Lysis solution for extracting nucleic acids and use thereof

Abstract

This invention provides a lysis buffer for nucleic acid extraction and its application. The lysis buffer comprises: 0.6M–0.8M NaH₂PO₄, 0.042–0.06M Na₂HPO₄, 0.007–0.009M NaCl, 0.03–0.05M Tris, 0.01–0.02M SDS, and 10%–20% anhydrous ethanol; wherein the pH of the lysis buffer is 5–6; and the nucleic acid is DNA and / or RNA. Using the lysis buffer of this invention, the sample can be efficiently lysed, effectively releasing and protecting the nucleic acid molecules in the sample, significantly improving the nucleic acid extraction efficiency. This invention provides a new reference for extracting DNA and / or RNA from low-biomass samples, contributing to improved sequencing quality of low-biomass samples.

Description

Technical Field

[0001] This invention relates to the technical field of DNA and / or RNA extraction, and more specifically, to a lysis buffer for extracting nucleic acids and its application. Background Technology

[0002] In recent years, with the deepening of deep-sea research and increased attention to marine ecosystems, metagenomics and metatranscriptomics studies of marine sediments have gradually become a hot topic. Marine sediments contain rich microbial communities and potential functional genes. Metagenomics and metatranscriptomics studies can reveal the diversity and function of marine microorganisms, expanding our understanding of biodiversity and function. Simultaneously, studying the impact of environmental factors on microbial communities through metagenomics and metatranscriptomics helps us understand the structure and function of marine ecosystems, as well as the role of microorganisms in global change and ecosystem function. These studies are of great significance for the protection and management of marine ecosystems, the development of marine biological resources, and the application of biotechnology.

[0003] Current reports on the co-extraction of DNA and RNA mainly focus on samples with large biomass, such as conventional soil and gut microbiota. Methods for simultaneously obtaining metagenomic and metatranscriptomic data from marine sediments are rarely reported. Existing column-based methods suffer significant nucleic acid loss, making them unsuitable for low-biomass marine sediments, especially deep-sea cold seep sediments. Furthermore, existing techniques suffer from low purity of the obtained nucleic acids, resulting in insufficient yields to support downstream purification. Currently, DNA and RNA are primarily obtained separately from marine sediments. DNA acquisition methods are diverse, while RNA acquisition mainly involves the classic Trizol lysis extraction and reagent-based methods primarily using column purification. The process involves lysis, column / extraction, and isopropanol precipitation. The lysis reagent is primarily Trizol; commercially available lysis reagents are not publicly available and are expensive. Reported co-extraction methods for DNA and RNA are complex and generally only applicable to sediment areas with large biomass.

[0004] Compared to obtaining only DNA or RNA for subsequent library construction, simultaneously obtaining DNA and RNA from the same sediment sample provides more comprehensive genomic and transcriptomic information. This method reduces experimental errors and technical variations, improves data consistency, and facilitates in-depth research into the mechanisms of gene expression regulation. Simultaneously, this method conserves samples and improves resource utilization efficiency. However, the co-extraction of DNA and RNA from marine sediments faces several challenges. First, marine sediments, especially deep-sea cold seep sediments, have low biomass and contain far fewer microorganisms compared to other environmental samples, making nucleic acid extraction difficult. Furthermore, marine sediments contain numerous impurities, such as mineral particles and carbonates, salts and metal ions, and organic matter. These impurities can exacerbate RNA degradation and interfere with the extraction and purification processes of DNA and RNA, affecting subsequent data analysis and interpretation. Currently, there are no patents regarding the co-extraction of DNA and RNA from marine sediments, and existing co-extraction methods are complex and cumbersome, generally only applicable to sediment areas with high biomass. Therefore, developing an efficient method for simultaneously extracting DNA and RNA from low-biomass samples such as marine sediments is crucial. Summary of the Invention

[0005] The main objective of this invention is to provide a lysis buffer for nucleic acid extraction and its application, in order to solve the problem of the lack of methods in the prior art for simultaneously extracting DNA and RNA from samples with low biomass (especially marine sediment samples).

[0006] To achieve the above objective, according to a first aspect of the present invention, a lysis buffer for extracting nucleic acids is provided, the lysis buffer comprising: 0.6M–0.8M NaH₂PO₄, 0.042–0.06M Na₂HPO₄, 0.007–0.009M NaCl, 0.03–0.05M Tris, 0.01–0.02M SDS, and 10%–20% (V / V, mL / mL) anhydrous ethanol; wherein the pH of the lysis buffer is 5–6.

[0007] To achieve the above objectives, according to a second aspect of the present invention, a method for extracting nucleic acids is provided, the method comprising:

[0008] S1, use the above-mentioned lysis buffer to lyse the sample to be tested to obtain the lysed solution;

[0009] S2, the DNA-RNA mixture is separated from the above lysed solution;

[0010] S3, isolate DNA from the above DNA-RNA mixture; and / or

[0011] RNA was isolated from the above DNA-RNA mixture.

[0012] Furthermore, the above S2 includes:

[0013] S21, the above-mentioned pyrolysis solution is extracted to obtain the extracted solution;

[0014] S22: Precipitate the solution after extraction to obtain a precipitate;

[0015] S23: Dissolve the above precipitate in water using DEPC treatment to obtain the above DNA-RNA mixture;

[0016] In step S22, the above-extracted solution is precipitated using ethanol and optionally isopropanol and sodium acetate.

[0017] Preferably, S3 includes:

[0018] The ribonuclease and the above DNA-RNA mixture were mixed and subjected to digestion reaction 1 to obtain the above DNA; and / or

[0019] The deoxyribonuclease and the above DNA-RNA mixture were mixed and digested to obtain the above RNA;

[0020] Preferably, the lysis buffer and the sample to be tested are in a volume-to-mass ratio of (1:2) to (1:3);

[0021] Preferably, the biomass of the sample to be tested is 10^5 to 10^11 microorganisms / g; preferably, the sample to be tested is marine sediment.

[0022] Preferably, the marine sediments are samples with a biomass of 10^5 to 10^7 microorganisms / g.

[0023] Furthermore, after pyrolyzing the sample to be tested using the lysis buffer, before obtaining the pyrolyzed solution, S1 further includes the steps of vortexing and water bath heating.

[0024] Preferably, the vortex duration is 1–3 minutes;

[0025] Preferably, the water bath heating temperature is 70–80°C, and the time is 40–60 min.

[0026] Further, the above-mentioned pyrolyzed solution was extracted using an extraction reagent to obtain the above-mentioned extracted solution;

[0027] Preferably, the extraction reagent comprises chloroform and isoamyl alcohol;

[0028] More preferably, the volume ratio of the above-mentioned chloroform to the above-mentioned isoamyl alcohol is 24:1;

[0029] Preferably, the pyrolysis solution and the extraction reagent are mixed at a volume ratio of 1:1.

[0030] Further, step S22 above includes:

[0031] The above-extracted solution, the above-mentioned isopropanol, and the above-mentioned sodium acetate were mixed to obtain precipitate 1;

[0032] The above precipitate 1 and the above ethanol are mixed to obtain precipitate 2;

[0033] The above precipitate 2 and the above ethanol are mixed to obtain the above precipitate;

[0034] Preferably, the extracted solution, isopropanol, and sodium acetate are mixed in a volume ratio of 10:7:1; wherein the concentration of sodium acetate is 3M.

