Kit for extracting and sequencing mitochondrial genomic DNA of small arthropods, sequencing method, and use thereof

Abstract

The present invention relates to a kit for extracting and sequencing mitochondrial genomic DNA of small arthropods, a sequencing method and the use thereof. The kit comprises a reagent A, a reagent B, a reagent C and a Tn5 transposase system for preparing a sequencing library. The provided kit or method is directed at the particularity of small arthropods and can achieve library preparation and high-throughput sequencing by using trace-amount mitochondrial DNA which is extracted from an individual small insect by a one-step method, thereby providing a simple and efficient universal technology for mitochondrial whole genome sequencing analysis of a wide variety of small insects. By using the provided kit or method, the volume of sequencing data required for mitochondrial genome assembly can be reduced by tens or even thousands folds than the volume of data of existing total DNA methods, thereby overcoming the problem of incapability to carry out mitochondrial genome sequencing analysis using individual small insects, and not only reducing the waste of precious insect specimens but also significantly reducing the sequencing cost for individual specimens.

Description

Kit for small arthropod mitochondrial genome DNA extraction and sequencing, sequencing method and application thereof

[0001] CROSS-REFERENCE

[0002] The present application claims priority to the Chinese patent application No. 202411345968.8, filed on September 25, 2024, entitled "Kit for small arthropod mitochondrial genome DNA extraction and sequencing, sequencing method and application thereof", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of gene sequencing and biological detection, in particular to a kit for small arthropod mitochondrial genome DNA extraction and sequencing, a small arthropod mitochondrial genome DNA extraction and sequencing method, and their applications in preparing small arthropod mitochondrial genome base and structural mutation analysis products, small arthropod identification products, and small arthropod diversity analysis products. BACKGROUND

[0004] Unlike higher vertebrates, insects are a class of small arthropods with the most extensive distribution and the most abundant species, and more than 1 million species have been found, more than all other animal species combined. Insect mitochondrial DNA is a cytoplasmic DNA different from the nuclear genome DNA and exists independently in the mitochondrial organelle. Mitochondrial genome DNA contains a molecular clock of species development and evolution and is a main genetic marker molecule for species identification and evolutionary analysis. Mitochondrial genome sequencing is widely used in the evolution and species identification of various insects and crustaceans, and in the investigation and monitoring of biological diversity.

[0005] All animal mitochondrial genomes are small, circular, and multicopy DNA, and the quality accounts for only 0.1% or less of the total nuclear genome DNA. Since insects are a class of small, poikilothermic, and lower arthropods, the content of mitochondrial DNA in insect tissue cells is significantly reduced compared to other animals (i.e., extremely low), and the structure of mitochondrial genome shows great variation, and even the extreme phenomenon of mitochondrial DNA cracking from one large ring into multiple small rings. Therefore, the preparation and sequencing analysis of insect mitochondrial genome DNA have more challenges and difficulties than large vertebrates.

[0006] Because the content of insect mitochondrial DNA is extremely low, a small insect individual generally cannot provide enough DNA for conventional analysis methods, and often several dozen individuals of the same insect species need to be combined together to meet the requirements of preparation and analysis.

[0007] Currently, common insect tissue mitochondrial genome DNA sequencing analysis includes:

[0008] 1) Insect total DNA sequencing (i.e. whole genome DNA sequencing, WGS) method, insect whole genome sequencing method refers to the method of using total DNA extracted from insect tissue for library preparation and high-throughput sequencing. Since mitochondrial DNA accounts for only 0.1% or less of the nuclear genome DNA in total DNA, a small amount of mitochondrial genome sequencing data must also be extracted from the whole genome sequencing data through mitochondrial DNA-specific bait PCR and a series of bioinformatics analysis processes before it can be used for mitochondrial genome assembly and subsequent analysis. In addition, the extraction and assembly of mitochondrial genome data require a large amount of sequencing data of total DNA as a basis, i.e. about 2-5G total DNA sequencing data is required for the assembly of one mitochondrial genome.

[0009] 2) Species-specific PCR amplification method is the most commonly used method for secondary enrichment of mitochondrial DNA. It is a method of sequencing after secondary enrichment of mitochondrial DNA by specific PCR amplification. According to the number of primer pairs or amplified fragments, it can be divided into 1 whole genome fragment, 2 or more PCR fragments method. Due to the particularity of small arthropod mitochondrial DNA, the success rate of PCP method for amplifying mitochondrial whole genome is greatly reduced.

