Layered extraction method for ancient DNA (deoxyribonucleic acid) of plateau lake sediment

Abstract

The invention discloses a layering extraction method for paleoDNA of plateau lake sediments, which comprises the following steps: measuring the total organic carbon content TOC or humus content HM of the sediments, dividing the sediments into three layers, namely an initial layer, a middle layer and a deep layer according to the measured content, the initial layer is TOC > = 3.5 wt% or HM > = 30 g.kg <-1 >, the middle layer is TOC = 1.0-3.5 wt% or HM = 6-30 g.kg <-1 >, and the deep layer is TOClt; 1.0 wt% or HMlt; 6 g.kg <-1 >; different steps of pretreatment, splitting decomposition, impurity removal and purification are adopted for different layers, so that the yield and purity of the ancient DNA are improved. By combining pre-cleaning, LPA carrier precipitation and silica gel membrane purification technologies, the DNA yield (deep gt; 200ng) and the amplifiability of different layers are remarkably improved, and the sequencing requirements of the microbe metagenome in the lake are met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of DNA extraction, and particularly relates to a method for stratified extraction of ancient DNA of plateau lake sediment. BACKGROUND

[0002] In the research of microbial molecular ecology of lake ecosystem, the acquisition of high-quality DNA is the basis for analyzing the structure and function of lake microbial community. However, the lake sediment has unique physicochemical properties: it is rich in humic acid, polyphenols and other organic inhibitors, the decomposition products of plant residues can interfere with purification, the complex of clay minerals and organic matter has a significant adsorption effect on nucleic acid, and the microbial abundance of different depth sediments is different (such as high microbial biomass in the surface eutrophic area and low biomass in the deep anaerobic area). The existing DNA extraction methods (such as soil DNA extraction kit) face significant challenges when dealing with lake sediments: the co-extraction of impurities such as humus and DNA results in low purity, which inhibits the subsequent PCR reaction; the adsorption of organic-inorganic complex to DNA causes insufficient extraction, especially for deep low-biomass sediments, which is difficult to meet the demand of metagenomic sequencing. SUMMARY

[0003] In view of this, the embodiments of the present application provide a method for stratified extraction of ancient DNA of plateau lake sediment to solve the problems in the related art.

[0004] According to the embodiments of the present application, a method for stratified extraction of ancient DNA of plateau lake sediment is provided, comprising: S1: first, measure the total organic carbon content TOC or humus content HM of the sediment, and divide the sediment into three layers according to the measured content, i.e. the initial layer, the middle layer and the deep layer, wherein the initial layer is TOC≥3.5wt% or HM≥30 g·kg -1 , the middle layer is TOC=1.0-3.5wt% or HM=6-30 g·kg -1 , and the deep layer is TOC<1.0wt% or HM<6 g·kg -1 ; S2: sampling and pretreatment are performed on the sediment of the initial layer, beads are added for wetting and mixing, lysis and crushing are performed to obtain a first supernatant; CTAB treatment and centrifugation are performed on the first supernatant, organic phase extraction is added, and after clarification, centrifugation is performed to obtain a second supernatant; the second supernatant is combined with a column, and after washing, a DNA initial layer extraction solution is obtained; S3: sampling and pretreatment are performed on the sediment of the middle layer, lysis solution is added for incubation, beads are added for grinding, and centrifugation is performed to obtain a third supernatant; CTAB treatment and centrifugation are performed on the third supernatant, organic phase extraction is added, and then ethanol precipitation and washing are performed, the obtained liquid is combined with a column, and after washing, a DNA middle layer extraction solution is obtained; S4: sampling the deep layer of the sediment, adding lysis solution and zirconium beads to a shaker, then crushing and centrifuging to obtain a fourth supernatant; adding an organic phase to the fourth supernatant for extraction, and centrifuging to obtain a fifth supernatant; inverting and mixing the magnetic beads used, and using the magnetic beads to adsorb the fifth supernatant, and then washing and eluting with ethanol to obtain a DNA deep layer extraction solution.

[0005] Further, the organic carbon in the sediment is determined according to the potassium dichromate oxidation-spectrophotometric method for determining soil organic carbon, and the content of humus in the sediment is determined according to the potassium dichromate oxidation method.

[0006] Further, in S2, the initial layer of the sediment is treated, specifically including: The sediment sample is taken in a grinding tube, if the sample contains obvious carbonates, 0.5 mol·L -1 HCl is added at a sample mass ratio of 1:2-4 (g:mL), and then washed with nuclease-free water until the supernatant is neutral, centrifuged and the washing solution is discarded; if there are no carbonates, no treatment is required.

[0007] Further, in S2, the beads are added, mixed, lysed and crushed to obtain a first supernatant, specifically including: Zirconium beads, initial layer lysis solution and proteinase K are added to the grinding tube, and the sample is vortexed to wet it, the ratio of zirconium beads, initial layer lysis solution, proteinase K and sample is (100-150) µL:600 µL:5 µL:(0.2-0.5) g, the tube is incubated in a 65°C water bath, then ground in a bead mill or a tissue cell crusher, centrifuged after grinding, and all the first supernatant is transferred to a centrifuge tube, and the volume is recorded as V1.

[0008] Further, in S2, the first supernatant is treated with CTAB and centrifuged, then an organic phase is added for extraction, and after clarification, the second supernatant is obtained by centrifugation, the second supernatant is combined with a column, and after washing, a DNA initial layer extraction solution is obtained, specifically including: 1) 100 g·L -1 CTAB is added to the first supernatant, the volume ratio of the added CTAB to the volume V1 of the first supernatant is 0.25:1, and then incubated in a 65°C water bath, centrifuged and all the second supernatant is transferred, and the volume is recorded as V2; 2) an equal volume of chloroform-isoamyl alcohol solution as V2 is added to the centrifuge tube, inverted and mixed, centrifuged and all the supernatant is transferred to a centrifuge tube, repeated extraction is performed, and finally the volume of the second supernatant transferred is recorded as V3; 3) Add (0.2-0.5) g: 100 µL of cHTR Reagent to the second supernatant clear aqueous phase with a volume of V3, vortex mix, stand at room temperature and centrifuge, take the clear supernatant to a new tube, and record the volume as V4; 4) Add an equal volume of XP1 Buffer to the centrifuge tube, vortex mix; put the HiBind® DNA Mini Column adsorption column in a new centrifuge tube, and add the supernatant with a volume of V4 to the column in several portions, each portion ≤ 700 µL, centrifuge and discard the waste liquid until the liquid is completely transferred; 5) Put the just filtered HiBind® DNA Mini Column in a new tube, add (0.2-0.5) g: 700 µL of HBC Buffer, centrifuge and discard the waste liquid, and then add (0.2-0.5) g: 700 µL of DNA Wash to the column, centrifuge and discard the waste liquid; 6) After centrifuging the empty column, put it in a new centrifuge tube, and add 50–100 µL of Elution Buffer preheated to 65 ℃ to the center of the column membrane, stand for incubation, centrifuge to elute the DNA, discard the adsorption column to obtain the DNA eluate, and store the DNA primary layer extract at -80 ℃.

[0009] Further, in S3, the middle layer sediment is sampled and pretreated, lysis solution is added for incubation, and then beads are ground, the third supernatant is taken after centrifugation, and the specific steps include: Add the sediment sample, zirconium beads, and sodium pyrophosphate to the grinding tube, gently invert, then add the lysis buffer containing PPVP and protease K, vortex mix, and then incubate in a water bath, and then put it in a bead mill or a tissue cell crusher for grinding, after completion, centrifuge and transfer all the third supernatant to a centrifuge tube, and record the volume as V5. The ratio of zirconium beads, sodium pyrophosphate, lysis solution, protease K, and sample is (100–150) µL: (5–10) µL: 650 µL: 10 µL: (0.3-0.5) g.