[0035] Preferably, the above-mentioned precipitate 1 and the above-mentioned ethanol are mixed at a mass-volume ratio of (1:1000) to (1:5000), and the above-mentioned precipitate 2 and the above-mentioned ethanol are mixed at a mass-volume ratio of (1:1000) to (1:5000), wherein the concentration of the above-mentioned ethanol is 70% to 80% (V / V, mL / mL).

[0036] Further, the above-extracted solution, the above-mentioned isopropanol and the above-mentioned sodium acetate are mixed and allowed to stand to obtain the above-mentioned precipitate 1. The standing time is 30 min to 60 min, and the standing temperature is 4 to 25 °C.

[0037] Furthermore, the above DNA-RNA mixture is mixed with ribonuclease at a volume ratio of (25:1) to (50:1);

[0038] Preferably, the concentration of the above-mentioned ribonuclease is 600–900 U / mL;

[0039] More preferably, the digestion reaction 1 described above takes 30 to 45 minutes and is carried out at a temperature of 35 to 38°C.

[0040] Preferably, the above-mentioned DNA-RNA mixture is mixed with the above-mentioned deoxyribonuclease at a volume ratio of (25-1):(30-1);

[0041] More preferably, the concentration of the above-mentioned deoxyribonuclease is 2000-2500 U / mL;

[0042] More preferably, the digestion reaction 2 described above takes 30 to 50 minutes and is carried out at a temperature of 35 to 37°C.

[0043] To achieve the above objectives, according to a third aspect of the present invention, a method for constructing a nucleic acid library is provided, the method comprising:

[0044] RNA was reverse transcribed to obtain double-stranded cDNA;

[0045] Library construction was performed using the aforementioned double-stranded cDNA to obtain an RNA library; and / or

[0046] DNA libraries are constructed to obtain DNA libraries;

[0047] The RNA mentioned above is RNA obtained according to the method described above; the DNA mentioned above is DNA obtained according to the method described above.

[0048] To achieve the above objectives, according to a fourth aspect of the present invention, a method for sequencing nucleic acids is provided, which sequences DNA libraries and / or RNA libraries obtained in the above-described nucleic acid library construction method.

[0049] By applying the technical solution of this invention, and using the lysis buffer of this invention (comprising 0.6M–0.8M NaH₂PO₄, 0.042–0.06M Na₂HPO₄, 0.007–0.009M NaCl, 0.03–0.05M Tris, 0.01–0.02M SDS, and 10%–20% anhydrous ethanol, pH 5–6) to fully lyse the sample, the sample can be lysed efficiently, effectively releasing and protecting nucleic acid molecules (including DNA and RNA) in the sample, significantly improving the nucleic acid extraction efficiency. Furthermore, compared with traditional lysis buffers, when using the lysis buffer of this application for nucleic acid extraction from low-biomass samples, especially marine sediment samples, it is possible to simultaneously obtain DNA and RNA with high integrity and purity from the same sample, greatly increasing the probability of successful construction of metagenomic and metagenome libraries, thereby contributing to increasing the feasibility and accuracy of obtaining marine sediment metagenomic and metagenome data. Attached Figure Description

[0050] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0051] Figure 1 The electrophoretic detection results of DNA-RNA co-extraction according to Example 1 of the present invention are shown; wherein, channel 1 represents lysis buffer 1 (pH 7.5), channel 2 represents lysis buffer 2 (pH 5.5), and channel M1 represents Takaraλ-Hind III digest Marker.

[0052] Figure 2The image shows an Agilent 2100 detection plot of RNA in lysis buffer 1 (pH 7.5) according to Example 1 of the present invention.

[0053] Figure 3 The image shows an Agilent 2100 detection plot of RNA in lysis buffer 2 (pH 5.5) according to Example 1 of the present invention.

[0054] Figure 4 The results of DNA-RNA co-extraction electrophoresis detection of two nearshore sediments (nearshore sediment 1 and nearshore sediment 2) and two deep-sea cold seep sediments (deep-sea cold seep sediment 1 and deep-sea cold seep sediment 2) according to embodiments of the present invention are shown; wherein channel 1 represents nearshore sediment 1, channel 2 represents nearshore sediment 2, channel 3 represents deep-sea cold seep sediment 1, channel 4 represents deep-sea cold seep sediment 2, channel M1 represents Takaraλ-Hind III digest Marker, and channel M2 represents Takara DL2000 Marker.

[0055] Figure 5 An Agilent 2100 RNA detection map of nearshore sediment 1 according to the present invention is shown.

[0056] Figure 6 An Agilent 2100 RNA detection map of nearshore sediment 2 according to the present invention is shown.

[0057] Figure 7 A graph showing the quality values ​​of each base from the metatranscriptome sequencing results of nearshore sediment 1 according to the present invention is presented.

[0058] Figure 8 A graph showing the quality score of each sequence from the metatranscriptome sequencing results of nearshore sediment 1 according to the present invention is displayed.

[0059] Figure 9 A graph showing the metagenomic sequencing results of nearshore sediment 1 according to the present invention is presented, illustrating the quality values ​​of each base.

[0060] Figure 10 A graph showing the quality score of each sequence from metagenomic sequencing results of nearshore sediment 1 according to the present invention is displayed.

[0061] Figure 11 A graph showing the quality values ​​of each base from the metatranscriptome sequencing results of nearshore sediment 2 according to the present invention is presented.

[0062] Figure 12 A graph showing the quality score of each sequence from the metatranscriptome sequencing results of nearshore sediment 2 according to the present invention is displayed.

[0063] Figure 13A graph showing the quality values ​​of each base from metagenomic sequencing of nearshore sediment 2 according to the present invention is presented.

[0064] Figure 14 A graph showing the quality score of each sequence from metagenomic sequencing results of nearshore sediment 2 according to the present invention is displayed.

[0065] Figure 15 An Agilent 2100 RNA detection map of deep-sea cold seep sediment 1 according to the present invention is shown.

[0066] Figure 16 An Agilent 2100 RNA detection map of deep-sea cold seep sediment 2 according to the present invention is shown.

[0067] Figure 17 A graph showing the quality values ​​of each base from the metrological transcriptome sequencing results of deep-sea cold seep sediment 1 according to the present invention is presented.

[0068] Figure 18 The quality score of each sequence in the metatranscriptome sequencing results of deep-sea cold seep sediment 1 according to the present invention is shown in the figure.

[0069] Figure 19 A statistical graph of the quality values ​​of each base from metagenomic sequencing results of deep-sea cold seep sediment 1 according to the present invention is shown.

[0070] Figure 20 The quality score of each sequence in the metagenomic sequencing results of deep-sea cold seep sediment 1 according to the present invention is shown in the figure.

[0071] Figure 21 A graph showing the quality values ​​of each base from the metrological transcriptome sequencing results of deep-sea cold seep sediment 2 according to the present invention is presented.

[0072] Figure 22 The quality score of each sequence in the metatranscriptome sequencing results of deep-sea cold seep sediment 2 according to the present invention is shown in the figure.