[0010] 3) Probe capture method is another method for secondary enrichment of mitochondrial genome DNA. Its principle is to capture small fragments of mitochondrial DNA from total DNA by a large number of species-specific mitochondrial DNA probes, and then use it for library preparation and high-throughput sequencing analysis.

[0011] 4) Some other non-PCR methods for mitochondrial DNA enrichment for library preparation and high-throughput sequencing, but these attempts can only use high-quality isolated cells and cultured cells of humans, and have been proven to be unable to achieve practical results (Scientific Reports, 2018, 8:2261).

[0012] The methods commonly used in insect mitochondrial whole genome sequencing each have limitations.

[0013] Firstly, the above-mentioned various methods all face the common problem of being unable to perform mitochondrial genome sequencing analysis at the level of small insects. This is because insects are small, poikilothermic, and low-grade arthropods, and the content of mitochondrial DNA in insect tissue cells is greatly reduced compared to other animals (i.e. very low), and a small insect individual often cannot provide enough total DNA for conventional analysis methods. Therefore, many rare small insects in the natural environment still cannot be sequenced at the individual level for mitochondrial genome sequencing analysis and species identification. Capturing multiple samples of the same insect for extraction of sufficient total DNA for sequencing analysis is currently a commonly used method, which is not only very difficult but also a serious waste of natural resources.

[0014] Secondly, the method of sequencing total DNA or whole genome sequencing to obtain mitochondrial genome data will cause a lot of waste, and has the problems of high cost, low efficiency and poor accuracy. This is because a large amount of raw sequencing data is required for total DNA, and the mitochondrial genome must be indirectly assembled by filtering out 99.9% or more of the nuclear genome sequencing data through various bioinformatics methods. The quality of the assembly is affected by a variety of factors, including the limited amount of mitochondrial genome data, pseudogenes in nuclear DNA, and assembly software. The phenomenon that the complete mitochondrial genome cannot be assembled due to insufficient sequencing quality or data volume is a common problem of this method. In addition, mitochondrial-specific bait PCR is an important step in the assembly of sequencing data for new species, which still requires partial mitochondrial genome information of the species to be tested to complete the assembly and analysis.

[0015] Thirdly, the methods of PCR amplification and probe capture for secondary enrichment of mitochondrial DNA also have many limitations in the application of small insects and arthropods. Both methods require prior knowledge of the sequence information of the mitochondrial genome of the species to be tested in order to design species-specific PCR primers or capture probes, so they can only be limited to a few common species. The vast majority of insect species do not have mitochondrial genome information and cannot be analyzed using PCR amplification and probe capture methods. For example, CN111206104A discloses a universal primer and method for efficiently and simply obtaining the mitochondrial genome of the total family of woodlice and its application. According to the known mitochondrial genome sequences of different woodlice families in the existing total family of woodlice, universal primers are designed based on the conserved regions of the known mitochondrial genome to obtain mitochondrial genome information of woodlice populations with close genetic distance. It can only be used for woodlice insects, which is a huge gap from the needs of more than 1 million different insects.

[0016] Fourthly, even for a few insect species with mitochondrial genome sequence information, it is a technically demanding, tedious and costly process to design effective PCR primers and amplify the desired PCR fragments. For example, it is very difficult to design universal primers for the more than 3000 species of the total family of woodlice mentioned in CN111206104A. The difficulty of this PCR amplification is particularly prominent for small insects, as the DNA provided by a single small insect individual is often insufficient to meet the basic requirements of PCR amplification. In addition, PCR amplification-based mitochondrial DNA enrichment is susceptible to amplification errors and biases introduced by in vitro amplification and mitochondrial pseudogenes (NUMTs) in nuclear DNA.

[0017] Therefore, there is an urgent need for a new method to overcome the problem of low mitochondrial content in small insect tissues and the lack of effective methods for mitochondrial whole genome sequencing. SUMMARY

[0018] Objectives of the Invention

[0019] The present application aims to provide a kit for small arthropod mitochondrial genome DNA extraction and sequencing, a small arthropod mitochondrial genome DNA extraction and sequencing method, and their applications in preparing small arthropod mitochondrial genome base and structural mutation analysis products, small arthropod identification products, and small arthropod diversity analysis products. The present application can prepare a library and perform high-throughput sequencing on trace amounts of mitochondrial DNA extracted from individual small insects, providing a simple and efficient universal technology for mitochondrial whole genome sequencing analysis of a large number of small insects. The amount of sequencing data required for mitochondrial genome assembly using the kit or method of the present application can be reduced by tens of times or even thousands of times compared to the amount of data of the existing total DNA method, greatly reducing the sequencing cost and analysis difficulty.