[0010] Further, in S3, the third supernatant is subjected to CTAB treatment and centrifugation, an organic phase is added for extraction, and then ethanol precipitation and washing are performed, and the obtained liquid is combined on a column, and after washing, the DNA middle layer extract is obtained, and the specific steps include: 1) Add 100 g·L -1 CTAB to the third supernatant, and the volume ratio of CTAB to the third supernatant is 0.11:1, incubate in a 65 ℃ water bath, centrifuge and transfer all the second supernatant, and record the volume as V6; 2) Add the same volume of chloroform-isoamyl alcohol solution as V6 to the supernatant, mix well by inverting and centrifuge, transfer all the third supernatant to the centrifuge tube, and record the volume as V7; 3) Add 1.0 mg·mL -1 LPA, 5.0 mol·L -1 NaCl and anhydrous ethanol, the volume ratio of LPA, NaCl, anhydrous ethanol and water phase is 0.02:02:2.5:1, invert and mix after avoiding light at 4℃ for ≥2 h, discard the supernatant after centrifugation, wash the precipitate with 70% ethanol twice and centrifuge, dry the precipitate at room temperature for a short time until the surface is matte, dissolve the precipitate in Tris-Cl EB, record the volume V8 after dissolution and add it to a new centrifuge tube; 4) Add four times the volume of V8 of XP1 Binding Buffer in the centrifuge tube, vortex mix, record the volume as V9, put the HiBind® DNA Mini Column adsorption column in a new centrifuge tube, add the supernatant with a volume of V9 to the adsorption column in several times, each addition amount is not more than 700µL, centrifuge and discard the waste liquid until the liquid is completely transferred, add HBC Buffer with a sample mass ratio of (0.3-0.5) g:700µL, centrifuge and discard the waste liquid, and transfer the adsorption column to a new centrifuge tube; 5) Add DNA Wash Buffer with a sample mass ratio of (0.3-0.5) g:700µL, centrifuge and discard the waste liquid and repeat the operation once, then centrifuge and empty, transfer the adsorption column to a new centrifuge tube, add preheated Elution Buffer with a sample mass ratio of (0.3-0.5) g:30~50µL to the center of the column membrane, stand after centrifugation to obtain the first eluate, add the first eluate to the center of the column membrane again, centrifuge to obtain the final DNA extraction solution, and obtain the DNA middle layer extraction solution, which is stored at -80℃.

[0011] Further, in S4, the deep sediment is sampled, the lysis solution and zirconium beads are placed on the shaker for light shaking, then broken and centrifuged to obtain the fourth supernatant, which specifically includes: Add sediment sample, zirconium beads, deep layer lysis buffer, 0.5 mol·L -1 sodium pyrophosphate, 0.5 mol·L -1 EDTA and proteinase K to the grinding tube, the ratio of zirconium beads, deep layer lysis buffer, sodium pyrophosphate, EDTA, proteinase K to sample is (100-150)µL:700µL:100µL:12µL:15µL:(0.4~0.7)g, place the grinding tube on the shaker for low-speed light shaking, then grind it on the bead mill or tissue cell crusher, centrifuge and obtain the fourth supernatant.

[0012] Further, in S4, the fourth supernatant is added with an organic phase for extraction, and the fifth supernatant is obtained by centrifugation. The used magnetic beads are mixed well by inverting, and the supernatant is adsorbed by the magnetic beads. Ethanol is used for washing and elution to obtain the DNA deep extraction liquid, which specifically comprises: 1) Add an equal volume of chloroform-isoamyl alcohol solution to the centrifugal tube as the volume V10 of the fourth supernatant. After mixing well by inverting, centrifuge and transfer all the second supernatant to a new tube. The volume is V11. 2) Mix the AMPure XP tube well. Prepare 80% ethanol. Calculate the volume of magnetic beads to be added as BeadsVol = 1.6 × V11. Take out the volume of magnetic beads BeadsVol to be added and directly add it to the centrifugal tube containing the supernatant with a volume of V11. Mix well by blowing up and down and stand at room temperature. 3) Place the tube in the magnetic stand and stand until the solution is completely clear. Suck out the supernatant and discard. Keep the tube on the magnetic stand, add 80% ethanol, and the ratio of magnetic beads to 80% ethanol is BeadsVol:200 µL. After standing, the washing solution is sucked off. Repeat the washing and air dry at room temperature. 4) Take the tube off the magnetic stand, add preheated EB in a ratio of (0.4-0.7) g:(30-50) µL to the sample, blow after mixing, and stand at room temperature. Place the tube in the magnetic stand until the solution is clear. Transfer the clear liquid to a new storage tube. If a parallel sample is taken during sampling, first add preheated EB to the first tube to elute the DNA in the first tube. After elution, the eluate is directly added to the second tube for the same elution step. Until all samples are eluted, the DNA deep extraction liquid is obtained. The DNA deep extraction liquid is stored at -80°C.

[0013] Further, the primary lysis solution used for lysis in S2 comprises: Tris-HCl, EDTA, guanidine hydrochloride, Triton X-100, DDT, and enzyme-free water, 1.0 mol·L -1 Tris-HCl: 0.5 mol·L -1 EDTA: 6.0 mol·L -1 Guanidine hydrochloride: 10% v / v Triton X-100: 1.0 mol·L -1 DDT: Enzyme-free water = 16:32:67.5:60:1:323.5, liquid unit is µL, mix well and adjust the pH to 10.2. Adjust the pH with 1.0 mol·L -1 NaOH or 1.0 mol·L -1 HCl is titrated in small amounts and recorded; The middle layer lysis solution used in S3 includes: Tris-HCl, EDTA, Guanidine-HCl, Triton X-100, PVPP, enzyme-free water, 1.0 mol / L -1 Tris-HCl: 0.5 mol / L -1 EDTA: 6.0 mol / L -1 Guanidine-HCl: 10% v / v Triton X-100: PVPP (solid): Enzyme-free water = 9: 18: 40: 30: 3: 203, liquid unit is µL, solid unit is mg, mix well and adjust the pH to 10.0, adjust the pH with 1.0 mol / L -1 NaOH or 1.0 mol / L -1 HCl is titrated in small amounts and recorded.

[0014] The deep layer lysis solution used in S4 includes: Tris-HCl, EDTA, Guanidine-HCl, Triton X-100, PVPP, enzyme-free water, 1.0 mol / L -1 Tris-HCl: 0.5 mol / L -1 EDTA: 6.0 mol / L -1 Guanidine-HCl: 10% v / v Triton X-100: PVPP (solid): Enzyme-free water = 2.8: 5.6: 13.2: 8: 1: 70.4, liquid unit is µL, solid unit is mg, mix well and adjust the pH to 9.8, adjust the pH with 1.0 mol / L -1 NaOH or 1.0 mol / L -1 HCl is titrated in small amounts and recorded.

[0015] The technical scheme provided by the embodiments of the present application can include the following beneficial effects: As can be seen from the above embodiments, the present application adjusts the amount of reagent according to the physicochemical properties of sediments at different depths of Dianchi Lake, effectively solves the problems of surface humus interference, middle layer clay adsorption, and deep layer low abundance degradation. The recovery rate of ancient DNA is 30%-50% higher than that of traditional methods, and more than 100 ng of ancient DNA can be extracted from deep sediments, meeting the demand of metagenomic sequencing. Through fractional purification, the OD260 / 280 ratio of the DNA sample is stable at 1.8-2.0, and there is no obvious inhibitor, which can be directly used for PCR and sequencing reaction.

[0016] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. DETAILED DESCRIPTION

[0017] Hereinafter, exemplary embodiments will be described in detail.

