-XT RCA Kit E1603).

[0088] Table 4

[0089] Components volume Template DNA 6μL phi29-XT Reaction Buffer, 5X 4μL dNTP mix, 10 mM 2μL Anti-exonuclease random primers 500 μM 2μL Nuclease-free water Add to 18 μL Total volume 40μL

[0090] 7. Vortex and mix for 5 seconds, centrifuge briefly and add to the sample tube, pipette and mix gently 5 times, and centrifuge briefly for 1 second.

[0091] 8. Place in PCR instrument and react at 95℃ for 3 minutes.

[0092] 9. Add 2 μl of phi29-XT DNA polymerase to the above reaction system, react at 42°C for 2 h, 65°C for 10 min, and store at 12°C. After the reaction is complete, centrifuge for 1 second.

[0093] 10. Transfer the above samples to a new 1.5 mL centrifuge tube and add 90 μL of AMPure XP (leave AMPure XP at room temperature for at least 30 minutes in advance and shake thoroughly before use).

[0094] 11. After mixing on a four-dimensional mixer for 5 minutes, place it on a magnetic rack. Wait until the magnetic beads are adsorbed on the magnetic rack and the supernatant becomes transparent. Discard the supernatant and do not absorb the magnetic beads.

[0095] 12. Add 200 μL of 80% alcohol (freshly prepared) to the sample tube to wash the magnetic beads. After standing for 1 minute, remove the alcohol and use a 10 μL pipette to absorb the residual alcohol. Repeat twice.

[0096] 13. Allow to dry for 30 seconds to 1 minute to avoid over-drying that affects DNA yield. Allow to dry until the magnetic beads are no longer reflective. Add 50 μL of nuclease-free water, pipette gently to mix 5 times, and react at room temperature for 2 minutes.

[0097] 14. After the reaction is completed, place it on the magnetic stand and transfer it to a new 1.5mL tube after the solution is clarified to avoid attracting the magnetic beads.

[0098] 15. Prepare a 0.2 mL PCR tube and add the reagents listed in Table 5.

[0099] Table 5

[0100] Components volume 5ug of DNA (DNA purified as above) 24μL NEB Buffer 2 (NEB, B7002S) 3μL T7 Endonuclease I (NEB, M0689) 3μL Total volume 30μL

[0101] 16. Mix by pipetting gently 5 times, centrifuge for 1 second, place in PCR instrument, react at 37℃ for 60 minutes. After the reaction is complete, centrifuge for 1 second.

[0102] 17. Transfer 30 μL of DNA to a new 1.5 mL centrifuge tube and make up to 50 μL. Mix by gently pipetting 5 times. Add 35 μL of magnetic beads prepared with buffer (NEB, B6004S) (prepared in advance and shaken to mix).

[0103] 18. After mixing on a four-dimensional mixer for 10 minutes, place it on a magnetic rack. Wait until the magnetic beads are adsorbed on the magnetic rack and the supernatant becomes transparent. Discard the supernatant and do not absorb the magnetic beads.

[0104] 19. Add 200 μL of 80% alcohol (freshly prepared) to the sample tube to wash the magnetic beads. After standing for 1 minute, remove the alcohol and absorb the residual alcohol. Repeat twice.

[0105] 20. Dry for 30s-1min to avoid over-drying that affects the DNA yield. Dry until the magnetic beads are no longer reflective. Add 49μL of nuclease-free water, gently pipette and mix 5 times, react at 50℃ for 1min, and then at RT for 2min.

[0106] 21. After the reaction is completed, place it on the magnetic stand and transfer it to a new 1.5mL tube after the solution is clarified to avoid attracting the magnetic beads.

[0107] 22. DNA amplification result detection: total amount (Qubit) and band detection (AATI detection).

[0108] Example 3

[0109] Nanopore sequencing library construction, the steps are as follows:

[0110] The process of amplifying the DNA fragments includes: using NEBNext FFPE DNA Repair Mix and NEBNext End Repair / dA Tailing Module to repair and add dA tails to the DNA ends; then connecting the sequencing adapters provided in the SQK-LSK114 kit to the DNA ends. The library is quality checked (Qubit and AATI) to ensure the quality of the library. The library quality check results are as follows: Figure 1 shown.

[0111] The results show that the library established by this application meets the requirements of Nanopore sequencing. The third-generation library constructed by the above-mentioned library construction method after extraction and amplification was run on PromethION 48 for 72 hours, and the output data volume of a single chip reached 65G, with an N50 of 7.8K.

[0112] Example 4

[0113] The only difference between this comparative example and Example 1 is that in the first incubation, the temperature is 55° C. and the incubation time is 120 min.

[0114] Example 5

[0115] The only difference between this comparative example and Example 1 is that in the second incubation, the incubation was carried out at 65° C. overnight (16 h).

[0116] Example 6

[0117] The only difference from Example 2 is that step 5 is omitted, that is, the pre-reaction at 20° C. for 30 min is not performed.

[0118] Comparative Example 1

[0119] The difference between this comparative example and Example 1 is that only the first incubation is performed, and the second incubation is not performed.

[0120] Comparative Example 2

[0121] The difference between this comparative example and Example 1 is that only the first incubation is performed, the amount of added protease is increased to 100 μL, and the second incubation is not performed.