[0020] Solutions

[0021] To achieve the above-mentioned objectives, the present application provides the following technical solutions:

[0022] In a first aspect, the present application provides a kit for small arthropod mitochondrial genome DNA extraction and sequencing, comprising the following components:

[0023] 1) Reagent A: including cell lysis and balancing reagent, used for lysing small arthropod tissues to obtain a lysis solution containing total DNA or preserving tissue lysis solution;

[0024] 2) Reagent B: including 1.7-2.5M sodium acetate;

[0025] 3) Reagent C: including phenol, chloroform and isoamyl alcohol in a volume ratio of (120-130):(19-29):1;

[0026] Among them, reagents B and C are used to mix with the lysis solution containing total DNA and obtain the aqueous phase containing mitochondrial DNA by layering;

[0027] 4) Tn5 transposase system, used for sequencing library preparation.

[0028] Further, in reagent A, the cell lysis and balancing reagent includes 3.25-4.75M guanidine isothiocyanate, 0.65-0.85M sodium citrate and 5-15v / v% N-lauroylsarcosine sodium salt; optionally including 3.85-4.25M guanidine isothiocyanate, 0.7-0.8M sodium citrate and 5-10v / v% N-lauroylsarcosine sodium salt; optionally including 3.85-4.1M guanidine isothiocyanate, 0.7-0.74M sodium citrate and 5-10v / v% N-lauroylsarcosine sodium salt.

[0029] Further, in reagent B, the concentration of sodium acetate is 1.8-2M.

[0030] Further, in reagent C, the volume ratio of phenol, chloroform and isoamyl alcohol is (120-127):(22-26):1.

[0031] In a second aspect, a method for extracting and sequencing mitochondrial DNA of small arthropods is provided, which uses the kit of the first aspect, and comprises the following steps:

[0032] I) Extracting mitochondrial DNA from the tissue or organ sample of small arthropods, mixing reagent B and reagent C with the tissue lysis solution of reagent A, separating the layers, and precipitating to obtain mitochondrial DNA;

[0033] II) Taking the mitochondrial DNA of step I) and mixing it with the Tn5 transposase system to perform cleavage reaction, and using the cleavage reaction product for sequencing.

[0034] Further, in step I), the method for extracting mitochondrial DNA is as follows:

[0035] 1) Taking the tissue or organ of small arthropods;

[0036] 2) Mixing the tissue or organ sample with reagent A to lyse the cells and release free nucleic acids to obtain a lysis solution;

[0037] 3) Adding reagent B and reagent C to the lysis solution of step 2) and mixing thoroughly, and then separating the water phase and the organic phase;

[0038] 4) Taking the water phase containing mitochondrial DNA in step 3) and precipitating with alcohol to recover the mitochondrial DNA of small arthropods.

[0039] Further, in step 1), the tissue or organ of small arthropods includes one or more of the head, cephalothorax, compound eye, and arthropod part. Soft tissue or soft tissue containing shell, or soft tissue with removed exoskeleton or shell can be used.

[0040] Further, in step 1), the sample amount is 1-20 mg.

[0041] Further, the tissue or organ is processed into small pieces by cutting or crushing.

[0042] Further, in step 1), the sample is preserved with reagent A, which can effectively prevent the degradation of nucleic acid molecules in the tissue, can be stored at room temperature, and can be processed in real time when sampling, and can be stored for at least one week.

[0043] Further, the tissue or organ is freshly collected, or frozen, or preserved with ethanol, or preserved with reagent A.

[0044] Further, in step 3), 7-13% reagent B is added by volume fraction of the lysis solution.

[0045] Further, in step 3), the amount of reagent C added is 1-1.5 times the volume of the lysis solution.

[0046] Further, in steps 2) and 3), the mixing method includes shaking.

[0047] Further, in step 3), the aqueous phase and the organic phase are accelerated to separate by low-temperature high-speed centrifugation. Generally, low-temperature high-speed centrifugation refers to centrifugation at 4-6°C at a speed of 10000-14000 rpm / min.