[0018] Embodiment 1: A method for stratified extraction of ancient DNA from highland lake sediments, comprising: S1: first determine the total organic carbon content TOC or humus content HM of the sediment, according to the measured content, the sediment is divided into three layers, namely the initial layer, the middle layer and the deep layer, the initial layer is TOC≥ 3.5wt% or HM≥ 30 g·kg -1 , the middle layer is TOC=1.0–3.5wt% or HM=6–30 g·kg -1 , and the deep layer is TOC<1.0wt% or HM<6 g·kg -1 ; First, collect the required sediment sample from the highland lake, cut and stratify immediately after collection according to the specific length of the collected sediment and the needs of the experimental scheme design, take the center soil sample of the sediment column as the DNA extraction soil sample, and put the DNA extraction soil sample into a 50mL centrifuge tube, the peripheral soil sample is put into a sample bag and sealed as a chemical determination sample, and the cut sample of each layer is labeled, such as the first layer DNA extraction soil sample and the first layer chemical determination sample. After sealing the DNA extraction soil sample and the chemical determination sample, they are stored in a temperature of-80℃ for freezing preservation, and are extracted as soon as possible.

[0019] According to the standard of the second national soil survey, the organic matter content≥ 30 g·kg -1 corresponds to the "rich" or "extremely rich" level, which is usually found in the recently deposited surface layer, i.e. the initial layer of the sediment, in the study of lake or river sediments, the surface layer TOC is usually >3%, here we set 3.5wt% as the upper threshold value, which can effectively distinguish the transition from the middle layer and ensure the ecological distinction of the layer, therefore we set HM≥ 30 g·kg -1 and TOC>3.5wt% of the soil sample as the deep sediment sample. The organic matter of the deep sediment has been highly mineralized, and the standard HM<6 g·kg -1 indicates that the organic matter is extremely poor, and TOC<1.0wt% corresponds to a low level of organic matter <1.7%, which is usually found in the deep part of the sediment core column, indicating that long-term burial leads to loss of organic carbon. Therefore we set HM<6 g·kg -1 and TOC<1.0wt% of the soil sample as the deep sediment sample.

[0020] Therefore, according to the above description, the sediment is divided into three types: initial layer, middle layer and deep layer, the initial layer is TOC≥ 3.5wt% or HM≥ 30 g·kg-1 , the middle layer is TOC = 1.0-3.5wt% or HM = 6-30 g·kg -1 , the deep layer is TOC <1.0wt% or HM <6 g·kg -1 The DNA extraction soil sample and chemical determination sample are taken out from the environment of-80℃, first placed in the environment of-20℃, then placed in the environment of 4℃ for step-by-step thawing, first the chemical determination sample is used to determine the total organic carbon content TOC or humus content HM of the sediment, the experimental method is determined according to the 'Determination of soil organic carbon potassium dichromate oxidation-spectrophotometric method' (HJ 615-2011), the content of organic carbon in the sediment is determined according to the potassium dichromate oxidation method. According to the organic carbon content or humus content determined by the chemical determination sample, the DNA extraction sample corresponding to the chemical determination sample is classified according to the classification standard we prepared, and the DNA sample is extracted according to the processing method of each sediment sample after classification.

[0021] S2: sampling and pretreating the sediment of the initial layer, adding beads to moisten and mix evenly, lysing and crushing to obtain a first supernatant; performing CTAB treatment and centrifugation on the first supernatant, then adding an organic phase to extract, and after clarification, centrifuging to obtain a second supernatant; combining the second supernatant on a column, and after washing, obtaining a DNA initial layer extraction solution; First, the initial layer sediment is treated: take 0.2 g of sediment sample in a 2 mL grinding tube, if the sample contains obvious carbonate, add 0.4 mL of 0.5 M HCl to wash for 30-60 s, then wash with 1.0 mL of nuclease-free water until the supernatant is neutral, use pH paper or a pH meter to confirm, and centrifuge in a high-speed centrifuge at 12,000-15,000 x g for 1-2 min to discard the wash. If there is no carbonate, this step can be omitted.

[0022] The initial layer lysis solution is configured in advance, and the formula is: 22.4 µL of 1.0 mol·L -1 Tris-HCl, 44.8 µL of 0.5 mol·L -1 EDTA, 94.5 µL of 6.0 mol·L -1 guanidine hydrochloride, 84 µL of 10%v / v Triton X-100, 1.4µL of 1.0 mol·L -1 DDT, 452.9 µL of nuclease-free water, mix evenly and adjust the pH to about 10.2, adjust the pH with 1.0 mol·L -1 NaOH or 1.0 mol·L -1 HCl micro titration and record.

[0023] Add 100 µL of 1.8 mm Zirconium beads, 600 µL of the first layer lysis solution and 5 µL of proteinase K into the centrifuge tube, vortex for 10 s to wet the sample, and incubate in a 65 °C water bath for 10 min. Put the grinding tube in a bead mill or tissue cell crusher for grinding, set the grinding intensity to 5 m / s, and grind for 30 s, pause for 30 s, and repeat for 2 cycles. After grinding, centrifuge at 12,000 x g for 2 min at room temperature in a high-speed centrifuge, and transfer all of the first supernatant to a 2-mL centrifuge tube, and record the volume as V1.

[0024] Add 100 g·L -1 Add CTAB to the supernatant at a volume ratio of 0.25:1 to V1, so that the final concentration of the supernatant is 2%. After adding, incubate at 65 °C for 10 min, centrifuge at 12,000 x g for 1 min at room temperature in a high-speed centrifuge, and transfer all of the supernatant to a 2-mL centrifuge tube, and record the volume as V2.

[0025] Add an equal volume of chloroform-isoamyl alcohol solution to the centrifuge tube, the ratio of chloroform-isoamyl alcohol solution is 26:1, invert and mix for 30-60 s, centrifuge at 12,000 x g for 1 min at room temperature in a high-speed centrifuge, transfer all of the supernatant to a 2-mL centrifuge tube, repeat the extraction operation for a total of 2 times to remove more organic inhibitors, and record the volume of the last transferred supernatant as V3.

[0026] The subsequent steps require the use of E.Z.N.A. Soil DNA Kit (HiBind Add 100 µL of cHTR Reagent to the clarified aqueous phase, vortex to mix, and stand at room temperature for 2 min, centrifuge at 13,000 x g for 2 min, and take the clarified supernatant to a new 2-mL centrifuge tube, if the supernatant color is still deep, repeat the operation once, and record the final obtained supernatant volume as V4.

[0027] Add an equal volume of XP1 Buffer to the centrifuge tube, vortex to mix; put a HiBind® DNA Mini Column in a new 2-mL centrifuge tube, add the just obtained supernatant to the HiBind® DNA Mini Column in several portions, each addition ≤ 700 µL, centrifuge at 12,000 x g for 1 min at room temperature, and discard the waste liquid until the liquid is completely transferred.

[0028] Place the just filtered HiBind® DNA Mini Column into a new 2 mL centrifuge tube, add 700 µL HBC Buffer, centrifuge at 12,000 x g for 1 min at room temperature, and discard the waste. The HBC Buffer needs to be diluted with isopropanol according to the instruction.

[0029] Add 700 µL DNA Wash to the HiBind® DNA Mini Column, centrifuge at 12,000 x g for 1 min at room temperature, and discard the waste. The DNA Wash needs to be diluted with absolute ethanol according to the instruction.

[0030] Centrifuge the HiBind® DNA Mini Column empty column at 12,000 x g for 2 min at room temperature to dry the column. Place the dried column into a new 2 mL centrifuge tube, add 50 µL Elution Buffer preheated to 65 °C to the center of the column membrane, incubate at room temperature for 5 min, centrifuge at 12,000 x g for 1 min at room temperature to elute the DNA, discard the HiBind® DNA Mini Column, and label the finished DNA for storage at -80 °C to reduce DNA degradation.