[0073] Figure 23 A statistical graph showing the quality values ​​of each base in the metagenomic sequencing results of deep-sea cold seep sediment 2 according to the present invention is shown.

[0074] Figure 24 The quality score of each sequence in the metagenomic sequencing results of deep-sea cold seep sediment 2 according to the present invention is shown in the figure.

[0075] Figure 25 The electrophoretic detection results of DNA-RNA co-extraction according to Example 4 of the present invention are shown; wherein, channel 1 represents lysis buffer, channel 2 represents lysis buffer 2, and channel M1 represents Takaraλ-Hind III digestMarker.

[0076] Figure 26 The image shows an Agilent 2100 detection plot of RNA in lysis buffer 1 according to Example 4 of the present invention.

[0077] Figure 27 The image shows an Agilent 2100 detection plot of RNA in lysis buffer 2 according to Example 4 of the present invention. Detailed Implementation

[0078] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0079] Terminology Explanation:

[0080] Marine sediments: refers to the general term for seabed sediments formed by various marine sedimentation processes. Sediments are classified according to depth into nearshore (sea) sediments (0-20 meters), shallow sea sediments (20-200 meters), semi-deep sea sediments (200-2000 meters), and deep sea sediments (greater than 2000 meters).

[0081] Deep-sea cold seep sediments: Sediments accumulated in cold seep areas at the bottom of the deep sea. These cold seep areas are usually rich in organic matter such as methane and hydrogen sulfide, supporting unique ecosystems.

[0082] As mentioned in the background section, existing technologies lack methods for simultaneously extracting DNA and RNA from samples with low biomass (e.g., the marine sediment biomass of this application is approximately 10^5-10^7 microorganisms / g). In this invention, the inventors, through various attempts (including but not limited to adjusting the lysis time, changing the lysis temperature, and adding lysozyme to promote lysis), discovered that by controlling the composition of the lysis buffer (i.e., adding a certain volume of anhydrous ethanol) and pH, efficient extraction of DNA and RNA from samples with low biomass can be achieved. Therefore, a series of protective measures for this invention have been proposed.

[0083] In a first typical embodiment of the present invention, a lysis buffer for extracting DNA and / or RNA is provided, the lysis buffer comprising: 0.6M–0.8M NaH₂PO₄, 0.042–0.06M Na₂HPO₄, 0.007–0.009M NaCl, 0.03–0.05M Tris, 0.01–0.02M SDS and 10%–20% (V / V, mL / mL) anhydrous ethanol; wherein the pH of the lysis buffer is 5–6.

[0084] The lysis buffer of this invention can efficiently lyse test samples, effectively release and protect nucleic acid molecules in the sample, and significantly improve nucleic acid extraction efficiency. Taking marine sediments as an example, marine sediments have low biomass and contain various impurities (including mineral particles and carbonates, salts and metal ions, and organic matter, etc.). These impurities can adsorb nucleic acids and exacerbate RNA degradation, ultimately affecting the co-extraction of DNA and RNA. This invention, by adding ethanol and a suitable ratio of phosphate to the lysis buffer, can effectively maximize the recovery of free nucleic acids after lysis. The lysis buffer of this invention not only allows for complete lysis of organisms in marine sediments, releasing nucleic acid molecules (including DNA and RNA) and resulting in high nucleic acid yield, but also eliminates interference from other impurities in marine sediments, ensuring high purity and integrity of the released nucleic acid molecules. This facilitates library construction and the acquisition of mRNA data to support downstream analysis.

[0085] Furthermore, the pH of the lysis buffer in existing technologies is typically 7.5–8.5, which is unfavorable for nucleic acid extraction. This invention, by optimizing the pH of the lysis buffer, found that a pH of 5–6 enables efficient nucleic acid extraction. The extracted nucleic acid molecules are not only abundant but also highly intact (see...). Figure 1 ).

[0086] In a second typical embodiment of the present invention, a method for extracting nucleic acids is provided, the method comprising:

[0087] S1, use the above-mentioned lysis buffer to lyse the sample to be tested to obtain the lysed solution;

[0088] S2, the DNA-RNA mixture is separated from the above lysed solution;

[0089] S3, isolate DNA from the above DNA-RNA mixture; and / or

[0090] RNA was isolated from the above DNA-RNA mixture.

[0091] The nucleic acid extraction method of this invention can simultaneously and efficiently extract intact DNA and RNA. For samples with low biomass, such as marine sediments, the nucleic acids extracted using this method can help study the impact of environmental factors on microbial communities through metagenomics and metatranscriptomics, thereby contributing to the understanding of the structure and function of marine ecosystems and the role of microorganisms in global change and ecosystem function.

[0092] In the lysis step S1 described above, the ratio of the sample to the lysis buffer can be adjusted and selected according to the sample quality. In a preferred embodiment, the volume-to-volume ratio of the lysis buffer to the sample is (1:2) to (1:3). This volume-to-volume ratio helps to obtain DNA and RNA with high integrity, high purity, and high content.

[0093] In a preferred embodiment of the present invention, the sample to be tested is a low biomass sample. More preferably, the sample to be tested is a sample with a biomass of 10^5 to 10^11 microorganisms / g (e.g., marine sediments have 10^5 to 10^7 microorganisms / g; another example is soil with approximately 10^10 to 10^11 microorganisms per gram of wet weight). More preferably, the sample to be tested is marine sediment. Even more preferably, the marine sediment is a sample with a biomass of 10^5 to 10^7 microorganisms / g. The nucleic acid extraction method of the present application can achieve co-extraction of DNA and RNA from the same sample to be tested with such low biomass as described above, and the extraction integrity and purity are high.

[0094] To ensure uniform mixing of the lysis buffer and the sample to be tested, and to further ensure complete dissolution of the sample, in a preferred embodiment of the present invention, after lysing the sample with the aforementioned lysis buffer, before obtaining the lysed solution, step S1 further includes a vortexing and water bath heating step; preferably, the vortexing time is 1–3 min. Vortexing under these conditions helps to thoroughly mix the lysis buffer and the sample to be tested and promotes the lysis of the sample. Preferably, the water bath heating temperature is 70–80°C; the time is 40–60 min. Water bath heating under these conditions (high temperature) helps the sample to undergo vigorous lysis, thereby fully releasing nucleic acid molecules (including DNA and RNA), resulting in high purity and high yield of nucleic acid molecules.

[0095] In a preferred embodiment of the present invention, S2 includes:

[0096] S21: Extract the above-mentioned pyrolysis solution to obtain the extracted solution;

[0097] S22: Precipitate the solution after extraction to obtain a precipitate;

[0098] S23: Dissolve the above precipitate in water using DEPC treatment to obtain the above DNA-RNA mixture; wherein, in step S22, the above extracted solution is precipitated using ethanol and optionally isopropanol and sodium acetate.

[0099] In a preferred embodiment of the present invention, an extraction reagent is used for the above extraction. The role of the extraction reagent is to help completely separate the nucleic acid from other components (such as proteins, salts, and other impurities) in the lysis system after nucleic acid release, thereby achieving nucleic acid purification. In a preferred embodiment of the present invention, the extraction reagent contains chloroform and isoamyl alcohol; preferably, the volume ratio of chloroform to isoamyl alcohol is 24:1. Using the extraction reagent in the above ratio can effectively and completely extract nucleic acid, improving the purity of nucleic acid. In a preferred embodiment of the present invention, the lysed solution and the extraction reagent are mixed at a volume ratio of 1:1. Extraction according to the above ratio can effectively improve the purity of nucleic acid.