[0048] Further, in step 4), anhydrous ethanol or isopropanol is used for alcohol precipitation.

[0049] Further, in step 4), the mitochondrial DNA obtained by precipitation is dissolved in TE solution.

[0050] Further, in step II), the sequencing method includes: library preparation: the cleavage product of Tn5 transposase is added into a universal sequencing adapter by PCR amplification, purification, multiplex sequencing and analysis by mixing multiple mitochondrial DNA libraries; the purification method is optional and is magnetic bead purification.

[0051] In a third aspect, the kit of the first aspect or the method of the second aspect is used for preparing a small arthropod mitochondrial genome de novo assembly analysis product, or a resequencing analysis product, or a mutation analysis product, which optionally includes a base and structure mutation analysis product, or a method for performing small arthropod mitochondrial genome de novo assembly analysis, or resequencing analysis, or mutation analysis using the kit of the first aspect or the method of the second aspect, and the mutation analysis includes base and structure mutation analysis.

[0052] In a fourth aspect, the kit of the first aspect or the method of the second aspect is used for preparing a small arthropod species identification and / or diversity analysis product, or a method for performing small arthropod species identification and / or diversity analysis using the kit of the first aspect or the method of the second aspect. Advantages

[0053] (1) The present application can be used for the extraction of mitochondrial DNA from small insects for library preparation and high-throughput sequencing. The present application provides a simple and efficient method for the sequencing of mitochondrial genomes of small insects. The Tn5 transposase method of the present application can be used to convert the small amount of circular mitochondrial DNA extracted from small insect tissues into a DNA sequencing library. This method overcomes the difficulty of sequencing mitochondrial genomes from a single small insect and has important practical significance in the identification of small insect species and biodiversity surveys. Using the kit or method of the present application, only 10-500 Mb of sequencing data is required to assemble a complete mitochondrial genome, which is 10-500 times less than the amount of total DNA sequencing data. In the case of resequencing analysis, 0.36 Mb of sequencing data can produce a mitochondrial genome sequencing depth of 1400x or more, which is at least 10,000 times less than the amount of data required for the same sequencing depth using the existing total DNA sequencing method, greatly reducing the sequencing cost and analysis difficulty.

[0054] (2) The present application is a method for extracting high-purity mitochondrial DNA from small arthropods (such as insects) and using it for genome sequencing. The method does not require the preparation of total DNA from insect tissues, followed by PCR amplification or probe capture for secondary enrichment of mitochondrial DNA. Therefore, the method of the present application is not only simple and fast, but also avoids the cumbersome technical operations and high experimental costs of PCR amplification or probe capture.

[0055] (3) Compared with insect total DNA sequencing data, the content or ratio of mitochondrial DNA in the sequencing products is increased from about 0.1% in the prior art to 50-99% in the present application, with a 100-fold improvement in enrichment efficiency. Since the present application greatly improves the efficiency of mitochondrial DNA sequencing, it not only overcomes the limitations of total DNA sequencing methods that cannot utilize a single small insect individual for sequencing, but also allows the simultaneous determination of dozens or more mitochondrial DNA samples with a sequencing data amount equivalent to a total DNA sample. This not only reduces the waste of rare insect samples, but also greatly reduces the cost of sequencing individual samples.

[0056] (4) The present application does not require prior knowledge of the mitochondrial genome of the species to be tested, and is a species-independent method that provides an effective method for the extraction and library preparation of mitochondrial DNA for a wide variety of insects lacking mitochondrial genome information.

[0057] (5) The sequencing data of the mitochondrial genome generated by the present application is high-fidelity and high-sequencing-depth. High-fidelity means that it avoids the PCR errors and bias brought by PCR amplification enrichment or the influence of pseudogenes in nuclear DNA, and high-sequencing-depth overcomes the problem that the complete mitochondrial genome cannot be assembled due to insufficient target sequencing data. In summary, the present application provides a general scheme for sequencing the mitochondrial genome of the most diverse small insects in a species-independent manner, and especially solves the technical problem of the feasibility of species identification and biodiversity investigation of a single small insect. BRIEF DESCRIPTION OF DRAWINGS

[0058] One or more embodiments are illustrated by way of example in the figures that form a part of this disclosure and which are shown by way of illustration in the accompanying drawings and are not intended to limit the scope of the embodiments to the embodiments specifically described herein. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0059] Figure 1 is the sequencing coverage of the mitochondrial genome of Pieris rapae of Example 1 of the present application (reference genome OL890650).

