A method for extracting DNA from difficult marine biological samples and constructing libraries suitable for third-generation sequencing

Abstract

The present invention relates to a method for extracting and constructing a library of difficult samples of marine organisms suitable for three-generation sequencing, including using an optimized CTAB method to perform a crude extraction of DNA, followed by adding a CTAB secondary lysate and RNase and proteinase K to fully remove RNA and other impurities that may be carried, and further using a special PEG / NaCl / magnetic bead system to remove low-solubility impurities, especially at A260, there is also an "invisible" impurity with absorbance, and finally, the present invention uses a specific concentration of PEG / NaCl system to enrich 10-20kb fragments for the construction of HiFi libraries in three-generation sequencing, achieving better results. Compared to the data output of basically 0 before optimization, when the DNA extraction and library construction method of the present invention performs three-generation sequencing, the data output can be as high as 1000Gb, with broad application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a method for extracting DNA from difficult marine biological samples and constructing a library suitable for third-generation sequencing. Background Art

[0002] Among the genomic DNA extraction from animal tissues, the extraction of DNA from marine organisms such as shrimps and crabs is the most difficult, and muscle tissue is more difficult to extract. Marine shrimps and crabs are arthropods that live in the ocean and belong to the arthropod soft shell class. There are many species. However, due to their special growth environment and their own characteristics of high protein content and astaxanthin and chitin, their tissue composition is relatively complex and DNA is difficult to extract. Sea cucumber is a general term for organisms of the class Holothuroidea of ​​the phylum Echinodermata. Sea cucumbers have existed since the Cambrian period more than 600 million years ago. They have a soft body and some sea cucumbers have warty feet around their bodies. They have been widely known as precious medicinal materials and nourishing ingredients since ancient times. Sea cucumbers are not only rich in protein, essential amino acids for the human body, various trace elements, vitamins, etc., but also contain many biologically active substances, such as sea cucumber polysaccharides, sea cucumber saponins, collagen, sea cucumber peptides and cerebrosides, etc. These active substances have good effects in anti-tumor, anti-fungal, anti-coagulation, lowering blood pressure, lowering blood lipids and enhancing the body's immune function.

[0003] The third-generation sequencing technology represented by PacBio has the characteristics of long read length and high throughput. At the same time, the third-generation sequencing platform also puts forward higher requirements for the quality of DNA. However, the fragments obtained by traditional DNA extraction technology are relatively short, only about 50Kb, and the purity is poor, which cannot meet the requirements of the third-generation sequencing platform for DNA extraction quality. Studies have found that even if the quality of DNA fragments obtained by traditional extraction methods meets the requirements of OD260 / 280, OD260 / 230, concentration, etc., they cannot achieve good sequencing results in actual machine third-generation sequencing, and even no data output, because there are some contaminants that are invisible to the ultraviolet spectrum. In short, whether it is PaBio's latest Revio platform or Oxford Nanopore's PromethION platform, they have extremely high requirements for DNA quality. However, marine organisms have many metabolites. Even if the extracted DNA meets the platform requirements, DNA samples obtained by some extraction methods cannot achieve good sequencing results. The reaction affects the polymerase activity on the Revio platform, ultimately resulting in low sequencing output for a single cell; the nanopore sequenced on the PromethION platform rapidly inactivates and has no activity, ultimately resulting in low sequencing output. Summary of the invention

[0004] In order to solve the above problems, the present invention aims to develop a method for extracting and constructing a library of difficult marine biological samples suitable for third-generation sequencing. Specifically, it includes: homogenizing the sample tissue, adding a specific enzyme mixture (chitinase, lipase, phospholipase) to permeabilize the cell membrane, separating and purifying the cell nucleus, and then lysing it through the modified CTAB and precipitation method. After obtaining the crude DNA, it is subjected to secondary CTAB purification and RNase and proteinase K re-treatment, followed by removal of low-solubility impurities and magnetic bead purification. The final DNA has greatly improved data output in actual machine sequencing. In addition, a special NaCl / PEG system is used for fragment screening in the construction of the third-generation sequencing library, which effectively improves data output.

[0005] The present invention provides a method for extracting DNA from difficult marine biological samples and constructing a library suitable for third-generation sequencing, comprising the following steps:

[0006] S1. Processing of marine biological samples.