[0031] S3: Sample and pretreat the middle layer sediment, add lysis solution and incubate, then add beads and grind, centrifuge to obtain the third supernatant; perform CTAB treatment and centrifugation on the third supernatant, add an organic phase for extraction, and then perform ethanol precipitation and washing to obtain a liquid, which is combined on a column, washed, and then DNA in the middle layer extract is obtained; Process the middle layer sediment sample: first, prepare the middle layer lysis buffer, which has the following formula: 22.5 µL 1.0 mol·L -1 Tris-HCl, 45.0 µL 0.5 mol·L -1 EDTA, 100.0 µL 6.0 mol·L -1 Guanidine Hydrochloride, 75.0 µL 10% v / v Triton X-100, 7.5 mg PVPP solid, 507.5 µL enzyme-free water, mix well, and adjust the pH to about 10.0. Adjust the pH with 1.0 mol·L -1 NaOH or 1.0 mol·L -1 HCl titration in small amounts and record.

[0032] Take 0.3 g of sediment sample in a 2 mL centrifuge tube, add 100 µL of 2.0 mm zirconium beads, and add 5 µL of 0.5 mol·L-1 Sodium pyrophosphate, invert gently 2–5 min to initially disperse clay-organic complexes; add 650 µL of lysis buffer containing PPVP and 10 µL of proteinase K, vortex 10 s to wet the sample, and incubate in a 65 °C water bath for 10 min. Place the grinding tube in a bead beater or tissue cell disrupter and grind at a setting of 4.5 m / s for 30 s, pause for 30 s, and repeat for 2 cycles. After grinding, centrifuge the sample at 12,000 x g for 2 min at room temperature, transfer the entire supernatant to a 2-ml centrifuge tube, and record the volume as V5.

[0033] To the supernatant with a volume of V5, add 100 g·L -1 CTAB, add at a volume ratio of 0.11:1 to the volume V5 of the supernatant, after adding, incubate at 65 °C for 10 min, centrifuge at 12,000 x g for 1 min at room temperature, transfer the entire supernatant to a 2-ml centrifuge tube, and record the volume as V6.

[0034] To the supernatant with a volume of V6, add a chloroform-isoamyl alcohol solution at a volume ratio of 24:1 to V6, invert and mix for 30~60 s, centrifuge at 12,000 x g for 1 min at room temperature, transfer the entire supernatant to a 2-ml centrifuge tube, and record the volume of the supernatant at this time as V7.

[0035] To the supernatant with a volume of V7, add 1.0 mg·mL -1 LPA, 5.0 mol·L -1 NaCl and anhydrous ethanol, the volume ratio of LPA, NaCl, anhydrous ethanol, and water phase is 0.02:02:2.5:1, so that the final concentration is 20 µg·mL -1 Immediately invert and mix 6–8 times, precipitate at 4 °C for ≥ 2 h in the dark, centrifuge at 15,000 x g, 4 °C, for 5 min, discard the supernatant, wash the precipitate with 70% ethanol 500 µL twice, each time for 5 min, and centrifuge at 15,000 x g. Dry the precipitate at room temperature for a short time, about 3–8 min, until the surface is matte but not too dry, dissolve the precipitate in 10 mM Tris-Cl EB, record the dissolution volume V8, and add it to a new 2-ml centrifuge tube.

[0036] The E.Z.N.A. Soil DNA Kit (HiBind) is required in the following steps.

[0037] Add four times V8 volume of XP1 Binding Buffer, which has been diluted with an equal volume of isopropanol according to the instructions, to the centrifuge tube, vortex to mix, put the HiBind® DNA Mini Column into a new 2 mL centrifuge tube, add the supernatant obtained just now to the HiBind® DNA Mini Column in several portions, each portion not exceeding 700 µL, centrifuge at 12,000 x g at room temperature for 1 min, discard the waste liquid until the liquid is completely transferred, add 700 µL of HBC Buffer diluted with an equal volume of isopropanol, centrifuge at 12,000 x g for 1 min, discard the waste liquid, and transfer the HiBind® DNA Mini Column to a new 2 mL centrifuge tube.

[0038] Add 700 µL of DNA Wash Buffer, centrifuge at 12,000 x g for 1 min, discard the waste liquid, and repeat this operation once, wherein the DNA Wash Buffer needs to be diluted with four volumes of anhydrous ethanol. After the last washing, spin at 13,000 x g for 2 min to remove residual ethanol.

[0039] Transfer the HiBind® DNA Mini Column to a new 2 mL centrifuge tube, add 30 µL of Elution Buffer preheated to 65 ℃ to the center of the column membrane, stand for 2-5 min, centrifuge at 13,000 x g for 1 min, re-add the eluted liquid to the center of the column membrane, centrifuge at 13,000 x g for 1 min again to increase the elution rate, and store the extracted DNA at -80 ℃ to reduce DNA degradation.

[0040] S4: Sample the deep sediment, add lysis solution and zirconium beads to the shaker, shake gently, break and centrifuge to obtain a fourth supernatant; add an organic phase to the fourth supernatant for extraction, centrifuge to obtain a fifth supernatant; invert the magnetic beads used to mix, and the fifth supernatant is adsorbed by magnetic beads, washed with ethanol, and eluted to obtain a deep extraction of DNA; Process the deep sediment sample: first configure the deep lysis solution, the formula of which is 22.4 µL of 1.0 mol·L -1 Tris-HCl, 44.8 µL of 0.5 mol·L -1 EDTA, 105.3 µL of 6.0 mol·L -1Guanidine hydrochloride, 64.0 μΐ 10% v / v Triton X-100, 8.0 mg PVPP solid, 563.5 μΐ enzyme-free water, mix and adjust pH to approximately 9.8, adjust pH with 1.0 mol L -1 NaOH or 1.0 mol L -1 HCl micro titration and record.

[0041] Take 0.4 g sample in 2 mL centrifuge tube, add to grinding tube, sediment sample, zirconium beads, deep lysis buffer, 0.5 mol L -1 Sodium pyrophosphate, 0.5 mol L -1 EDTA and proteinase K, zirconium beads, deep lysis buffer, sodium pyrophosphate, EDTA, proteinase K to sample ratio 100 μΐ: 700 μΐ: 100 μΐ: 12 μΐ: 15 μΐ: 0.4 g, place on a shaker at room temperature or 4°C for 5-10 min at low speed, then grind in a bead beater or tissue cell disrupter, set grinding parameters to 3 m / s, 30 s, stop 30 s, cycle 2 times, centrifuge at 13,000 x g for 1-2 min, transfer supernatant to a new 2 mL centrifuge tube, record volume as V10. If the DNA content in the sediment is low, 3-5 parallel samples can be taken and mixed into one tube at this step of elution.

[0042] Add to the centrifuge tube a volume equal to V10 of chloroform-isoamyl alcohol solution in a ratio of 24:1, mix well by inverting for 30-60 s, centrifuge at room temperature in a high-speed centrifuge at 12,000 x g for 1 min, transfer all supernatant to a 2 mL centrifuge tube, record volume as V11.

[0043] Mix the AMPure XP tube well by inverting thoroughly at room temperature or shaking on a vortexer for 30 s, make sure the beads are completely and uniformly suspended before taking, do not take the supernatant of the precipitate; prepare 80% ethanol, prepare fresh each time, at the same time, place the EB in a water bath at 65°C to preheat; prepare the magnetic stand and place it on the workbench ready for use.