[0060] Figure 2 is the assembly map of the de novo assembly of the mitochondrial genome of Pieris rapae according to the sequencing results of Example 1 of the present application.

[0061] Figure 3 is the sequencing coverage of the mitochondrial genome of Caridina cantonensis of Example 2 of the present application (reference genome MZ781227.1).

[0062] Figure 4 is the assembly map of the de novo assembly of the mitochondrial genome of Caridina cantonensis according to the sequencing results of Example 2 of the present application.

[0063] Figure 5 is the sequencing coverage of the mitochondrial genome of Helicoverpa armigera extracted from Comparative Example 1 of the present application (reference genome KF701043.1).

[0064] Figure 6 is the assembly map of the de novo assembly of the mitochondrial genome of Helicoverpa armigera according to the sequencing results of Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0065] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are some of the embodiments of the present application but not all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall into the scope of the present application. Unless otherwise clearly indicated, in the entire specification and claims, the term “comprise” or its variants such as “contain” or “include” should be understood as including the stated elements or components, and not excluding other elements or components.

[0066] In addition, in order to better illustrate the present application, a large number of specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means and the like which are well known to those skilled in the art are not described in detail, in order to highlight the main idea of the present application.

[0067] The present application will be described in detail below.

[0068] In order to overcome the many limitations of low mitochondrial content in small insects and the lack of effective methods for mitochondrial whole genome sequencing, the present application provides a new method and kit, which can not only isolate a small amount of high-purity mitochondrial DNA from insect tissue organs in one step, but also realize rapid library preparation and genome sequencing of a single small insect. The method includes sampling of a single insect tissue organ, one-step extraction and preparation of mitochondrial DNA, and a complete set of solutions for mitochondrial DNA library preparation based on Tn5. The main steps of one-step extraction and preparation of mitochondrial genome DNA are as follows:

[0069] 1. Insect tissue organ sample collection and preservation:

[0070] (1) Any live insect captured can be freshly removed from the head or head-thorax using a sterile clock and forceps according to its individual size, and placed in a sterile 2ml microtube.

[0071] (2) Freshly collected insect tissue organs can be immediately used for the following sample preparation, or stored at low temperature with dry ice or added to a preservation solution until the following sample preparation, preserved with reagent A (other reagents can also be used for preservation), which can effectively prevent the degradation of nucleic acid molecules in the tissue, and can be stored at room temperature, facilitating real-time processing during sampling.

[0072] 2. One-step rapid extraction and preparation of mitochondrial genome DNA from insect tissue organs

[0073] (1) Use sterile small surgery to cut the head or head and thorax of insects into small pieces and place them into a 2 mL microtube. Add 600 μL of reagent A and shake well to promote the complete lysis of cells and form a uniform cell lysate containing various free nucleic acids.

[0074] Among them, reagent A is a tissue cell lysis and balancing reagent, which includes 3.25-4.75 M guanidine isothiocyanate, 0.65-0.85 M sodium citrate and 5-15 v / v% N-lauroylsarcosine sodium salt.

[0075] (2) High-speed short-time centrifugation of the microtube containing the tissue lysate. Transfer all the supernatant (i.e. lysate) after centrifugation to another sterile 2 mL microtube.

[0076] (3) In the lysate of step (2) above, add 7-13% reagent B and an equal volume of reagent C to the lysate, then mix well to promote the separation of the aqueous phase and the organic phase, and effectively separate the mitochondrial DNA and nuclear DNA in the two liquid phases by layering.

[0077] Among them, reagent B is a buffer reagent for promoting nucleic acid separation, which includes 1.7-2.5 M sodium acetate; reagent C is an organic reagent for promoting liquid phase separation, which includes phenol, chloroform and isopropyl alcohol, and the volume ratio of phenol, chloroform and isopropyl alcohol is (120-130):(19-29):1.

[0078] (4) Low-temperature high-speed centrifugation of the microtube in step (3) above to effectively separate the aqueous phase and the organic phase, and transfer the aqueous phase containing mitochondrial DNA to a new sterile microtube, then precipitate and recover the mitochondrial DNA by isopropyl alcohol or anhydrous ethanol.