[0007] S2. Crude extraction and purification of DNA:

[0008] A. Add CTAB lysis buffer with the composition of 50-150mM Tris, 10-30mM EDTA, 2% w / v CTAB, 1.2M NaCl, proteinase K, and RNase, add equal volume of chloroform and isoamyl alcohol for extraction, invert to mix, centrifuge to precipitate impurities, aspirate the supernatant and add one to three times the volume of CTAB precipitation buffer with the composition of 50-150mM Tris, 10-30mM EDTA, 2% w / v CTAB, mix, centrifuge to obtain particle precipitation, wash with ethanol, centrifuge to remove supernatant, dry and dissolve to obtain crude DNA;

[0009] B. Add the obtained crude DNA to CTAB secondary lysis buffer, which consists of Tris 100mM, EDTA 20mM, CTAB 5% w / v, NaCl 1.2M, 1% PVP, pH=8, add RNase and proteinase K for incubation, add an equal amount of chloroform and isoamyl alcohol, invert and mix, centrifuge to remove the supernatant, add sodium acetate and mix evenly, then precipitate the DNA with an equal volume of isopropanol, collect the DNA by centrifugation, remove the supernatant, and wash with ethanol to obtain the purified DNA;

[0010] S3. Further purification of DNA:

[0011] Add 1V of pollutant precipitant to the DNA solution, wherein the components of the pollutant precipitant are 0.5-1M NaCl, 5-15% PEG 6000, Tris-HCl (pH=8), EDTA (pH=8) and magnetic beads, mix thoroughly, place on a magnetic rack for static adsorption, add the supernatant after the magnetic beads are adsorbed to a new test tube, add the prepared magnetic bead solution until the final DNA:magnetic bead volume ratio is 1:1, adjust the PEG and NaCl concentrations to NaCl=2M, PEG 6000=20-30%, and then perform magnetic bead purification;

[0012] S4, construction of HiFi library;

[0013] S5, sequencing.

[0014] Furthermore, in step S4, a 1 M NaCl, 0.5 mM EDTA, 10 mM Tris-HCl, 15% PEG 6000 system is used to enrich fragments of a specific size.

[0015] Further, step S1 is: take about 0.5g of tissue and cut it into blocks less than 3mm on a clean culture dish placed on ice; add 3ml of pre-cooled HB buffer, the ingredients of the HB buffer are 5-15mM Tris, 5-15mMEDTA, 5-20mM MgCl2, 0.1% NP 40, homogenize at 20000-30000rpm 4-5 times, stop for 40s each time for 20s to avoid heating, add pre-cooled HB buffer and specific enzyme mixture to 5ml, mix well, the specific enzyme mixture contains chitinase, lipase and phospholipase, centrifuge at 800g for 5 minutes at 4°C, remove the supernatant, add 3ml of HB buffer, mix well with a wide-mouth pipette tip, wash the cell nucleus, and centrifuge at 800g for 10 minutes.

[0016] Furthermore, in step S2, twice the volume of CTAB precipitation buffer is added.

[0017] Further, step S2 is:

[0018] A. Add CTAB lysis buffer with the composition of 50-150mM Tris, 10-30mM EDTA, 2% w / v CTAB, 1.2M NaCl and 25-75μL proteinase K and incubate for 1-2 hours, add 0.1%-1% volume of RNase and incubate for 0.5-1 hour, add an equal volume of chloroform isoamyl alcohol to extract, invert to mix, centrifuge at 5000g for 10 minutes to precipitate impurities, aspirate the supernatant and add one to three times the volume of CTAB precipitation buffer, whose composition is 50-150mM Tris, 10-30mM EDTA, 2% w / v CTAB, 55℃ for 30min, mix, centrifuge at 16000g for 5 minutes to obtain particle precipitation, wash with 70% ethanol, centrifuge at 16000g, remove supernatant, dry, dissolve in EB to obtain crude DNA;

[0019] B. Add the obtained crude DNA into CTAB secondary lysis buffer, whose components are Tris 100mM, EDTA 20mM, CTAB 5% w / v, NaCl 1.2M, 1% PVP, pH=8, and add 5μL RNase at the same time, incubate for 1 hour, then add 5-20μL proteinase K, incubate for 1 hour, add an equal amount of chloroform isoamyl alcohol, mix by inversion, centrifuge at 5000g for 10 minutes, aspirate the supernatant, add 0.1 times the volume of 3M sodium acetate and mix well, then precipitate the DNA with an equal volume of isopropanol, collect the DNA by centrifugation at 5000g for 10 minutes, remove the supernatant, wash the precipitate twice with 70% ethanol, and dissolve it to obtain the purified DNA.