[0044] Calculate the volume of magnetic beads to be added as BeadsVol = 1.6 x V11, mix the AMPure XP tube well with a pipette, invert up and down for at least 30 s, quickly take out BeadsVol with a pipette gun, and add it directly to the centrifuge tube containing the sample with a volume of V11. Mix gently with a pipette by blowing up and down 10-15 times, make sure the beads and sample are in good contact. Let stand at room temperature for 10 min to allow the DNA to bind to the magnetic beads.

[0045] Place the tube in the magnetic stand and let it sit for 5 min until the solution is completely clear and the supernatant is transparent. Carefully pipette as much of the supernatant as possible without disturbing the bead and discard. Keep the tube in the magnetic stand and add 200 µL of 80% ethanol. Let it sit for 30 s, then discard the wash, again without disturbing the bead. Repeat the addition of 200 µL of 80% ethanol once. Remove as much of the visible alcohol as possible from the magnetic stand. Keep the tube in the magnetic stand and let it air dry for 2 min at room temperature. Observe the bead surface change from shiny wet to dull.

[0046] Remove the tube from the magnetic stand and add 30 µL of EB preheated to 65°C. Gently pipette 8-10 times to fully wet and dissolve the bound DNA. Let it sit for 2 min at room temperature. Place the tube in the magnetic stand and let it sit for 2-4 min until the solution is clear. Carefully transfer the clear liquid to a new nuclease-free storage tube. If a parallel sample was taken, this step is done first in the first tube to elute the DNA from the first tube. After elution, pipette the eluate directly into the second tube to perform the same elution step. After elution, pipette the eluate directly into the third tube to perform the elution, and so on until all samples are eluted. Label the extracted DNA and store it at -80°C to reduce DNA degradation.

[0047] Example 2: A method for high-altitude lake sediment ancient DNA stratified extraction, comprising: S1: first determine the total organic carbon content TOC or humus content HM of the sediment, according to the measured content, the sediment is divided into three layers, namely the first layer, the middle layer and the deep layer, the first layer is TOC≥ 3.5wt% or HM≥ 30 g·kg -1 , the middle layer is TOC=1.0–3.5wt% or HM=6–30 g·kg -1 , and the deep layer is TOC<1.0wt% or HM<6 g·kg -1 ; First, collect the required sediment sample from the high-altitude lake. After collection, cut and stratify immediately according to the specific length of the collected sediment and the needs of the experimental scheme design. Take the center soil sample of the sediment column as the DNA extraction soil sample, and place it in a 50mL centrifuge tube. The soil sample in the outer circle is placed in a sample bag and sealed as a chemical determination sample. Label each layer of the cut sample, such as the first layer of DNA extraction soil sample and the first layer of chemical determination sample. After sealing the DNA extraction soil sample and the chemical determination sample, store them in a -80°C temperature for freezing preservation, and extract as soon as possible.

[0048] According to the national second soil survey standard, the organic matter content is ≥ 30 g·kg -1For the "rich" or "very rich" level, usually found in the recent deposition of the surface layer, i.e. the first layer of soil samples, in the study of lake or river sediments, the surface layer TOC is usually > 3%, and here we set 3.5wt% as the upper threshold value, which can effectively distinguish the transition from the middle layer and ensure that the level has ecological distinction, so we set HM≥ 30 g·kg -1 and TOC>3.5wt% of soil samples belong to deep sediment samples. The organic matter of deep sediment has been highly mineralized, and the standard HM<6 g·kg -1 indicates that the organic matter is extremely poor, and TOC<1.0wt% corresponds to a low level of organic matter <1.7%, which is often found in the deep part of the sediment core column, indicating that long-term burial leads to the loss of organic carbon. Therefore, we set HM<6 g·kg -1 and TOC<1.0wt% of soil samples belong to deep sediment samples.

[0049] Therefore, according to the above description, the sediments are divided into three types: i.e. the first layer, the middle layer and the deep layer, the first layer is TOC≥ 3.5wt% or HM≥ 30 g·kg -1 , the middle layer is TOC=1.0–3.5wt% or HM=6–30 g·kg -1 , and the deep layer is TOC<1.0wt% or HM<6 g·kg -1 . The DNA extraction soil samples and chemical determination samples were taken out from the environment of-80℃, and then put into the environment of-20℃ and 4℃ for stepwise thawing. First, the total organic carbon content TOC or humus content HM of the sediment was determined using the chemical determination sample, and the experimental method was determined according to "Determination of Soil Organic Carbon Potassium Dichromate Oxidation-Spectrophotometric Method" (HJ 615-2011). The content of organic carbon or humus was determined according to the potassium dichromate oxidation method. According to the organic carbon content or humus content determined by the chemical determination sample, the DNA extraction sample corresponding to the chemical determination sample was classified according to the classification standard we proposed, and the DNA sample was extracted according to the processing method of each sediment sample after classification.

[0050] S2: Sampling and pretreating the first layer of sediment, adding beads to moisten and mix, lysing and crushing to obtain the first supernatant; CTAB treatment and centrifugation were performed on the first supernatant, and then organic phase extraction was added, and after clarification, centrifugation was performed to obtain the second supernatant; the second supernatant was combined and washed to obtain the DNA first layer extract; The first layer of sediment is treated first: 0.5 g of sediment sample is taken in a 2 mL grinding tube, if the sample contains obvious carbonates, 2.0 mL of 0.5 M HC1 is added to clean for 30-60 s, then 1.0 mL of nuclease-free water is added to wash until the supernatant is neutral, and the pH is confirmed with pH paper or a pH meter. The sample is centrifuged at 12,000-15,000 x g in a high-speed centrifuge for 1-2 min, and the wash is discarded. If there is no carbonate, this step can be omitted.

[0051] The first layer of lysate is configured in advance, and the formula is: 22.4 μL of 1.0 mol·L -1 Tris-HCl, 44.8 μL of 0.5 mol·L -1 EDTA, 94.5 μL of 6.0 mol·L -1 GuHCl, 84 μL of 10% v / v Triton X-100, 1.4 μL of 1.0 mol·L -1 DDT, 452.9 μL of nuclease-free water, mix well and adjust the pH to about 10.2, adjust the pH with 1.0 mol·L -1 NaOH or 1.0 mol·L -1 HCl titrate in small amounts and record.

[0052] Add 150 μL of 1.8 mm zirconium beads, 600 μL of the first layer of lysate, and 5 μL of proteinase K to the centrifuge tube, vortex for 10 s to wet the sample, and incubate in a 65°C water bath for 10 min. The grinding tube is placed in a bead mill or tissue cell crusher for grinding, with a grinding intensity of 5 m / s and a time of 30 s, a pause of 30 s, and a cycle of 2 times. After grinding, centrifuge at 12,000 x g at room temperature for 2 min, and transfer all the first supernatant to a 2 ml centrifuge tube, and the volume is recorded as V1.

[0053] Add 100 g·L-1CTAB to the supernatant, with a volume ratio of 0.25:1 to V1, to make the final concentration of the supernatant 2%. After adding, incubate at 65°C for 10 min, centrifuge at 12,000 x g at room temperature for 1 min, and transfer all the supernatant to a 2 ml centrifuge tube, and the volume is recorded as V2.

[0054] Add equal volume of chloroform-isoamyl alcohol solution to the centrifuge tube, the ratio of chloroform-isoamyl alcohol solution is 26:1, invert to mix for 30-60s, centrifuge at 12000xg for 1 min at room temperature, transfer all supernatant to a new 2ml centrifuge tube, repeat the extraction operation for 2 times to remove more organic inhibitors, the volume of the last transferred supernatant is recorded as V3.

[0055] The following steps need to use E.Z.N.A. Soil DNA Kit (HiBind) Add 100 μL cHTR Reagent to the clear aqueous phase, vortex to mix, stand at room temperature for 2 min, centrifuge at 13,000xg for 2 min, take the clear supernatant to a new 2ml centrifuge tube, if the supernatant color is still deep, repeat the operation once, and the final obtained supernatant volume is V4.