[0079] (5) Quantification and quality control of mitochondrial DNA. Use NanoDrop and Qubit instruments in combination to determine the concentration of nucleic acids and DNA.

[0080] 3. Tn5 single-tube rapid library preparation:

[0081] (1) Tn5 transposase cleavage reaction: mix 3-10 μL of insect circular mitochondria with Tn5 transposase (RK20547, ABclone) to prepare a 20 μL reaction system, treat at 55°C for 5 min, and then add 5 μL of termination liquid.

[0082] (2) PCR amplification reaction with sequencing adapters: directly add 10 μL of universal primer pair containing sequencing adapters (sequencing through primer i5 / i7 primer) and 15 μL of PCR amplification mixture (RK20726, ABclone) to the above-mentioned 25 μL cleavage reaction system to form a 50 μL PCR reaction system.

[0083] (3) PCR reaction condition: 72°C for 3 min, then 10-12 cycles of 95°C / 10 sec, 55°C / 30 sec, 72°C / 30 sec, and finally 72°C / 5 min.

[0084] (4) Magnetic bead purification (AMpure XP (Agencourt)) to obtain high-quality mitochondrial DNA library, Qubit and 2100 library quality control.

[0085] (5) Multiplex combination of mitochondrial DNA library and high-throughput sequencing: each mitochondrial DNA library has a unique identification code, multiple DNA libraries can be mixed in equal amounts, and then multiplex sequencing and analysis can be performed using any model of illumine high-throughput sequencer.

[0086] Some specific embodiments are as follows:

[0087] Example 1 One-step extraction and sequencing of mitochondrial genome DNA of Pieris rapae

[0088] 1. Sampling of Pieris rapae

[0089] (1) Collect Pieris rapae outdoors.

[0090] (2) Sample the head of the Pieris rapae sample with a sterile clock pin and transfer it to a sterile microtube containing 500 μL of reagent A (containing 3.85 M guanidine isothiocyanate, 0.7 M sodium citrate and 5% N-lauroylsarcosine sodium salt), shake and mix, obtain Pieris rapae tissue preservation solution, which can be stored at room temperature or 4°C for a short time (storage time can be at least one week) until the following extraction.

[0091] 2. Extraction and preparation of mitochondrial genome DNA of Pieris rapae

[0092] (1) Transfer 250 μL of the above Pieris rapae tissue preservation solution to a sterile 2 mL microtube, then add 350 μL of reagent A, the total volume is 600 μL, vortex and shake for 1-2 minutes to completely lyse the Pieris rapae tissue, and obtain a Pieris rapae tissue lysate.

[0093] (2) In the Pieris rapae tissue lysate, add 10% reagent B (2 M sodium acetate) and 600 uL of reagent C (phenol-chloroform-isoamyl alcohol with a volume ratio of 123:22:1) according to the volume fraction of the Pieris rapae tissue lysate, vortex and shake for 1 minute, and then short static after short static, low-temperature high-speed centrifugation to separate the water phase and the organic phase, wherein the water phase contains mitochondrial DNA and the organic phase contains nuclear DNA, so that they can be effectively separated.

[0094] (3) Carefully transfer the upper aqueous phase (containing Pieris mitochondria DNA) into another sterile 1.5 mL microtube, add an equal volume of isopropanol, and let stand briefly.

[0095] (4) Low-temperature high-speed centrifugation, collect the DNA precipitate at the bottom of the tube, wash with 75% ethanol, and recover into 13 uL TE solution to prepare the Pieris mitochondria DNA.

[0096] (5) Measure the concentration of the extracted nucleic acid using the NanoDrop and Qubit kits.

[0097] 3. Rapid library preparation and high-throughput sequencing of high-purity Pieris mitochondria DNA:

[0098] (1) Tn5 transposase cleavage reaction: Take 5 uL of the above Pieris mitochondria DNA and mix with Tn5 transposase (RK20547, ABclone) to prepare a 20 uL reaction system, treat at 55°C for 5 min, and add 5 uL of termination solution.

[0099] (2) PCR amplification reaction with sequencing adapters: Directly add 10 uL of universal primer pair containing sequencing adapters (sequencing through primer i5 / i7 primer) and 15 uL of PCR amplification mixture (RK20726, ABclone) to the above 25 uL cleavage reaction to form a 50 uL PCR reaction system.