[0020] Further, the magnetic bead purification in step S3 is specifically as follows: the DNA sample to be purified is mixed with the magnetic bead suspension in a volume ratio of 1:1, and incubated at room temperature for 15 minutes; after being separated instantly, it is allowed to stand for several minutes until all the magnetic beads are adsorbed on the magnetic rack, and then aspirated and temporarily stored in a new EP tube; freshly prepared 75% ethanol is added to the EP tube, and it is allowed to stand for 30-60 seconds, and the supernatant is aspirated and discarded; the 75% ethanol washing is repeated once; the EP tube is separated instantly, and then the EP tube is put back on the magnetic rack; all residual ethanol is aspirated; it is allowed to stand in a metal bath at 37°C for 30-60 seconds to allow the ethanol in the EP tube to completely evaporate; an appropriate amount of EB is added to the EP tube. Buffer, flick to completely resuspend the magnetic beads; place the EP tube in a 37℃ metal bath and incubate for 1 hour to elute the DNA. Since the magnetic beads are very easy to settle, during the incubation period, flick the EP tube every 5-10 minutes to fully resuspend the magnetic beads; place the EP tube on the magnetic rack and let it stand for several minutes until all the magnetic beads are adsorbed on the magnetic rack. Then, aspirate the supernatant containing DNA from the EP tube, wash twice with 70% ethanol, dry thoroughly, and dissolve with EB.

[0021] Furthermore, the size of the magnetic beads used in the magnetic bead purification is 10 μm, and 0.3% Tween-20 is added.

[0022] Furthermore, the platform used for sequencing in step S5 is the Revio platform or the PromethION platform.

[0023] The present invention also provides the application of any of the above methods in DNA extraction and / or third-generation sequencing.

[0024] Beneficial effects:

[0025] 1. In the conventional CTAB extraction method in the prior art, CTAB salt solution is only added in the initial lysis step, and the polarity difference formed by isopropanol and water subsequently causes the DNA to precipitate naturally. The present invention adopts an optimized CTAB extraction method based on DNA precipitation, which uses CTAB to form a complex with proteins and polysaccharides in a high-concentration ion solution, and can bind to nucleic acids in a low-concentration ion solution. It first uses a high-concentration ion environment to remove impurities, and then creates a low-ion condition to precipitate DNA, which effectively increases the DNA concentration, is simple to operate, non-toxic and harmless, and avoids the introduction of new impurities.

[0026] 2. The present invention selects to add CTAB secondary lysis solution as well as RNase and proteinase K to fully remove RNA and other possible impurities. The results show that although the total amount of DNA obtained by the optimized method is reduced, the overall purity is higher. The OD value before optimization (conventional CTAB method) is poor, and it is significantly better after optimization.

[0027] 3. The present invention adopts a special PEG / NaCl / magnetic bead system to remove low-solubility impurities, that is, at low NaCl / PEG concentrations, the contaminants are first precipitated onto the magnetic beads, and then by increasing the NaCl / PEG concentration and adding more magnetic beads, DNA can be precipitated from the remaining supernatant. Therefore, contaminants with high affinity for SPRI magnetic beads and lower solubility than DNA can be removed from the solution, significantly improving the output of sequencing data.

[0028] 4. The method for extracting DNA from difficult marine biological samples suitable for third-generation sequencing described in the present invention can remove "invisible" impurities, such as impurities with absorbance at A260, and thereby obtain high-purity long-fragment DNA with a DNA length greater than 150 kb, thereby significantly improving the output of sequencing data.

[0029] 5. The present invention uses a PEG / NaCl system of a specific concentration to enrich 10-20 kb fragments for the construction of HiFi libraries in third-generation sequencing, achieving better results.

[0030] 6. When the optimized DNA extraction and library construction method described in the present invention is used for third-generation sequencing, the data output is as high as 1000Gb, while the output before optimization is basically 0. That is, by optimizing the extraction method and library construction method, the sequencing data output is significantly improved, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 This is a cell image after DAPI staining. The cell nucleus appears blue in the dark field, indicating that a relatively pure cell nucleus can be obtained. In the bright field, some cell fragments can be seen, which can be removed through subsequent lysis treatment and other operations.

[0033] Figure 2 This is a direct current 1H electrophoresis: the first and second left images are markers, the third left image is crude DNA, and the fourth left image is DNA after adding C TAB secondary lysis buffer, RNase and proteinase K.

[0034] Figure 3 This is pulsed-field 14H electrophoresis: the first on the right is the Marker (50Kb ladder), the first on the left is the crude DNA, and the second on the left is the DNA after adding CTAB secondary lysis buffer, RNase and proteinase K.

[0035] Figure 4 This is the sequencing data output diagram (Revio platform) of the method before optimization (Comparative Example 1).