[0100] Using the above ratio helps ensure that DNA and RNA are fully dissolved in the extraction reagent. In S3 above, the precipitating reagent is used to precipitate DNA and RNA. This precipitating reagent includes ethanol, and optionally isopropanol and sodium acetate. Ethanol alters the affinity of DNA for water, causing DNA molecules to precipitate from the solution. This allows DNA to be separated by methods such as centrifugation for subsequent experimental procedures. Isopropanol facilitates the transfer of DNA and RNA between the aqueous and organic phases, while sodium acetate helps form a precipitate. Precipitation separates DNA and RNA from the extraction reagent, facilitating subsequent experimental procedures.

[0101] To ensure sufficient precipitation of DNA and RNA, in a preferred embodiment of the present invention, step S22 includes: mixing the extracted solution, isopropanol, and sodium acetate to obtain precipitate 1; mixing precipitate 1 with ethanol to obtain precipitate 2; and mixing precipitate 2 with ethanol to obtain the precipitate. In a preferred embodiment of the present invention, the extracted solution, isopropanol, and sodium acetate are mixed in a volume ratio of 10:7:1, wherein the concentration of sodium acetate is 3M.

[0102] In a preferred embodiment of the present invention, precipitate 1 and ethanol are mixed at a mass-to-volume ratio of (1:1000) to (1:5000), and precipitate 2 and ethanol are mixed at a mass-to-volume ratio of (1:1000) to (1:5000), wherein the concentration of ethanol is 70% to 80%. In this step, the nucleic acid is washed twice with ethanol, allowing DNA and RNA to precipitate fully and dissolving other residual impurities in the ethanol, thereby purifying DNA and RNA.

[0103] In a preferred embodiment of the present invention, the extracted solution, isopropanol, and sodium acetate are mixed and allowed to stand to obtain precipitate 1. The standing time is 30 min to 60 min, and the standing temperature is 4 to 25°C. Standing under the above conditions has the beneficial effect of allowing nucleic acids to precipitate fully.

[0104] In a preferred embodiment of the present invention, S3 includes: mixing ribonuclease and the DNA-RNA mixture for digestion reaction 1 to obtain the DNA; and / or mixing deoxyribonuclease and the DNA-RNA mixture for digestion reaction 2 to obtain the RNA. The role of ribonuclease (e.g., RNase A) is to remove RNA from the DNA-RNA mixture. By selecting an appropriate amount of ribonuclease and appropriate digestion time and temperature, the optimal RNA removal effect can be obtained. In a preferred embodiment of the present invention, the DNA-RNA mixture and ribonuclease are mixed at a volume ratio of (25:1) to (50:1); preferably, the concentration of the ribonuclease is 600 to 900 U / mL; preferably, the digestion reaction 1 time is 30 to 45 min; and the temperature is 35 to 38°C.

[0105] The role of deoxyribonuclease (e.g., DNase I) is to remove DNA from the DNA-RNA mixture. Selecting an appropriate amount of deoxyribonuclease, digestion time, and temperature can achieve the best DNA removal effect. In a preferred embodiment of the present invention, the DNA-RNA mixture is mixed with deoxyribonuclease at a volume ratio of (25:1) to (30:1); preferably, the concentration of the deoxyribonuclease is 2000–2500 U / mL; preferably, the digestion reaction 2 takes 30–45 min; and the temperature is 35–38°C.

[0106] Before dissolving the precipitate with DEPC-treated water, the precipitate needs to be properly dried to remove any adhering ethanol, which facilitates subsequent experiments. In some embodiments, the drying time is 2–5 minutes, and the temperature is 25–37°C. DEPC-treated water is added to ensure complete dissolution of DNA and RNA; the amount of DEPC-treated water added is determined based on the required concentrations of DNA and RNA.

[0107] In a third typical embodiment of the present invention, a method for constructing a nucleic acid library is provided. The method includes: reverse transcription of RNA to obtain double-stranded cDNA; constructing a library using the double-stranded cDNA to obtain an RNA library; and / or constructing a library using DNA to obtain a DNA library; wherein the RNA is RNA obtained by the above-described methods for extracting DNA and / or RNA; and the DNA is DNA obtained by the above-described methods for extracting DNA and / or RNA.

[0108] The library construction method of this invention, by utilizing the aforementioned lysis buffer to extract DNA and RNA with high integrity and purity, can successfully achieve simultaneous DNA and RNA library construction for the same sample, thereby helping to obtain more complete and accurate metagenomic and metatranscriptomic sequencing data.

[0109] In a fourth typical embodiment of the present invention, a nucleic acid sequencing method is provided, which sequences the DNA library and / or RNA library obtained in the above-described nucleic acid library construction method. Using the sequencing method of the present invention, metagenomic and metagenomic sequencing are performed simultaneously on the same sample, resulting in higher sequencing efficiency and relatively more complete and accurate sequencing data.

[0110] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0111] Example 1: Effect of different pH lysis buffers on extraction efficiency

[0112] Cell lysis: Weigh 0.5g of fresh marine sediment into two 2ml sterile centrifuge tubes, and add 1ml of co-extraction buffer (pH 7.5 and pH 5.5, respectively) to extract the lysis buffer. Vortex for 1 min to mix the sediment with the solution. Incubate the sediment suspension in a 70℃ water bath for 40 min, inverting the centrifuge tubes every 10 min during this period.

[0113] The co-extraction lysis buffer No. 1, with a pH of 7.5, consisted of the following reagents (final concentration): 0.718 M NaH₂PO₄, 0.052 M Na₂HPO₄, 0.008 M NaCl, 0.04 M Tris, 0.012 M SDS, and 15% (v / v, mL / mL) anhydrous ethanol. The pH was adjusted to 7.5 with NaOH.

[0114] The co-extraction lysis buffer No. 2 with pH 5.5 above includes the following reagents (final concentration): 0.718M NaH2PO4, 0.052M Na2HPO4, 0.008M NaCl, 0.04M Tris, 0.012M SDS, 15% anhydrous ethanol, and the pH is adjusted to 5.5 with hydrochloric acid.

[0115] DNA-RNA co-extraction: Centrifuge the tube from the previous step at 10000g for 10 min, and transfer 900 μL of supernatant to another 2 ml centrifuge tube. Add 900 μL of extraction reagent (chloroform and isoamyl alcohol 24:1), and mix thoroughly by inverting. Centrifuge at 10000g for 10 min at 4℃.