[0100] (3) PCR reaction conditions: 72°C for 3 min, then 10-14 cycles of 95°C / 10 sec, 55°C / 30 sec, 72°C / 30 sec, and finally 72°C / 5 min.

[0101] (5) Magnetic bead purification (AMpure XP (Agencourt)) to obtain high-quality mitochondria DNA library, and Qubit and 2100 library quality control.

[0102] (6) High-throughput sequencing of Pieris mitochondria DNA library: Mix multiple library samples containing independent adapters in equal amounts or as needed, and then use any model of illumine high-throughput sequencer for multiplex sequencing and analysis.

[0103] 4. Primary analysis, de novo assembly, and annotation analysis of Pieris sequencing data of the mitochondria genome:

[0104] (1) Align the sequencing data of the Pieris mitochondria genome with the mitochondria reference genome of its closest species, perform variation analysis and resequencing analysis (resequencing analysis refers to data alignment analysis using the existing species mitochondria genome information, without assembly analysis), and the results are shown in Tables 1, 2, and Fig. 1.

[0105] (2) Mitochondrial genome de novo assembly analysis process: using MitoZ software (Nucleic Acids Research, 2019) for quality control of sequencing data of each sample, de novo assembly and annotation analysis of mitochondrial genome, etc. The results are shown in Table 3 and Figure 2.

[0106] Table 1, the mitochondrial genome sequencing parameters of the sample of Pieris rapae analyzed in the present application

[0107] According to Table 1, the ratio of mtDNA% to total DNA extracted in this embodiment is 92.70%, indicating that the kit of the present application can extract mitochondrial DNA with very high purity. The sequencing coverage reaches 100%, and the average sequencing depth reaches 22328x, indicating that the sequencing data can cover all sites of the genome, with very high sequencing coverage.

[0108] The sequencing data of the mitochondrial genome of Pieris rapae in the present embodiment is compared with the reference genome OL890650, and the results of the variant sites and frequencies are shown in Figure 1 and Table 2.

[0109] The results of Figure 1 and Table 2 show that this embodiment can produce high-quality sequencing data, and the present application can produce high-quality sequencing data of Pieris rapae and effectively detect various variant sites and mutation rates. Not only can it detect low-frequency variant sites, but it can also effectively detect mutations with an abundance of 1.7%, and it can efficiently detect SNP variant sites, greatly improving the analysis efficiency.

[0110] Table 2, the variant sites and frequencies of the mitochondrial DNA of Pieris rapae in this embodiment compared with the reference genome (OL890650)

[0111] This embodiment can extract mitochondrial DNA of Pieris rapae with high purity, and when performing sequencing analysis, it can de novo assemble and annotate the complete mitochondrial genome sequence of Pieris rapae without any sequence information of the test sample. The mitochondrial assembly map is shown in Figure 2, and the sequencing, assembly and annotation analysis are shown in Table 3.

[0112] Table 3, sequencing, assembly and annotation analysis of the sample of Pieris rapae in this embodiment

[0113] The results of Figure 2 and Table 3 show that the total sequencing bases (data amount) generated by the present embodiment is 445.4 Mb (which is actually much more than the data amount required for assembling and annotating an unknown mitochondrial genome), and the mitochondrial genome with a coverage depth of 15426x can be assembled, and all 37 genes can be completely annotated. Even if 10 Mb of the sequencing data amount (i.e., the data amount is reduced by 43 times) is selected for assembly analysis, all 37 genes can still be assembled and annotated, and a coverage depth of 368x can be reached.

[0114] The present application does not need to design PCR primers for enrichment from total DNA, which reduces the difficulty of data analysis of sequencing, and the mitochondrial genome sequencing data generated by the extracted mitochondrial DNA of the present application is a high-fidelity data, which avoids the influence of PCR errors, biases and pseudo genes in nuclear DNA brought by PCR amplification enrichment, and greatly reduces the sequencing error rate.

[0115] Example 2 One-step extraction and sequencing of mitochondrial genome DNA of crustacean (Macrobrachium rosenbergii)

[0116] 1. Sampling of Macrobrachium rosenbergii

[0117] (1) Macrobrachium rosenbergii was purchased from the market.