[0056] Add equal volume of XP1 Buffer to the centrifuge tube, vortex to mix; Put the HiBind® DNA Mini Column in a new 2ml centrifuge tube, add the just obtained supernatant to the HiBind® DNA Mini Column, the amount of each addition ≤ 700 μL, centrifuge at 12,000xg for 1 min at room temperature, discard the waste liquid until the liquid is completely transferred.

[0057] Put the just filtered HiBind® DNA Mini Column in a new 2ml centrifuge tube, add 700 μL HBC Buffer, centrifuge at 12,000xg for 1 min at room temperature, discard the waste liquid. The HBC Buffer needs to be diluted with isopropanol according to the instructions.

[0058] Add 700 μL DNA Wash to the HiBind® DNA Mini Column, centrifuge at 12,000xg for 1 min at room temperature, discard the waste liquid. The DNA Wash needs to be diluted with absolute ethanol according to the instructions.

[0059] Centrifuge the HiBind® DNA Mini Column empty column at 12,000 x g for 2 min at room temperature to spin down the column. Place the spun down column into a new 2 mL centrifuge tube, add 100 µL of Elution Buffer preheated to 65 °C to the center of the column membrane, incubate at room temperature for 5 min, centrifuge at 12,000 x g for 1 min at room temperature to elute the DNA, discard the HiBind® DNA Mini Column, and label the extracted DNA and store at -80 °C to reduce DNA degradation.

[0060] S3: Sample and pretreat the middle layer sediment, add lysis solution and incubate, then add beads and grind, centrifuge to obtain the third supernatant; perform CTAB treatment and centrifugation on the third supernatant, add an organic phase for extraction, and then perform ethanol precipitation and washing to obtain the liquid, which is combined on a column to obtain the DNA middle layer extract after washing; Process the middle layer sediment sample: first, prepare the middle layer lysis buffer, which has the following formula: 22.5 µL of 1.0 mol·L -1 Tris-HCl, 45.0 µL of 0.5 mol·L -1 EDTA, 100.0 µL of 6.0 mol·L -1 GuHCl, 75.0 µL of 10% v / v Triton X-100, 7.5 mg of PVPP solid, 507.5 µL of enzyme-free water, mix well, and adjust the pH to about 10.0 using 1.0 mol·L -1 NaOH or 1.0 mol·L -1 HCl, titrate in small amounts, and record.

[0061] Take 0.5 g of the sediment sample into a 2 mL centrifuge tube, add 150 µL of 2.0 mm zirconium beads, add 10 µL of 0.5 mol·L -1 sodium pyrophosphate, gently invert 2-5 min to preliminarily disperse the clay-organic complex; add 650 µL of lysis buffer containing PPVP and 10 µL of proteinase K, vortex for 10 s to wet the sample, and incubate in a 65 °C water bath for 10 min. Place the grinding tube in a bead mill or tissue cell crusher for grinding, set the grinding intensity to 4.5 m / s, and grind for 30 s, pause for 30 s, and repeat for 2 cycles. After grinding, centrifuge in a high-speed centrifuge at 12,000 x g for 2 min at room temperature, transfer all the supernatant to a 2 mL centrifuge tube, and record the volume as V5.

[0062] To the supernatant with a volume of V5, add 100 g·L -1CTAB, the ratio of the volume added to the volume of the supernatant V5 is 0.11:1, after adding, incubate at 65 °C for 10 min, centrifuge at 12000 x g in a high-speed centrifuge at room temperature for 1 min, transfer all the supernatant to a 2 ml centrifuge tube, and record the volume as V6.

[0063] Add a chloroform-isoamyl alcohol solution with a volume ratio of 24:1 to the supernatant with a volume of V6, mix well by inverting for 30-60 s, centrifuge at 12000 x g in a high-speed centrifuge at room temperature for 1 min, transfer all the supernatant to a 2 ml centrifuge tube, and record the volume of the supernatant at this time as V7.

[0064] Add 1.0 mg·mL -1 LPA, 5.0 mol·L -1 NaCl and anhydrous ethanol, the volume ratio of LPA, NaCl, anhydrous ethanol and water phase is 0.02:02:2.5:1, and the final concentration is 20 µg·mL -1 Immediately invert and mix well for 6-8 times, avoid light, precipitate at 4 °C for ≥ 2 h, centrifuge at 15,000 x g, 4 °C, 5 min, discard the supernatant, wash the precipitate with 70% ethanol 500 µL twice, 5 min each time, 15,000 x g centrifugation. Dry the precipitate at room temperature for a short time, about 3-8 min, until the surface is matte but not too dry, dissolve the precipitate in 10 mM Tris-Cl EB, record the dissolution volume V8 and add it to a new 2mL centrifuge tube.

[0065] The E.Z.N.A. Soil DNA Kit (HiBind) is required in the following steps.

[0066] Add four times the volume of V8 of XP1 Binding Buffer that has been diluted with an equal volume of isopropanol according to the instructions in the centrifuge tube, vortex to mix, put the HiBind® DNA Mini Column in a new 2mL centrifuge tube, add the supernatant obtained just now to the HiBind® DNA Mini Column in several portions, each addition not more than 700 µL, centrifuge at 12,000 x g at room temperature for 1 min, discard the waste liquid until the liquid is completely transferred, add 700 µL of HBC Buffer diluted with an equal volume of isopropanol, centrifuge at 12,000 x g for 1 min, discard the waste liquid, and transfer the HiBind® DNA Mini Column to a new 2mL centrifuge tube.

[0067] Add 700 µL DNA Wash Buffer, centrifuge at 12,000 x g for 1 min, discard the supernatant, and repeat this step once with the DNA Wash Buffer diluted 1:4 with absolute ethanol. After the final wash, spin down the column at 13,000 x g for 2 min to remove residual ethanol.

[0068] Transfer the HiBind® DNA Mini Column to a new 2 mL centrifuge tube, add 50 µL of Elution Buffer preheated to 65 ℃ to the center of the column membrane, let stand for 2–5 min, centrifuge at 13,000 x g for 1 min, reapply the eluted liquid to the center of the column membrane, and centrifuge at 13,000 x g for 1 min to increase the elution rate. Label the extracted DNA and store it at -80 ℃ to reduce DNA degradation.

[0069] S4: Sample the deep sediment, add lysis solution and zirconium beads to the shaker, break and centrifuge to obtain the fourth supernatant; add organic phase extraction to the fourth supernatant, centrifuge to obtain the fifth supernatant; invert the magnetic beads and mix evenly, adsorb the fifth supernatant with magnetic beads, wash with ethanol, and elute to obtain the DNA deep extraction liquid; Process the deep sediment sample: first, configure the deep lysis solution, the formula of which is 22.4 µL 1.0 mol·L -1 Tris-HCl, 44.8 µL 0.5 mol·L -1 EDTA, 105.3 µL 6.0 mol·L -1 GuHCl, 64.0 µL 10% v / v Triton X-100, 8.0 mg PVPP solid, 563.5 µL enzyme-free water, mix well and adjust the pH to about 9.8, adjust the pH with 1.0 mol·L -1 NaOH or 1.0 mol·L -1 HCl microtitration and record.