[0116] DNA-RNA co-precipitation: Transfer 900 μL of the supernatant from the previous step to a new 2 ml centrifuge tube, add 630 μL of isopropanol and 90 μL of 3M sodium acetate, mix thoroughly by inverting, and incubate at 4°C for 30 min. Then centrifuge at 10000g for 10 min at 4°C, discard the supernatant, and retain the precipitate. Add 500 μL of 70% ethanol to the precipitate and centrifuge at 8000g for 5 min. Repeat this step to obtain the DNA-RNA precipitate. Centrifuge the DNA-RNA precipitate at 10000g for 3 min at 4°C, remove any remaining liquid from the centrifuge tube using a pipette, and dry at room temperature for 3 min to obtain the DNA-RNA precipitate. Add 100 μL of DEPC-treated water to the DNA-RNA precipitate to dissolve it. Determine the DNA and RNA concentrations using Qubit. Perform agarose gel electrophoresis (see...). Figure 1 Where 1 represents lysis buffer 1 with pH 7.5, and 2 represents lysis buffer 2 with pH 5.5, and RNA integrity was determined using an Agilent 2100 assay (see...). Figure 2 and Figure 3 ).

[0117] The results of the agarose gel electrophoresis experiment are as follows: Figure 1 As shown: RNA extracted using lysis buffer 2 (pH 5.5) clearly shows 16S rRNA and 23S rRNA, while RNA extracted using lysis buffer 1 (pH 7.5) does not clearly show 16S rRNA and 23S rRNA.

[0118] RIN indicates RNA integrity, ranging from 0 to 10. A higher RIN value indicates better RNA quality and higher integrity. Additionally, a higher RIN value results in a flatter baseline and a sharper main peak. Generally, an RIN value ≥ 5 indicates good RNA integrity. (RNA detection graph from Agilent 2100) Figure 2 The results showed that the RIN value of RNA extracted using lysis buffer 1 (pH 7.5) was 3.6, which is less than 5, indicating poor RNA integrity. The elution peaks of 16S rRNA and 23S rRNA were incomplete. Figure 3 The RNA extracted using lysis buffer 2 (pH 5.5) had a RIN value of 6.8, which is greater than 5, indicating good RNA integrity. The elution peaks of 16S rRNA and 23S rRNA were relatively intact.

[0119] Example 2: Procedures for Obtaining Metagenomics and Metatranscriptomes from Two Nearshore Sediments

[0120] Cell lysis: Weigh 0.5 g of fresh marine sediment into a 2 ml sterile centrifuge tube and add 1 ml of co-extraction lysis buffer. Vortex for 1 min to mix the sediment with the solution. Place the sediment suspension in a 70°C water bath for 40 min, inverting the centrifuge tube every 10 min during this period. The co-extraction lysis buffer contained the following reagents and final concentrations: 0.6 M NaH₂PO₄, 0.042 M Na₂HPO₄, 0.007 M NaCl, 0.03 M Tris, 0.01 M SDS, and 10% anhydrous ethanol. The pH of the lysis buffer was 6.

[0121] DNA-RNA co-extraction: Centrifuge the tube from the previous step at 10000g for 10 min, and transfer 900 μL of supernatant to another 2 ml centrifuge tube. Add 900 μL of extraction reagent (chloroform and isoamyl alcohol 24:1), and mix thoroughly by inverting. Centrifuge at 10000g for 10 min at 4℃.

[0122] DNA-RNA co-precipitation: Transfer 900 μL of the supernatant from the previous step to a new 2 ml centrifuge tube, add 630 μL of isopropanol and 90 μL of 3M sodium acetate, mix thoroughly by inverting, and incubate at 4°C for 30 min. Then centrifuge at 10000g for 10 min at 4°C, discard the supernatant, and retain the precipitate. Add 500 μL of 70% ethanol to the precipitate and centrifuge at 8000g for 5 min. Repeat this step to obtain the DNA-RNA precipitate. Centrifuge the DNA-RNA precipitate at 10000g for 3 min at 4°C, remove any remaining liquid from the centrifuge tube using a pipette, and dry at room temperature for 3 min to obtain the DNA-RNA precipitate. Add 100 μL of DEPC-treated water to the DNA-RNA precipitate to dissolve it. Determine the DNA and RNA concentrations using Qubit. Perform agarose gel electrophoresis (…). Figure 4 ) and Agilent 2100 to determine RNA integrity ( Figure 5 and Figure 6 ).

[0123] The results of the agarose gel electrophoresis experiment are as follows: Figure 4 The results showed that 16S rRNA and 23S rRNA were clearly visible in the two RNA samples extracted from nearshore sediments. Figure 4 The 1 in the text represents nearshore sediment 1; Figure 4 The 2 in the text represents nearshore sediment 2; Figure 4 The 3 in the text represents deep-sea cold seep sediment 1. Figure 4In the diagram, 4 represents deep-sea cold seep sediment 2. The Agilent 2100 RNA analysis shows that nearshore sediment 1 has a RIN value of 6.5, which is greater than 5, indicating good integrity. Its 16S rRNA and 23S rRNA peaks are also relatively intact (see [link to analysis]). Figure 5 The RIN value of nearshore sediment 2 was 6.4, greater than 5, indicating high integrity. Its 16S rRNA and 23S rRNA peaks were also relatively intact (see [reference]). Figure 6 ).

[0124] DNA-RNA separation: Take 50 μl of the DNA-RNA mixture obtained in the previous step, add 2 μl of RNase A (10 mg / ml), digest at 37°C for 30 min, and take 200 ng of the obtained DNA according to the BGI Genomics Easy_Fast_Enzyme Digestion Library Preparation Kit. Use this for metagenomic library construction. Take the remaining 50 μl of the DNA-RNA mixture obtained in the previous step, add 2 μl of DNase I (2000 U / ml), digest at 37°C for 30 min, and inactivate the enzyme at 65°C for 20 min. Use the obtained RNA for metagenomic library construction according to subsequent steps.

[0125] RNA reverse transcription: 1 μg of RNA was used to synthesize cDNA first strand using TIANGEN's FastKing gDNA Dispelling RTSuperMix cDNA first strand synthesis reagent (KR118). The reverse transcription reaction system was prepared according to Table 1. Reverse transcription was performed according to Table 2.

[0126] Table 1. Two-chain reaction system

[0127]

[0128]

[0129] Table 2 Reverse Transcription Procedure

[0130] reaction temperature reaction time 42℃ 15min 95℃ 3min

[0131] cDNA Second Stroke Generation: Take 15 μL of the first-stroke product from the previous step and add it to 35 μL of the second-stroke synthesis reagent. The second-stroke reaction reagent is prepared according to the table below. Prepare the second-stroke synthesis reaction system as shown in Table 3, and carry out the cDNA second-stroke synthesis reaction according to Table 4.

[0132] Table 3. Two-chain reaction system

[0133] Composition Usage 5×second strand buffer 10μL dNTP mix (10mM) 2μL RNaseH (5 U / μL) 0.5μL DNA polymerase I (10 U / μL) 2.5μL Nuclease free water 20μL

[0134] Table 4. Reaction Table for Two-Chain Synthesis

[0135] reaction temperature reaction time 16℃ 2h 4℃ ∞

[0136] Following the two-strand synthesis reaction, the cDNA product is purified. The cDNA product purification process is as follows.

[0137] 1. Mix the DNA Clean Beads. Pipette 100 μL of DNA Clean Beads into the sample tube from the previous step. Gently pipette at least 10 times until all magnetic beads are suspended. On the last pipette, make sure all liquid and magnetic beads in the pipette tip are pipetted into the tube.

[0138] 2. Incubate at room temperature for 10 minutes.

[0139] 3. Centrifuge the centrifuge tube briefly, then place it on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Carefully aspirate the supernatant and discard it.