[0122] Test Example 1

[0123] The AATI test was performed on the DNA of the FFPE samples of Examples 1, 4, 5 and Comparative Examples 1 and 2. The results are shown in Table 6. The DNA quality inspection result diagrams of Examples 1, 4, 5 and Comparative Examples 1 and 2 are shown in Table 6. Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 shown.

[0124] Table 6

[0125]

[0126] In Table 6, the main DNA peak (bp) reflects the integrity of the DNA. The larger the main DNA peak, the better the DNA integrity and the less severe degradation.

[0127] A260 / A280 reflects the purity of DNA; the A260 / A280 ratio of DNA samples with higher purity is generally around 1.8. If it is lower than 1.8, it means that the sample may be contaminated with proteins or phenolic substances.

[0128] A260 / A230 is used to assess whether there is contamination such as carbohydrates and salts in the sample; the A260 / A230 ratio of DNA samples with higher purity is usually greater than 2.0. If the ratio is lower than 2.0, it indicates that the above-mentioned contamination may exist in the sample.

[0129] NC / QC is a ratio of the concentration measured by NanoDrop to that measured by Qubit, which is used to assess whether the extracted DNA is rough or contaminated with RNA.

[0130] Test Example 2

[0131] The MDA amplification results of Example 2 and Example 6 were subjected to AATI detection, and the results are shown in Table 7. The library quality inspection result diagrams of Example 2 and Example 6 are shown in Table 7. Figure 7 and Figure 8 shown.

[0132] Table 7

[0133] Group Qbuit concentration (ng / μL) Total amount (μg) Main peak of gene fragment (bp) Example 2 68.3 3.07 10158 Example 6 20.5 0.923 6331

[0134] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: using the library construction method for FFPE samples in this application, relatively complete FFPE sample DNA can be extracted, and using the MDA amplification system of this application, FFPE DNA fragments with a length of more than 7000bp with high total concentration and large total amount can be amplified, and the constructed library is of high quality and meets the requirements of third-generation sequencing. Moreover, the library construction method of this application is simple and easy, low in cost and good in repeatability, and has wide applicability and promotion value. Through third-generation sequencing, the genome structure and variation information of FFPE samples can be more accurately analyzed, providing strong support for disease diagnosis, drug development, personalized medicine and other fields.

[0135] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for building a library of FFPE samples, characterized in that: The library construction method comprises: S1) extracting DNA from the FFPE sample to obtain DNA from the FFPE sample; S2) pre-treating the DNA of the FFPE sample and then amplifying it to obtain a sequencing library of the FFPE sample; Wherein, before the DNA extraction, the FFPE sample is subjected to a first incubation and a second incubation using a protease; The pretreatment includes placing the DNA of the FFPE sample at 15 to 45° C. for 10 to 120 minutes; Before performing the second incubation, performing a third incubation on the product of the first incubation using RNASE A; The amplification comprises multiple displacement amplification.

2. The library construction method according to claim 1, characterized in that: The first incubation temperature is 50-60° C., and the first incubation time is 24-30 hours; The temperature of the second incubation is 60-70° C., and the time of the second incubation is 30-120 min; Preferably, the protease comprises proteinase K; Preferably, in the first incubation, the concentration of the protease in the first incubation system is 45-60 mg / mL; Preferably, in the second incubation, the concentration of the protease in the second incubation system is 15-30 mg / mL.

3. The library construction method according to claim 1, characterized in that: The third incubation temperature is 15-30°C and the time is 1-5 minutes; Preferably, the concentration of the RNASE A in the third incubation system is 5-20 mg / mL.

4. A method for extracting DNA from FFPE samples, characterized in that: The extraction method comprises: After performing a first incubation and a second incubation on the FFPE sample using a protease, extracting DNA from the FFPE sample; Before the second incubation, the product of the first incubation is subjected to a third incubation using RNASE A.

5. The method for extracting DNA according to claim 4, characterized in that The first incubation temperature is 50-60° C., and the first incubation time is 24-30 hours; The temperature of the second incubation is 60-70° C., and the time of the second incubation is 30-120 min; Preferably, the protease comprises proteinase K.

6. The method for extracting DNA according to claim 5, characterized in that In the first incubation, the concentration of the protease in the first incubation system is 45-60 mg / mL; In the second incubation, the concentration of the protease in the second incubation system is 15-30 mg / mL.

7. The method for extracting DNA according to claim 4, characterized in that: Preferably, the temperature of the third incubation is 15-30°C and the time is 1-5 minutes; Preferably, the concentration of the RNASE A in the third incubation system is 5-20 mg / mL.

8. A method for amplifying DNA from FFPE samples, characterized in that: The amplification method comprises: After pre-treating the DNA of the FFPE sample obtained by the method for extracting DNA from the FFPE sample according to any one of claims 4 to 7, a pre-treated product is obtained; performing multiple displacement amplification on the pre-treated product; The pretreatment includes placing the DNA of the FFPE sample at 15 to 45° C. for 10 to 120 minutes.

9. A sequencing library of FFPE samples, characterized in that: The sequencing library is a sequencing library of FFPE samples constructed using the library construction method described in any one of claims 1 to 3.

10. A high-throughput sequencing method, characterized in that: The method comprises constructing a sequencing library of the FFPE sample using the library construction method described in any one of claims 1 to 3; and sequencing the sequencing library of the FFPE sample on a machine.

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