[0070] Take 0.7 g of the sample in a 2 mL centrifuge tube, add sediment sample, zirconium beads, deep lysis buffer, 0.5 mol·L -1 sodium pyrophosphate, 0.5 mol·L -1EDTA and proteinase K, zirconium beads, deep lysis buffer, sodium pyrophosphate, EDTA, proteinase K and sample ratio is 150 μL: 700 μL: 100 μL: 12 μL: 15 μL: 0.7g, placed on a shaker at room temperature or 4°C for 5-10 min, then placed in a bead mill or tissue cell crusher for grinding, the grinding parameters are set to 3 m / s, 30 s, stop 30 s, cycle 2 times, centrifuged at 13,000 x g for 1-2 min, take the supernatant to a new 2ml centrifuge tube, and the volume is V10. If the DNA content in the sediment is low, 3-5 parallel samples can be taken and mixed in one tube at this step.

[0071] Add an equal volume of V10 chloroform-isoamyl alcohol solution with a ratio of 24:1 to the centrifuge tube, mix well for 30-60s, centrifuge at 12000 x g at room temperature for 1 min in a high-speed centrifuge, and transfer all the supernatant to a 2ml centrifuge tube, and the volume is V11.

[0072] Mix the AMPure XP tube thoroughly at room temperature or place it in a vortex for 30s, make sure the beads are completely and uniformly suspended before use, do not take the supernatant of the precipitate; prepare 80% ethanol, prepare it immediately before use, and at the same time, place the EB in a water bath at 65°C for preheating; prepare the magnetic stand and place it on the workbench for standby.

[0073] Calculate the volume of magnetic beads added as BeadsVol = 1.6 x V11, mix the AMPure XP tube thoroughly with a pipette, upside down for at least 30s, quickly take out BeadsVol with a pipette gun, and add it directly to the centrifuge tube containing the sample with a volume of V11. Mix gently with a pipette for 10-15 times, make sure the beads and sample are in contact. Let it stand at room temperature for 10 min to allow the DNA to bind to the magnetic beads.

[0074] Place the tube in the magnetic stand and let it stand for 5 min until the solution is completely clear and the supernatant is transparent. Carefully aspirate the supernatant with a pipette and discard it without touching the bead mass; keep the tube in the magnetic stand, add 200 μL of 80% ethanol, stand for 30 s, then discard the wash, also without touching the bead mass; repeat the addition of 200 μL of 80% ethanol once. Remove all visible alcohol on the magnetic stand. Keep the tube in the magnetic stand and air dry at room temperature for 2 min, and observe the surface of the beads from wet bright to matte.

[0075] Remove the tube from the magnetic stand, add 50 µL of pre-warmed EB at 65°C, gently flick 8-10 times to fully wet the beads and dissolve the bound DNA, let stand at room temperature for 2 min, place the tube in the magnetic stand and let stand for 2-4 min until the solution is clear, carefully transfer the clear liquid to a new nuclease-free storage tube. If a parallel sample was taken, this step is done first on the first tube to elute the DNA from the first tube, after elution the eluate is directly added to the second tube to perform the same elution step, after elution is complete it is added to the third tube to perform the elution, and so on until all samples are eluted.

[0076] The extracted DNA is labeled and stored at -80°C to reduce DNA degradation.

[0077] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the specification and examples be considered as exemplary only.

Claims

1. A method for ancient DNA stratigraphic extraction of high-altitude lake sediments, characterized by, Comprise: S1: first determine the total organic carbon content TOC or humus content HM of the sediment, and according to the measured content, the sediment is divided into three layers, i.e. a first layer, a middle layer and a deep layer, wherein the first layer is TOC≥3.5wt% or HM≥30 g·kg -1 , the middle layer is TOC=1.0–3.5wt% or HM=6–30 g·kg -1 , and the deep layer is TOC < 1.0wt% or HM < 6 g·kg -1 ; S2: sample and pretreat the sediment of the initial layer, add beads to wet and mix, lyse and crush to obtain the first supernatant; CTAB treatment and centrifugation are performed on the first supernatant, then organic phase extraction is added, and the second supernatant is obtained after centrifugation; the second supernatant is combined with a column, and the DNA initial layer extract is obtained after washing; S3: sample and pretreat the sediment of the middle layer, add lysis solution and incubate, then add beads to grind, centrifuge to obtain the third supernatant; CTAB treatment and centrifugation are performed on the third supernatant, then organic phase extraction is added, and the DNA middle layer extract is obtained after ethanol precipitation and washing; S4: sample the sediment of the deep layer, add lysis solution and zirconium beads to a shaker, then crush and centrifuge to obtain the fourth supernatant; add organic phase extraction to the fourth supernatant, centrifuge to obtain the fifth supernatant; mix the magnetic beads used, and use the magnetic beads to adsorb the fifth supernatant, then wash and elute with ethanol to obtain the DNA deep layer extract.

2. The method for extracting ancient DNA from high-altitude lake sediments according to claim 1, characterized in that, Determine the organic carbon in the sediment according to the potassium dichromate oxidation-spectrophotometric method for determining soil organic carbon, and determine the content of humus in the sediment according to the potassium dichromate oxidation method.

3. The method for extracting ancient DNA from high-altitude lake sediments according to claim 1, characterized in that, In S2, the sediment of the initial layer is treated, specifically including: Take sediment sample in a grinding tube, if the sample contains obvious carbonate, add 0.5 mol·L -1 HCl clean for 30-60 s, then wash with nuclease-free water until the supernatant is neutral, centrifuge and discard the washing liquid; if there is no carbonate, no treatment is required.

4. The method for extracting ancient DNA from high-altitude lake sediments according to claim 1, wherein, In S2, the beads are added to wet and mix, lyse and crush to obtain the first supernatant, specifically including: Add zirconium beads, initial layer lysis solution and proteinase K to the grinding tube, vortex to wet the sample, the ratio of zirconium beads, initial layer lysis solution, proteinase K and sample is (100-150) µL:600 µL:5 µL:(0.2-0.5) g, place the tube in a 65°C water bath incubator, then grind in a bead mill or tissue cell crusher, centrifuge after grinding, and transfer all the first supernatant to a centrifuge tube, and the volume is recorded as V1.

5. The method for extracting ancient DNA from high-altitude lake sediments according to claim 1, characterized in that, In S2, the first supernatant is treated with CTAB and centrifuged, then organic phase extraction is added, and the second supernatant is obtained after centrifugation, and the DNA initial layer extract is obtained after the second supernatant is combined with a column and washed, specifically including: 1) To the first supernatant, 100 g-L -1 CTAB was added at a ratio of 0.25:1 to the volume of the first supernatant, V1, and incubated in a water bath at 65 °C, centrifuged and the entire second supernatant was transferred, the volume of which was recorded as V2; 2) Add an equal volume of chloroform-isoamyl alcohol solution to the centrifuge tube, mix well by inverting, centrifuge and transfer all the supernatant to a centrifuge tube, repeat the extraction, and the volume of the second supernatant transferred finally is recorded as V3; 3) Add cHTR Reagent to the clarified aqueous phase of the second supernatant with a volume of V3 at a ratio of (0.2-0.5) g:100 µL to the sample mass, vortex to mix, stand at room temperature and centrifuge, take the clarified supernatant to a new tube, and record the volume as V4; 4) Add an equal volume of XP1 Buffer to the centrifuge tube, vortex to mix; put the HiBind® DNA Mini Column adsorption column in a new centrifuge tube, add the supernatant with a volume of V4 to the column in portions, each addition amount ≤ 700µL, centrifuge and discard the waste liquid until the liquid transfer is completed; 5) Put the just filtered HiBind® DNA Mini Column into a new tube, add (0.2-0.5) g:700 µL HBC Buffer, centrifuge and discard the waste, then add (0.2-0.5) g:700 µL DNA Wash to the column, centrifuge and discard the waste; 6) After centrifuging the empty column, put it into a new centrifuge tube, add 50-100 µL Elution Buffer preheated to 65℃ to the center of the column membrane, incubate, centrifuge to elute the DNA, discard the adsorption column to obtain the DNA eluent, and store the DNA eluent at -80℃.