[0140] 4. Keep the centrifuge tubes fixed on the magnetic rack, add 500 μL of 80% ethanol to rinse the magnetic beads and tube walls, let stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0141] 5. Repeat step 4 once. Try to remove as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, you can centrifuge the tube briefly. After separating the tubes on a magnetic rack, use a small-range pipette to remove the liquid from the bottom of the tube.

[0142] 6. Keep the centrifuge tubes fixed on the magnetic rack, open the tube caps, and allow them to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked. Over-drying (cracking) the magnetic beads will lead to reduced yield.

[0143] 7. Remove the centrifuge tube from the magnetic rack, add 50 μL of TE Buffer to elute the DNA, and gently pipette at least 10 times until all magnetic beads are suspended.

[0144] 8. Incubate at room temperature for 10 minutes.

[0145] 9. Centrifuge the centrifuge tube briefly, then place it on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Carefully aspirate 20 μL of the supernatant into a new 1.5 mL centrifuge tube.

[0146] Double-stranded cDNA library construction, sequencing, and analysis: The purified double-stranded cDNA product from the previous step was combined with nucleic acids from the DNA-RNA co-precipitation step using the MGIEasy Fast Enzyme Digestion Library Preparation Kit (catalog number: 940-001193-00) according to the manufacturer's instructions. Sequencing of the library was performed using MGIseq2000. Quality control analysis was conducted after data was processed. After obtaining the raw data's FastQ file, the data quality was verified using FastQC, as shown in Tables 5 and 10. Figures 7-14Table 5 shows the total number of reads, total number of rRNA reads, and total number of mRNA reads for the four samples (nearshore sediment 1, nearshore sediment 2, deep-sea cold seep sediment 1, and deep-sea cold seep sediment 2). Table 5 indicates that the RNA obtained using the method of this invention has a higher proportion of mRNA after library construction and sequencing.

[0147] Figure 7 A graph showing the statistical values ​​of each base in the metatranscriptome sequencing results of nearshore sediment 1. Figure 8 The quality score of each sequence in the metatranscriptome sequencing results of nearshore sediment 1 is shown in the figure. Figure 9 A graph showing the metagenomic sequencing results of nearshore sediment 1, with each base quality value statistically analyzed. Figure 10 Metagenomic sequencing results of nearshore sediment 1: quality score of each sequence. Figure 11 A graph showing the statistical values ​​of each base in the metatranscriptome sequencing results of nearshore sediment 2. Figure 12 The image shows the quality score of each sequence from the metatranscriptome sequencing results of nearshore sediment 2. Figure 13 A graph showing the metagenomic sequencing results of nearshore sediment 2, with statistical values ​​for each base. Figure 14 Metagenomic sequencing results of nearshore sediment 2: quality score of each sequence.

[0148] Figures 7-14 The Per base sequence quality module contains a BoxWhisker plot for each read position, displaying the quality score range for all bases at all read positions in the file. The x-axis represents the position in progress, and the y-axis displays the quality score. A higher quality score at a read position indicates higher sequencing quality at that site. By observing the quality score plot, one can determine the overall sequencing quality, whether there are any poor-quality base sites, and whether there is a trend of overall quality score decline.

[0149] The Per sequence quality scores module displays the distribution of the average quality score for each read position in the file. The x-axis shows the average quality score, and the y-axis represents the frequency of each quality score. For good data, this graph should have only one peak at the end. If other peaks appear, it may indicate that a subset of reads has quality issues.

[0150] Table 10 shows the proportion of bases with a sequencing quality of Q30 or higher for Read1, Read2, and Total reads in the metagenomic and metagenomic sequencing results of four samples (nearshore sediment 1, nearshore sediment 2, deep-sea cold seep sediment 1, and deep-sea cold seep sediment 2). A higher proportion of these bases indicates higher sequencing quality. Read1 and Read2 refer to the two reads obtained from both ends of the same DNA fragment in paired-end sequencing.

[0151] Table 10 and Figures 7-14 The results showed that the sequencing quality of the metatranscriptome and metasequencing genomes of nearshore sediments 1 and 2 was good.

[0152] Example 3: Procedure for Obtaining Metagenomics and Metatranscriptomes from Two Deep-Sea Cold Seep Sediments

[0153] Cell lysis: Weigh 0.5 g of fresh deep-sea cold seep sediment into a 2 ml sterile centrifuge tube and add 1 ml of co-extraction lysis buffer. Vortex for 1 min to mix the sediment with the solution. Place the sediment suspension in a 70°C water bath for 40 min, inverting the centrifuge tube every 10 min during this period. The co-extraction lysis buffer contained the following reagents and final concentrations: 0.8 M NaH₂PO₄, 0.06 M Na₂HPO₄, 0.009 M NaCl, 0.05 M Tris, 0.02 M SDS, and 20% anhydrous ethanol. The pH of the lysis buffer was 5.

[0154] DNA-RNA co-extraction: Centrifuge the tube from the previous step at 10000g for 10 min, and transfer 900 μL of supernatant to another 2 ml centrifuge tube. Add 900 μL of extraction reagent (chloroform and isoamyl alcohol 24:1), and mix thoroughly by inverting. Centrifuge at 10000g for 10 min at 4℃.

[0155] DNA-RNA co-precipitation: Transfer 900 μL of the supernatant from the previous step to a new 2 ml centrifuge tube, add 630 μL of isopropanol and 90 μL of 3M sodium acetate, mix thoroughly by inverting, and incubate at 4°C for 30 min. Then centrifuge at 10000g for 10 min at 4°C, discard the supernatant, and retain the precipitate. Add 500 μL of 70% ethanol to the precipitate and centrifuge at 8000g for 5 min. Repeat this step to obtain the DNA-RNA precipitate. Centrifuge the DNA-RNA precipitate at 10000g for 3 min at 4°C, remove any remaining liquid from the centrifuge tube using a pipette, and dry at room temperature for 3 min to obtain the DNA-RNA precipitate. Add 100 μL of DEPC-treated water to the DNA-RNA precipitate to dissolve it. Determine the DNA and RNA concentrations using Qubit. Agarose gel electrophoresis (see...) Figure 4 ) and Agilent 2100 to determine RNA integrity (see Figure 15 and 16 ).

[0156] The experimental results of agarose gel electrophoresis are as follows: Figure 4 ( Figure 4 The 1 in the text represents nearshore sediment 1; Figure 4 The 2 in the text represents nearshore sediment 2; Figure 4 The 3 in the text represents deep-sea cold seep sediment 1. Figure 4 As shown in Figure 4 (representing deep-sea cold seep sediment 2): RNA extracted from deep-sea cold seep sediments 1 and 2 clearly shows 16S rRNA and 23S rRNA. The RNA Agilent 2100 assay shows that the RIN value of deep-sea cold seep sediment 1 is 6.8, greater than 5, indicating good integrity, and its 16S rRNA and 23S rRNA peaks are relatively complete (see...). Figure 15 The RIN value of deep-sea cold seep sediment 2 was 6.7, greater than 5, indicating high integrity. Its 16S rRNA and 23S rRNA peaks were also relatively intact (see [link to relevant documentation]). Figure 16 ).