[0036] Figure 5 This is the output diagram of sequencing data after the optimized method (Revio platform). DETAILED DESCRIPTION

[0037] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in the present invention should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0038] Example 1 DNA extraction from difficult marine biological samples

[0039] S1. Processing of Difficult Marine Biological Samples

[0040] Take sea cucumber as an example, take about 0.5g tissue, cut into pieces less than 3mm on a clean culture dish placed on ice; add pre-cooled 3ml HB buffer (10mM Tris, 10mM EDTA, 10mM MgCl2, 0.1% NP 40), homogenize with a tissue homogenizer, homogenize at 25000rpm 4-5 times, 20s each time and stop for 40s to avoid heating. Add pre-cooled HB buffer and specific enzyme mixture to 5ml, mix well, the specific enzyme mixture contains chitinase, lipase and phospholipase. Centrifuge at 800g for 5 minutes at 4℃. The precipitate after centrifugation includes cell nuclei, and the supernatant contains cytoplasm, cell membrane and mitochondria. Add 3ml HB buffer, blow and mix with a wide-mouth pipette tip, wash the cell nuclei, and centrifuge at 800g for 10 minutes.

[0041] The results are as follows Figure 1 As shown, the cell nucleus stained with DAPI appears blue under dark field, and it can be seen that the obtained cell nucleus is relatively pure. Some residual cell fragments can be seen under bright field, and these impurities can be removed through subsequent processing and other operations.

[0042] S2. Crude extraction and preliminary purification of DNA

[0043] Different buffers and precipitants have different affinities for different pollutants, such as NaCl / PEG, isopropanol, ethanol or CTAB, which show different affinities for precipitating different pollutants. The present invention adopts an optimized CTAB extraction method based on DNA precipitation for lysis, specifically: obtaining a nuclear precipitate, utilizing the property of CTAB to combine proteins and polysaccharides in a high-concentration ion solution to form a complex, adding 5ml CTAB lysis solution (100mM Tris, 20mM EDTA, 2% w / v CTAB, 1.2M NaCl) and 50μL proteinase K (20mg / mL) 30rpm rotation and incubation at room temperature for 1.5 hours, adding 0.5% volume of RNase 30rpm rotation and incubation at room temperature for 0.5 hours, adding an equal volume of chloroform isoamyl alcohol for extraction, inverting and mixing, centrifuging at 5000g for 10 minutes, and precipitating impurities. Further, taking advantage of the ability of CTAB to bind nucleic acids in low-concentration ion solutions, aspirate the supernatant and add one to three volumes of CTAB precipitation buffer (100mM Tris, 20mM EDTA, 2% w / v CTAB), incubate at 55°C for 30min, rotate to mix (white crystals of CTAB-DNA complex can be observed floating in the tube), and centrifuge at 16000g for 5 minutes to obtain particle precipitation. Wash with 70% ethanol at room temperature for 15 minutes to remove excess salt and CTAB, centrifuge at 16000g for 5 minutes, and carefully remove the supernatant without disturbing the particles. Dry for 10-15min to remove ethanol, dissolve in 500μL EB to obtain crude DNA.

[0044] In the prior art, the general CTAB extraction method only adds CTAB salt solution in the initial lysis step, and then the polarity difference formed by isopropanol and water causes the DNA to precipitate naturally. The present invention adopts an optimized CTAB extraction method based on DNA precipitation, which uses CTAB to form a complex with proteins and polysaccharides in a high-concentration ion solution, and can bind to nucleic acids in a low-concentration ion solution. It first uses a high-concentration ion environment to remove impurities, and then creates a low-ion condition to precipitate DNA, which effectively increases the DNA concentration, is simple to operate, non-toxic and harmless, and avoids the introduction of new impurities.

[0045] The results are shown in Table 1. The results show that the optimized CTAB extraction method based on DNA precipitation according to the present invention can effectively precipitate DNA compared with the conventional CTAB method (Comparative Example 1), thereby obtaining a product with higher purity, and achieved better results in terms of OD260 / 280, OD260 / 230, purity, etc., among which a 2-fold volume of CTAB solution is the best.

[0046] Table 1

[0047]

[0048] Furthermore, the applicant also found that the addition of RNase to the lysis buffer in this optimization method cannot completely remove impurities such as RNA. Experiments have shown that residual RNA and other impurities that may be carried in crude DNA will lead to a low digestion rate for library construction, thereby reducing the sequencing output on the machine.

[0049] In order to fully remove RNA and other possible impurities, and to improve the data output during the third generation sequencing, the present invention selects to add CTAB secondary lysis solution and RNase and proteinase K for further treatment: 500μL CTAB secondary lysis buffer (Tris = 100mM, EDTA = 20mM, CTAB 5% w / v, NaCl = 1.2M, 1% PVP, pH 8), which increases the concentration of CTAB compared to the aforementioned CTAB lysis solution to better remove impurities such as polysaccharides. At the same time, 5μL RNase (100U / μL) was added to the 500μL crude DNA solution, incubated at 400rpm on a horizontal shaker at room temperature for 1 hour, followed by adding 10μL proteinase K (20mg / mL), and incubated at 400rpm on a horizontal shaker at room temperature for 1 hour. Add an equal amount of chloroform: isoamyl alcohol (24: 1) and mix by inversion. Centrifuge at 5000g for 10 minutes, and carefully place the upper aqueous phase into a new 15ml tube. To recover DNA from the aqueous phase, 0.1 volume of 3M sodium acetate (pH-5.2) was added to the aqueous phase and mixed well. Then, an equal volume of isopropanol was used to precipitate the DNA (when isopropanol is used to precipitate nucleic acids, the high concentration of salt will cause a large amount of polysaccharides to exist in the solution, thereby achieving the effect of removing polysaccharides). The DNA was collected by centrifugation at 5000g for 10 minutes. The supernatant was removed and the precipitate was washed twice with 70% freshly prepared ethanol. The residual alcohol was removed and 200 μL EB was added to dissolve. The results are shown in Table 2 and Figure 2-3 shown.