6. The method for extracting ancient DNA from high-altitude lake sediments according to claim 1, wherein, In S3, the middle layer of the sediment is sampled and pretreated, lysis solution is added and incubated, then beads are ground, the third supernatant is centrifuged, and the specific steps include: Add the sediment sample, zirconium beads, and sodium pyrophosphate to the grinding tube, gently invert, then add the lysis buffer containing PPVP and protease K, vortex to mix, and then incubate in a water bath, then put it into a bead mill or a tissue cell crusher for grinding, after completion, centrifuge and transfer all the third supernatant to a centrifuge tube, and record the volume as V5; the ratio of zirconium beads, sodium pyrophosphate, lysis solution, protease K, and sample is (100-150) µL:(5-10) µL:650 µL:10 µL:(0.3-0.5) g.

7. The method for extracting ancient DNA from high-altitude lake sediments according to claim 1, wherein, In S3, the third supernatant is treated with CTAB and centrifuged, organic phase extraction is added, then ethanol precipitation and washing are performed, and the obtained liquid is combined with the column, and the DNA middle layer extract is obtained after washing, and the specific steps include: 1) To the third supernatant was added 100 g-L -1 CTAB, at a ratio of the volume of CTAB to the volume of the third supernatant V5 of 0.11:1, incubated in a 65 °C water bath, centrifuged and the entire second supernatant was transferred, the volume of which was recorded as V6; 2) Add an equal volume of chloroform-isoamyl alcohol solution to the supernatant, invert to mix, and centrifuge, then transfer all the third supernatant to a centrifuge tube, and record the volume as V7; 3) Add 1.0 mg mL -1 LPA, 5.0 mol L -1 NaCl and anhydrous ethanol, LPA, NaCl, anhydrous ethanol and water phase volume ratio is 0.02:02:2.5:1, after mixing evenly in the dark at 4°C for ≥2 h, centrifugal after the supernatant, 70% ethanol wash the precipitate twice and centrifugal, room temperature short time dry the precipitate to the surface matte, dissolve the precipitate in Tris-Cl EB, record the volume V8 after dissolution and add it to a new centrifugal tube; 4) Add four times the volume of V8 to the centrifuge tube XP1 Binding Buffer, vortex to mix, and record the volume as V9, put the HiBind® DNA Mini Column adsorption column into a new centrifuge tube, add the supernatant with a volume of V9 to the adsorption column in several times, each addition amount is not more than 700 µL, centrifuge and discard the waste until the liquid is completely transferred, add (0.3-0.5) g:700 µL HBC Buffer, centrifuge and discard the waste, and transfer the adsorption column to a new centrifuge tube; 5) Add (0.3-0.5) g sample: 700 µL DNA Wash Buffer, centrifuge and discard the supernatant and repeat once, then centrifuge and empty the column, transfer the column to a new centrifuge tube, add (0.3-0.5) g sample: 30-50 µL preheated Elution Buffer to the center of the column membrane, centrifuge after standing to obtain the first eluent, re-add the first eluent to the center of the column membrane, centrifuge again to obtain the final DNA extract, and obtain the DNA middle layer extract, and store the DNA middle layer extract at -80°C.

8. The method for extracting ancient DNA from high-altitude lake sediments according to claim 1, wherein, In S4, the deep sediment is sampled, the lysis solution and zirconium beads are placed on a shaker for light shaking, then broken and centrifuged to obtain a fourth supernatant, which specifically includes: The sediment sample, zirconium beads, deep lysis buffer, 0.5 mol·L -1 Sodium pyrophosphate, 0.5 mol·L -1 EDTA and proteinase K, the ratio of zirconium beads, deep lysis buffer, sodium pyrophosphate, EDTA, proteinase K to the sample is (100-150) μL:700 μL:100 μL:12 μL:15 μL:(0.4~0.7) g, the grinding tube is placed on the shaker for low-speed light shaking, and then placed in the bead mill or tissue cell crusher for grinding, centrifuged and the fourth supernatant was obtained.

9. The method for extracting ancient DNA from high-altitude lake sediments according to claim 1, wherein, In S4, the fourth supernatant is added with an organic phase extraction, and the fifth supernatant is obtained by centrifugation. The magnetic beads are mixed by inverting, the supernatant is adsorbed by magnetic beads, ethanol is washed and eluted to obtain the DNA deep extract, which specifically includes: 1) Add an equal volume of chloroform-isoamyl alcohol solution to the centrifuge tube as the volume V10 of the fourth supernatant, mix well by inverting, and then centrifuge to transfer all the second supernatant to a new tube, and the volume is V11; 2) Mix the AMPure XP tube thoroughly, prepare 80% ethanol, calculate the volume of magnetic beads to be added BeadsVol = 1.6 x V11, take out the volume of magnetic beads BeadsVol to be added, and directly add it to the centrifuge tube containing the supernatant with a volume of V11, mix well by blowing up and down, and then stand at room temperature; 3) Place the tube in the magnetic stand and stand until the solution is completely clear, then discard the supernatant. Keep the tube on the magnetic stand, add 80% ethanol, and the ratio of magnetic beads to 80% ethanol is BeadsVol:200 µL. After standing, the washing solution is aspirated, and the washing is repeated, and the tube is dried at room temperature; 4) Take the tube off the magnetic stand, add (0.4-0.7) g sample: (30-50) µL preheated EB, blow and stand at room temperature, place the tube in the magnetic stand until the solution is clear, and then transfer the clear liquid to a new storage tube. If a parallel sample was taken during sampling, first add preheated EB to the first tube to elute the DNA in the first tube, then aspirate the eluent and add it to the second tube for the same elution step, until all samples are eluted, and the DNA deep extract is obtained. The DNA deep extract is stored at -80°C.

10. The method for extracting ancient DNA from high-altitude lake sediments according to claim 1, wherein, The primary lysis solution used in the lysis in S2 includes: Tris-HCl, EDTA, guanidine hydrochloride, Triton X-100, DDT, and enzyme-free water, 1.0 mol / L -1 Tris-HCl: 0.5 mol / L -1 EDTA: 6.0 mol / L -1 Guanidine hydrochloride: 10% v / v Triton X-100: 1.0 mol / L -1 DDT: Enzyme-free water = 16:32:67.5:60:1:323.5, liquid unit is µL, mix well and adjust the pH to 10.2, adjust the pH with 1.0 mol / L -1 NaOH or 1.0 mol / L -1 HCl is titrated in small amounts and recorded; The middle layer lysis solution used in S3 cracking includes: Tris-HCl, EDTA, Guanidine-HCl, Triton X-100, PVPP, enzyme-free water, 1.0 mol / L -1 Tris-HCl: 0.5 mol / L -1 EDTA: 6.0 mol / L -1 Guanidine-HCl: 10% v / v Triton X-100: PVPP: Enzyme-free water = 9: 18: 40: 30: 3: 203, liquid unit is µL, solid unit is mg, mix well and adjust pH to 10.0, adjust pH with 1.0 mol / L -1 NaOH or 1.0 mol / L -1 Titrate a small amount of HCl and record; The deep lysis solution used in cleavage in S4 includes: Tris-HCl, EDTA, Guanidine-HCl, Triton X-100, PVPP, enzyme-free water, 1.0 mol·L -1 Tris-HCl: 0.5 mol·L -1 EDTA: 6.0 mol·L -1 Guanidine-HCl: 10% v / v Triton X-100: PVPP: enzyme-free water = 2.8: 5.6: 13.2: 8: 1: 70.4, liquid units in µL, solid units in mg, mix well and adjust pH to 9.8, adjust pH with 1.0 mol·L -1 NaOH or 1.0 mol·L -1 HCl titrate in small amounts and record.