[0157] DNA-RNA separation: Take 50 μl of the DNA-RNA mixture obtained in the previous step, add 2 μl of RNase A (10 mg / ml), digest at 37°C for 30 min, and take 200 ng of the obtained DNA according to the BGI Genomics Easy_Fast_Enzyme Digestion Library Preparation Kit. Use this for metagenomic library construction. Take the remaining 50 μl of the DNA-RNA mixture obtained in the previous step, add 2 μl of DNase I (2000 U / ml), digest at 37°C for 30 min, and inactivate the enzyme at 65°C for 20 min. Use the obtained RNA for metagenomic library construction according to subsequent steps.

[0158] RNA reverse transcription: 1 μg of RNA was used to synthesize the first strand of cDNA using the TIANGEN FastKing gDNA Dispelling RTSuperMix cDNA first-strand synthesis reagent (KR118). The reverse transcription reaction system was prepared according to Table 6. The reverse transcription reaction was performed according to Table 7.

[0159] Table 6. Two-chain reaction system

[0160] Composition Usage 5×FastKing-RT SuperMix 4μL Total RNA 1μg RNase-free water Add to 20μL

[0161] Table 7 Reverse transcription reaction conditions

[0162] reaction temperature reaction time 42℃ 15min 95℃ 3min

[0163] cDNA Second-Strand Synthesis: Take 15 μL of the first-strand product from the previous step and add it to 35 μL of the second-strand synthesis reagent. The second-strand reaction reagent is prepared according to the table below. Prepare the second-strand synthesis reaction system as shown in Table 8, and carry out the cDNA second-strand synthesis reaction according to Table 9.

[0164] Table 8. Two-chain reaction system

[0165] Composition Usage 5×second strand buffer 10μL dNTP mix (10mM) 2μL RNaseH (5 U / μL) 0.5μL DNA polymerase I (10 U / μL) 2.5μL Nuclease free water 20μL

[0166] Table 9. Reaction Table for Dichain Synthesis

[0167] reaction temperature reaction time 16℃ 2h 4℃ ∞

[0168] Following the two-strand synthesis reaction, the cDNA product is purified. The cDNA product purification process is as follows.

[0169] 1. Mix the DNA Clean Beads. Pipette 100 μL of DNA Clean Beads into the sample tube from the previous step. Gently pipette at least 10 times until all magnetic beads are suspended. On the last pipette, make sure all liquid and magnetic beads in the pipette tip are pipetted into the tube.

[0170] 2. Incubate at room temperature for 10 minutes.

[0171] 3. Centrifuge the centrifuge tube briefly, then place it on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Carefully aspirate the supernatant and discard it.

[0172] 4. Keep the centrifuge tubes fixed on the magnetic rack, add 500 μL of 80% ethanol to rinse the magnetic beads and tube walls, let stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0173] 5. Repeat step 4 once. Try to remove as much liquid as possible from the tube. If a small amount of liquid remains on the tube wall, you can centrifuge the tube briefly. After separating the tubes on a magnetic rack, use a small-range pipette to remove the liquid from the bottom of the tube.

[0174] 6. Keep the centrifuge tubes fixed on the magnetic rack, open the tube caps, and allow them to dry at room temperature until the surface of the magnetic beads is no longer reflective and cracked. Over-drying (cracking) the magnetic beads will lead to reduced yield.

[0175] 7. Remove the centrifuge tube from the magnetic rack, add 50 μL of TE Buffer to elute the DNA, and gently pipette at least 10 times until all magnetic beads are suspended.

[0176] 8. Incubate at room temperature for 10 minutes.

[0177] 9. Centrifuge the centrifuge tube briefly, then place it on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Carefully aspirate 20 μL of the supernatant into a new 1.5 mL centrifuge tube.

[0178] Double-stranded cDNA library construction, sequencing, and analysis: The purified double-stranded cDNA product from the previous step was combined with nucleic acids from the DNA-RNA co-precipitation step using the MGIEasy Fast Enzyme Digestion Library Preparation Kit (catalog number: 940-001193-00) according to the manufacturer's instructions. Sequencing of the library was performed using MGIseq2000. Quality control analysis was conducted after data processing. The raw data files were then tested for quality using FastQC, as shown in Tables 5 and 10. Figures 17-24 As shown.

[0179] Figure 17 A graph showing the statistical values ​​of each base from the metatranscriptome sequencing results of deep-sea cold seep sediment 1 is presented. Figure 18 The quality score of each sequence in the metatranscriptome sequencing results of deep-sea cold seep sediment 1 is shown. Figure 19 A graph showing the metagenomic sequencing results of deep-sea cold seep sediment 1, with statistical values ​​for each base. Figure 20 Metagenomic sequencing results of deep-sea cold seep sediment 1: quality score of each sequence. Figure 21 A graph showing the statistical values ​​of each base in the metatranscriptome sequencing results of deep-sea cold seep sediment 2. Figure 22 The quality score of each sequence in the metatranscriptome sequencing results of deep-sea cold seep sediment 2 is shown. Figure 23 A graph showing the metagenomic sequencing results of deep-sea cold seep sediment 2, with statistical values ​​for each base. Figure 24 Quality score of each sequence from metagenomic sequencing results of deep-sea cold seep sediment 2.

[0180] Table 10 and Figures 17-24 The results showed that the sequencing quality of the metatranscriptome and metasequencing of deep-sea cold seep sediment 1 and deep-sea cold seep sediment 2 was good.

[0181] Table 5. Quality control results of metagenomic sequencing for 4 samples

[0182]

[0183]

[0184] Table 10 Sequencing quality statistics

[0185]

[0186] Example 4: Effect of lysis buffer component concentration on extraction efficiency

[0187] Cell lysis: Weigh 0.5g of fresh marine sediment into two 2ml sterile centrifuge tubes, and add 1ml of co-extraction lysate (pH 6) for each tube. Vortex for 1 min to mix the sediment and solution. Incubate the sediment suspension in a 70℃ water bath for 40 min, inverting the centrifuge tubes every 10 min during this period.

[0188] The co-extraction lysis buffer No. 1, with a pH of 6, consisted of the following reagents (final concentration): 0.718 M NaH₂PO₄, 0.052 M Na₂HPO₄, 0.01 M NaCl, 0.04 M Tris, 0.012 M SDS, and 15% (v / v, mL / mL) anhydrous ethanol. The pH was adjusted to 6 with hydrochloric acid.

[0189] The co-extraction lysis buffer No. 2 with pH 6 above includes the following reagents (final concentration): 0.9M NaH2PO4, 0.03M Na2HPO4, 0.008M NaCl, 0.06M Tris, 0.03M SDS, 40% anhydrous ethanol, and the pH is adjusted to 6 with hydrochloric acid.

[0190] DNA-RNA co-extraction: Centrifuge the tube from the previous step at 10000g for 10 min, and transfer 900 μL of supernatant to another 2 ml centrifuge tube. Add 900 μL of extraction reagent (chloroform and isoamyl alcohol 24:1), and mix thoroughly by inverting. Centrifuge at 10000g for 10 min at 4℃.