[0050] Table 2

[0051]

[0052] The results showed that although the total amount of DNA obtained by the optimized method decreased, the overall purity was higher, which was reflected in the OD260 / 280, OD260 / 230 and NC / QC indicators. The OD value before optimization (conventional CTAB method) was poor, but it was significantly better after optimization.

[0053] S3. Further purification of DNA

[0054] A. Removal of low-solubility impurities and magnetic bead purification

[0055] The applicant found that different buffers and precipitants have different affinities for different contaminants, such as CTAB, ethanol, NaCl / PEG, isopropanol, etc., so in theory, a cleaner DNA solution can be obtained by preferentially precipitating impurities. Specifically, when HMW DNA remains in the solution, at low NaCl / PEG concentrations, the contaminants are first precipitated onto the magnetic beads, and then by increasing the NaCl / PEG concentration and adding more magnetic beads, the DNA can be precipitated from the remaining supernatant, so contaminants with high affinity for SPRI magnetic beads and low solubility can be removed from the solution.

[0056] First, add pollutant precipitants (Tris-HCl (pH = 8), EDTA (pH = 8), NaCl, PEG, magnetic beads (1 μm)) of different concentrations (specific groups are shown in Table 3) at 1:1 v / v to the DNA solution, shake the test tube horizontally or gently move it for 5 minutes, mix it thoroughly, place it on a magnetic rack, and let it stand for adsorption. Add the supernatant after the magnetic beads are adsorbed to a new test tube, add the prepared magnetic bead solution, until the final DNA: magnetic beads ratio is 1:1 v / v, and adjust the PEG and NaCl concentrations to NaCl = 2M, PEG6000 = 25%. Then, perform magnetic bead purification: mix the DNA sample to be purified with the magnetic bead suspension at a ratio of 1:1 v / v, and gently tap the EP tube to mix them thoroughly; incubate the thoroughly mixed system at room temperature for 15 minutes; after a split second, place the EP tube containing the reaction system on a magnetic rack, let it stand for several minutes until all the magnetic beads are adsorbed on the magnetic rack, then aspirate and temporarily store it in a new EP tube; add freshly prepared 75% ethanol to the EP tube, let it stand for 30-60 seconds, aspirate and discard the supernatant; repeat the 75% ethanol wash once; split the EP tube, and then place the EP tube back on the magnetic rack. Aspirate all the residual ethanol; place in a 37°C metal bath for 30-60 seconds to allow the ethanol in the EP tube to completely evaporate; add an appropriate amount of EB Buffer to the EP tube and flick it to completely resuspend the magnetic beads; place the EP tube in a 37°C metal bath for 1 hour to elute the DNA. Since the magnetic beads are very easy to settle, during the incubation period, flick the EP tube every 5-10 minutes to fully resuspend the magnetic beads; place the EP tube on a magnetic rack and let it stand for several minutes until all the magnetic beads are adsorbed on the magnetic rack, then aspirate the supernatant containing the DNA from the EP tube. Wash twice with 70% ethanol, dry thoroughly, and dissolve the EB.

[0057] Table 3

[0058] Group NaCl PEG 6000 Tris-HCl (pH = 8) EDTA (pH = 8) 1 0.2M 5％ 10mM 0.5mM 2 0.2M 10％ 10mM 0.5mM 3 0.2M 15％ 10mM 0.5mM 4 0.5M 5％ 10mM 0.5mM 5 0.5M 10％ 10mM 0.5mM 6 0.5M 15％ 10mM 0.5mM 7 1M 5％ 10mM 0.5mM 8 1M 10％ 10mM 0.5mM 9 1M 15％ 10mM 0.5mM