[0191] DNA-RNA co-precipitation: Transfer 900 μL of the supernatant from the previous step to a new 2 ml centrifuge tube, add 630 μL of isopropanol and 90 μL of 3M sodium acetate, mix thoroughly by inverting, and incubate at 4°C for 30 min. Then centrifuge at 10000g for 10 min at 4°C, discard the supernatant, and retain the precipitate. Add 500 μL of 70% ethanol to the precipitate and centrifuge at 8000g for 5 min. Repeat this step to obtain the DNA-RNA precipitate. Centrifuge the DNA-RNA precipitate at 10000g for 3 min at 4°C, remove any remaining liquid from the centrifuge tube using a pipette, and dry at room temperature for 3 min to obtain the DNA-RNA precipitate. Add 100 μL of DEPC-treated water to the DNA-RNA precipitate to dissolve it. Determine the DNA and RNA concentrations using Qubit. Perform agarose gel electrophoresis (see...). Figure 25 (where 1 represents lysis buffer 1, 2 represents lysis buffer 2, and Agilent 2100 was used to determine RNA integrity (see...) Figure 26 and Figure 27 ).

[0192] The results of the agarose gel electrophoresis experiment are as follows: Figure 25As shown: RNA extracted with lysis buffer 1 clearly shows 16S rRNA and 23S rRNA, while RNA extracted with lysis buffer 2 does not clearly show 16S rRNA and 23S rRNA.

[0193] RIN indicates RNA integrity, ranging from 0 to 10. A higher RIN value indicates better RNA quality and higher integrity. Additionally, a higher RIN value results in a flatter baseline and a sharper main peak. Generally, an RIN value ≥ 5 indicates good RNA integrity. The RNA assay using Agilent 2100 shows that RNA extracted with lysis buffer 1 has an RIN value of 7.9, greater than 5, indicating good RNA integrity. The peaks of 16S rRNA and 23S rRNA are intact (see [link to assay]). Figure 26 ). Figure 27 The results showed that the RIN value of RNA extracted using lysis buffer 2 was 2.4, which is less than 5. The peaks of 6S rRNA and 23S rRNA were incomplete, indicating RNA degradation.

[0194] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The present invention provides a low-cost and simple method for simultaneously extracting DNA and RNA from marine sediments (low biomass and rich in various impurities). The extracted DNA and RNA are used to construct metagenomics and metagenomics, enabling the simultaneous acquisition of metagenomic and metatranscriptomical data from the same sample. This method is applicable to nearshore and deep-sea cold seep sediments. Through this method, RNA and DNA can be obtained simultaneously, and the same extraction product can be used for genomics and metagenomics studies, reducing experimental errors. The nucleic acid impurities obtained by this method are few and relatively complete, providing sufficient effective data to support subsequent analysis after library construction. The present invention also provides a new reference for extracting DNA and / or RNA from other low-biomass samples, helping to improve the sequencing quality of low-biomass samples.

[0195] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A lysis buffer for extracting nucleic acids, characterized in that, The lysis buffer comprises: 0.6M–0.8M NaH2PO4, 0.042–0.06M Na2HPO4, 0.007–0.009M NaCl, 0.03–0.05M Tris, 0.01–0.02M SDS, and 10%–20% anhydrous ethanol; The pH of the lysis solution is 5 to 6.

2. A method for extracting nucleic acids, characterized in that, The method includes: S1, the sample to be tested is lysed using the lysis buffer described in claim 1 to obtain a lysed solution; S2, Separate the DNA-RNA mixture from the lysed solution; S3, separating DNA from the DNA-RNA mixture; and / or RNA was isolated from the DNA-RNA mixture.

3. The method according to claim 2, characterized in that, S2 includes: S21: Extract the pyrolyzed solution to obtain the extracted solution; S22: Precipitate the extracted solution to obtain a precipitate; S23: Dissolve the precipitate in water using DEPC to obtain the DNA-RNA mixture; In step S22, the extracted solution is precipitated using ethanol and optionally isopropanol and sodium acetate. Preferably, S3 includes: The ribonuclease and the DNA-RNA mixture are mixed and digested to obtain the DNA; and / or The deoxyribonuclease and the DNA-RNA mixture were mixed and digested to obtain the RNA; Preferably, the lysis buffer and the sample to be tested are in a volume-to-mass ratio of (1:2) to (1:3); Preferably, the sample to be tested is a sample with a biomass of 10^5 to 10^11 microorganisms / g; Preferably, the sample to be tested is marine sediment; Preferably, the marine sediment is a sample with a biomass of 10^5 to 10^7 microorganisms / g.

4. The method according to claim 2, characterized in that, After the sample to be tested is lysed using the lysis buffer, before obtaining the lysed solution, step S1 further includes the steps of vortexing and water bath heating. Preferably, the vortex duration is 1–3 minutes; Preferably, the water bath heating temperature is 70–80°C, and the time is 40–60 minutes.

5. The method according to claim 3, characterized in that, The lysed solution was extracted using an extraction reagent to obtain the extracted solution. Preferably, the extraction reagent comprises chloroform and isoamyl alcohol; More preferably, the volume ratio of the trichloromethane to the isoamyl alcohol is 24:1; Preferably, the pyrolyzed solution and the extraction reagent are mixed at a volume ratio of 1:

1.

6. The method according to claim 3, characterized in that, Step S22 includes: The extracted solution, isopropanol, and sodium acetate were mixed to obtain precipitate 1. The precipitate 1 and the ethanol are mixed to obtain precipitate 2; The precipitate 2 and the ethanol are mixed to obtain the precipitate; Preferably, the extracted solution, isopropanol, and sodium acetate are mixed in a volume ratio of 10:7:1; wherein the concentration of sodium acetate is 3M. Preferably, the precipitate 1 and the ethanol are mixed at a mass-volume ratio of (1:1000) to (1:5000), and the precipitate 2 and the ethanol are mixed at a mass-volume ratio of (1:1000) to (1:5000), wherein the concentration of the ethanol is 70% to 80%.

7. The method according to claim 6, characterized in that, The extracted solution, isopropanol, and sodium acetate are mixed and allowed to stand to obtain precipitate 1. The standing time is 30 min to 60 min, and the standing temperature is 4 to 25 °C.

8. The method according to claim 3, characterized in that, The DNA-RNA mixture is mixed with the ribonuclease at a volume ratio of (25:1) to (50:1); Preferably, the concentration of the ribonuclease is 600–900 U / mL; More preferably, the digestion reaction 1 takes 30 to 45 minutes and is carried out at a temperature of 35 to 38°C. Preferably, the DNA-RNA mixture and the deoxyribonuclease are in a volume ratio of (25-1): (30~1) Mix; More preferably, the concentration of the deoxyribonuclease is 2000–2500 U / mL; More preferably, the digestion reaction 2 takes 30 to 50 minutes and is carried out at a temperature of 35 to 37°C.

9. A method for constructing a nucleic acid library, characterized in that, The method includes: RNA was reverse transcribed to obtain double-stranded cDNA; Library construction was performed using the double-stranded cDNA to obtain an RNA library; and / or DNA libraries are constructed to obtain DNA libraries; Wherein, the RNA is RNA obtained according to any one of claims 2-8; and the DNA is DNA obtained according to any one of claims 2-8.

10. A method for sequencing nucleic acids, characterized in that, The DNA library and / or RNA library obtained in the nucleic acid library construction method of claim 9 are sequenced.

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