[0059] The experimental results are shown in Table 4. It can be seen that the OD260 / 280 and OD260 / 230 of each experimental group basically meet the requirements of the third-generation sequencing. As the PEG concentration increases, the longer the DNA fragment, the more phosphate groups are exposed due to water loss. The sodium ions in the solution form a "salt bridge" with the carboxyl groups on the surface of the magnetic beads, allowing the DNA to adsorb to the surface of the magnetic beads. Therefore, when removing impurities, experimental group 9 will also lose some ultra-long fragment DNA, resulting in a decrease in concentration. In experimental groups 1-3, due to the low NaCl content, the amount of large fragment DNA lost under the same PEG concentration is less. Although its OD260 / 280 and OD260 / 230 theoretically meet the requirements of the third-generation sequencing, the removal effect of the "invisible" impurities therein is not good, resulting in a small amount of data obtained on the machine. After comprehensive comparison, the parameters of experimental group 5 were selected as the optimal proportion of pollutant precipitants: 0.5 M NaCl, 10% PEG 6000, 10 mM Tris-HCl (pH = 8), 0.5 mM EDTA (pH = 8) and small-sized magnetic beads.

[0060] Table 4

[0061]

[0062] In addition, the applicant found that the use of larger-sized magnetic beads (10 μm) can purify and recover larger fragments (150 kb), while the fragment size recovered by small-sized magnetic beads (1 μm) is relatively low (100 kb), that is, the large-sized magnetic beads have a better protection effect on large-fragment DNA. Further, the applicant observed that although the length of the recovered DNA fragments increased with the increase of PEG 6000 concentration, the high concentration of PEG 6000 also caused the DNA to curl excessively, resulting in an extended elution time, and ultimately a reduced yield. To solve this problem, the applicant found that the magnetic bead system with 0.3% Tween-20 added had a better recovery rate than the magnetic bead system without Tween-20 (Table 5), because the addition of Tween in the magnetic bead purification system will also increase the viscosity of the solution, reduce the shear force and physical effect on the DNA during the operation process, and thus help to improve the DNA recovery rate.

[0063] Table 5

[0064]

[0065] Example 2 Library construction method suitable for third-generation sequencing platform

[0066] On the PB Revio platform, after obtaining purified DNA, 15-20Kb HiFi library construction is required before sequencing can be performed. The steps include: 1) DNA shearing, 2) library construction (adding adapters), 3) fragment screening and enrichment, etc. Among them, DNA shearing is to use an instrument to break the purified DNA into 10-20Kb DNA fragments, and library construction is constructed using a dedicated kit; the purpose of fragment screening and enrichment is to remove small fragments and enrich 10-20Kb library fragments. The most commonly used method is to use an automated recovery instrument such as Bluepippin for automated electrophoresis recovery.

[0067] In this regard, the applicant conducted the following verification and optimization:

[0068] A. First, the library yield of the method before improvement (Comparative Example 1) is significantly lower than that of the method after improvement (i.e., the aforementioned method of the present invention), while the indicators of the method after improvement meet the quality control standards. The details are as follows:

[0069] Table 6

[0070]

[0071] The reason for this is that during the library construction process, there will be a damage repair enzyme repair process to repair DNA gaps and other damage; then the digestive enzyme digests the DNA with gaps on the DNA chain to remove single-stranded and double-stranded DNA libraries, with the goal of eventually forming a complete dumbbell-shaped double-stranded library. The quality control index in the sea cucumber method before optimization was significantly lower than that after optimization because there were certain impurities in the DNA, which affected the activity of the damage repair enzyme, resulting in a low yield of the subsequent digestive enzyme digestion of the gapped DNA.

[0072] After forming the dumbbell-shaped library, we began to prepare for the subsequent sequencing process: Taking the Revio platform as an example, the improved method can obtain higher sequencing data output than the unimproved method, as shown in Table 7 and Figure 4-5 shown.

[0073] Table 7

[0074]

[0075] The DNA obtained by the method before the improvement had a small initial amount of active ZMW pores during sequencing, and the decline rate was very fast. The data output yield was basically 0, and the P1% was unqualified and seriously low. After the improvement, the active ZMW returned to normal during sequencing, the decline rate was stable, and the P1% was qualified and met the standard.

[0076] B. Secondly, the present invention also optimizes the PEG / NaCl system with the best concentration in fragment screening and enrichment, which has low cost and good screening effect, specifically:

[0077] When HMW DNA stays in the solution, under the condition of high concentration of polymer compounds, different molecular weight DNA can be graded and precipitated from the solution, and the precipitate can be collected by centrifugation, so as to achieve the purpose of screening DNA fragments and obtain a 10-20Kb HiFi library for sequencing. The applicant found that the recovery rate of DNA and the length of the recovered fragments increased with the increase of PEG6000 concentration, but the high concentration of PEG6000 caused the DNA to curl up excessively, resulting in a prolonged elution time. Therefore, in order to explore the best screening system, different concentration gradients were set to verify the effect. The system is as follows: 1M NaCl, 0.5mM EDTA, 10mMTris-HCl, and Group A: 10% PEG6000, Group B: 12.5% ​​PEG6000, Group C: 15% PEG6000, Group D: 17.5% PEG6000. The specific results are shown in Tables 8-9.

[0078] The results showed that experimental group C had the best effect, with the most concentrated library fragments after screening, most in line with the HiFi library requirements (HiFi reads require the library length to be between 10-20Kb), and the highest output.

[0079] Table 8

[0080]

[0081] Table 9

[0082]

[0083] It can be seen that the present invention provides a simple, low-cost, and better screening system, namely 1M NaCl, 0.5mM EDTA, 10mM Tris-HCl, and 15% PEG6000, which has a better effect than the standard method (conventional gel excision method).

[0084] Comparative Example 1

[0085] This comparative example uses the conventional CTAB method to extract DNA from difficult marine biological samples, specifically: add an appropriate amount of liquid nitrogen to 0.5g of sample and quickly grind it into a uniform powder. Transfer the powder into a 1.5mL centrifuge tube, add 800μL CTAB separation buffer (2% (M / V) CTAB, 1.4M NaCl, 100mM Tris-Cl (pH8.0), 20mM EDTA, pH 8.0), turn the centrifuge tube upside down, and mix evenly. Place the centrifuge tube containing the sample in a 65℃ water bath for 30 minutes, and shake it gently every 3-4 minutes to mix. Centrifuge at 12000rpm for 5 minutes at low temperature, and transfer the upper aqueous phase to a new centrifuge tube. Add equal volumes of Tris-phenol and chloroform isoamyl alcohol, turn the centrifuge tube upside down, and mix evenly; centrifuge at 12000rpm for 5 minutes at low temperature, and transfer the upper aqueous phase to a new centrifuge tube. Add an equal volume of chloroform and isoamyl alcohol, invert the centrifuge tube and mix evenly; centrifuge at 12000rpm for 5 minutes at low temperature, and transfer the upper aqueous phase to a new centrifuge tube. Add 0.6 times the volume of isopropanol, mix gently, and place at -20℃ for precipitation for 30 minutes. Centrifuge at 12000rpm for 5 minutes at low temperature to remove the supernatant, add 500μL 70% ethanol to wash the precipitate, and centrifuge at 12000rpm for 2 minutes at room temperature to remove the supernatant. The precipitate should be dried as much as possible. Add an appropriate amount of TE (20μL) containing RNase to dissolve the precipitate, bathe at 37℃ for 30 minutes, digest RNA, and purify with 1X magnetic beads to obtain DNA. The purity was tested and used for third-generation sequencing. The results are shown in Tables 2, 6-7.

[0086] In summary, the present invention provides a method for extracting DNA from difficult marine biological samples and constructing a library suitable for third-generation sequencing, optimizes its process and parameters, and ultimately greatly improves data output in actual machine sequencing.

[0087] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for extracting DNA from difficult marine biological samples and constructing libraries suitable for third-generation sequencing, characterized in that: The steps include: S1. Processing of a marine biological sample, wherein the marine organism is a sea cucumber; S2. Crude extraction and purification of DNA: A. Add CTAB lysis buffer with the composition of 50-150mM Tris, 10-30mM EDTA, 2% w / v CTAB, 1.2M NaCl, proteinase K, and RNase, add equal volume of chloroform and isoamyl alcohol for extraction, invert to mix, centrifuge to precipitate impurities, aspirate the supernatant and add two to three times the volume of CTAB precipitation buffer with the composition of 50-150mM Tris, 10-30mM EDTA, 2% w / v CTAB, mix, centrifuge to obtain particle precipitation, wash with ethanol, centrifuge to remove supernatant, dry and dissolve to obtain crude DNA; B. Add the obtained crude DNA to CTAB secondary lysis buffer, which consists of Tris 100mM, EDTA 20mM, CTAB 5% w / v, NaCl 1.2M, 1% PVP, pH=8, and add RNase and proteinase K for incubation. Add an equal amount of chloroform and isoamyl alcohol, invert and mix, centrifuge and aspirate the supernatant, add sodium acetate and mix evenly, then precipitate the DNA with an equal volume of isopropanol, collect the DNA by centrifugation, remove the supernatant, and wash with ethanol to obtain the purified DNA. S3. Further purification of DNA: Add 1V of a pollutant precipitant to the DNA solution, wherein the components of the pollutant precipitant are 0.5-1M NaCl, 5-15% PEG 6000, Tris-HCl (pH=8), EDTA (pH=8) and magnetic beads with a size of 10 μm, mix thoroughly, place on a magnetic stand for static adsorption, add the supernatant after the magnetic beads are adsorbed to a new test tube, add the prepared magnetic bead solution until the final DNA:magnetic bead volume ratio is 1:1, adjust the PEG and NaCl concentrations to NaCl=2M, PEG 6000=20-30%, and then perform magnetic bead purification; S4, construction of HiFi library; S5, sequencing.

2. The method according to claim 1, characterized in that In step S4, 1 M NaCl, 0.5 mM EDTA, 10 mM Tris-HCl, and 15% PEG 6000 system is used to enrich fragments of a specific size.

3. The method according to any one of claims 1-2, characterized in that: Step S1 is: take 0.5g tissue and cut it into blocks less than 3mm on a clean culture dish placed on ice; add 3ml pre-cooled HB buffer, the ingredients of the HB buffer are 5-15mM Tris, 5-15mM EDTA, 5-20mM MgCl2, 0.1% NP 40, homogenize at 20000-30000rpm for 4-5 times, stop for 40s each time for 20s to avoid heating, add pre-cooled HB buffer and specific enzyme mixture to 5ml, mix well, the specific enzyme mixture contains chitinase, lipase and phospholipase, centrifuge at 800g for 5 minutes at 4°C, remove the supernatant, add 3ml HB buffer, mix well with a wide-mouth pipette tip, wash the cell nucleus, and centrifuge at 800g for 10 minutes.

4. The method according to any one of claims 1-2, characterized in that: Add twice the volume of CTAB precipitation buffer in step S2.

5. The method according to any one of claims 1-2, characterized in that: Step S2 is: A. Add CTAB lysis buffer with the composition of 50-150mM Tris, 10-30mM EDTA, 2% w / v CTAB, 1.2M NaCl and 25-75μL proteinase K and incubate for 1-2 hours, add 0.1%-1% volume of RNase and incubate for 0.5-1 hour, add an equal volume of chloroform isoamyl alcohol for extraction, invert and mix, centrifuge at 5000g for 10 minutes to precipitate impurities, aspirate the supernatant and add two to three times the volume of CTAB precipitation buffer, whose composition is 50-150mM Tris, 10-30mM EDTA, 2% w / v CTAB, 55℃ for 30min, mix, centrifuge at 16000g for 5 minutes to obtain particle precipitation, wash with 70% ethanol, centrifuge at 16000g, remove supernatant, dry, dissolve in EB to obtain crude DNA; B. Add the obtained crude DNA into CTAB secondary lysis buffer, whose components are Tris 100mM, EDTA 20mM, CTAB 5% w / v, NaCl 1.2M, 1% PVP, pH=8, and add 5μL RNase at the same time, incubate for 1 hour, then add 5-20μL proteinase K, incubate for 1 hour, add an equal amount of chloroform isoamyl alcohol, mix by inversion, centrifuge at 5000g for 10 minutes, aspirate the supernatant, add 0.1 times the volume of 3M sodium acetate and mix well, then precipitate the DNA with an equal volume of isopropanol, collect the DNA by centrifugation at 5000g for 10 minutes, remove the supernatant, wash the precipitate twice with 70% ethanol, and dissolve it to obtain the purified DNA.

6. The method according to any one of claims 1-2, characterized in that: The specific steps of magnetic bead purification in step S3 are as follows: the DNA sample to be purified is mixed with the magnetic bead suspension in a volume ratio of 1:1, and incubated at room temperature for 15 minutes; after a split second, the mixture is allowed to stand for several minutes until all the magnetic beads are adsorbed on the magnetic rack, and then aspirated and temporarily stored in a new EP tube; freshly prepared 75% ethanol is added to the EP tube, the mixture is allowed to stand for 30-60 seconds, and the supernatant is aspirated and discarded; the 75% ethanol washing is repeated once; the EP tube is split second, and the tube is placed back on the magnetic rack; all residual ethanol is aspirated; the mixture is allowed to stand in a metal bath at 37°C for 30-60 seconds to allow the ethanol in the EP tube to completely evaporate; an appropriate amount of EB is added to the EP tube. Buffer, flick to completely resuspend the magnetic beads; place the EP tube in a 37℃ metal bath and incubate for 1 hour to elute the DNA. Since the magnetic beads are very easy to settle, during the incubation period, flick the EP tube every 5-10 minutes to fully resuspend the magnetic beads; place the EP tube on the magnetic rack, let it stand for several minutes until all the magnetic beads are adsorbed on the magnetic rack, then aspirate the supernatant containing DNA from the EP tube, wash twice with 70% ethanol, dry thoroughly, and dissolve with EB.

7. The method according to claim 6, characterized in that Magnetic beads and 0.3% Tween-20 were added during the magnetic bead purification.

8. The method according to any one of claims 1-2, characterized in that: The platform used for sequencing in step S5 is the Revio platform or the PromethION platform.

9. Application of the method according to any one of claims 1 to 8 in third-generation sequencing.

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