[0118] (2) One of the compound eyes of Macrobrachium rosenbergii was sampled with a sterile clock watch forceps and small surgical scissors and then transferred to a 2 ml sterile microtube containing 500 uL of reagent A (containing 3.85 M guanidine isothiocyanate, 0.7 M sodium citrate and 5% N-lauroylsarcosine sodium salt), and after shaking and mixing, Macrobrachium rosenbergii tissue preservation solution was obtained, which can be placed at room temperature or 4°C for a short time until the following extraction is performed.

[0119] 2. Extraction and preparation of mitochondrial genome DNA of Macrobrachium rosenbergii

[0120] (1) 250 uL of the above Macrobrachium rosenbergii tissue preservation solution was transferred to a sterile 2 mL microtube, and then 350 uL of reagent A was added, and the total volume was 600 uL. The Macrobrachium rosenbergii tissue was fully lysed by vortexing for 1-2 minutes to obtain Macrobrachium rosenbergii tissue lysate.

[0121] The remaining steps of the extraction and preparation of the mitochondrial genome DNA of Macrobrachium rosenbergii refer to steps (2)-(5) of the extraction and preparation steps of step 2 of Example 1.

[0122] 3. Rapid library preparation and high-throughput sequencing of high-purity mitochondrial DNA of Macrobrachium rosenbergii:

[0123] (1) Tn5 transposase cleavage reaction: 5 μL of the above-mentioned mitochondrial DNA of rosy shrimp was mixed with Tn5 transposase (RK20547, ABclone), and a 20 uL reaction system was prepared, treated at 55°C for 5 min, and 5 ul of termination liquid was added.

[0124] The remaining steps refer to steps (2)-(6) of the rapid library preparation and high-throughput sequencing steps of step 3 of example 1.

[0125] 4. Primary analysis, de novo assembly and annotation analysis of the mitochondrial genome of rosy shrimp sequencing data:

[0126] (1) Align the sequencing data of the mitochondrial genome of rosy shrimp with the mitochondrial reference genome of its closest species, perform variant analysis and resequencing analysis (resequencing analysis refers to data alignment analysis using the mitochondrial genome information of the existing species, without assembly analysis), the results are shown in Table 4, Table 5 and Figure 3.

[0127] (2) Mitochondrial genome de novo assembly analysis process: using MitoZ software (Nucleic Acids Research, 2019) for quality control of sequencing data of each sample, de novo assembly and annotation analysis of mitochondrial genome, etc., the results are shown in Table 6 and Figure 4.

[0128] Table 4, mitochondrial genome sequencing parameters of rosy shrimp sample analyzed in the present application

[0129] According to Table 4, the ratio of mtDNA% to total DNA extracted in this example is 96.68%, indicating that the mitochondrial DNA of rosy shrimp with very high purity is extracted. The sequencing coverage reaches 100%, and the average sequencing depth reaches 11846x, indicating that the sequencing data can cover all sites of the genome, with very high sequencing coverage.

[0130] The results of the sequencing data of the mitochondrial genome of rosy shrimp in the variation site and frequency compared with the reference genome (MZ781227.1) are shown in Figure 3 and Table 5.

[0131] The results of Figure 3 and Table 5 show that this example can produce high-quality sequencing data, and the present application can produce high-quality rosy shrimp sequencing data and effectively detect various variation sites and variation rates, not only can detect low-frequency variation sites, but also can efficiently detect SNP variation sites, greatly improving the analysis efficiency.

[0132] Table 5, variation sites and frequencies of rosy shrimp mitochondrial DNA compared with the reference genome (MZ781227.1) in this example

[0133] The present embodiment can extract the mitochondrial DNA of Procambarus clarkii with high purity. When sequencing analysis is performed, the complete mitochondrial genome sequence of Procambarus clarkii can be assembled and annotated from scratch without any sequence information of the sample to be tested. The mitochondrial assembly map is shown in FIG. 4, and the sequencing, assembly and annotation analysis is shown in Table 6.

[0134] Table 6, sequencing, assembly and annotation analysis of the sample of Procambarus clarkii in Example 1

[0135] The results of FIG. 4 and Table 6 show that the total sequencing base number (data amount) generated in the present embodiment is 437.9 Mb (the sequencing data amount is actually much more than the data amount required for assembling and annotating an unknown mitochondrial genome), the mitochondrial genome with a coverage depth of 16550x can be assembled, and all 37 genes can be completely annotated. The sequencing data amount is actually much more than the data amount required for assembling and annotating an unknown mitochondrial genome. Even if 10 Mb of the data amount (i.e., the data amount is reduced by 42 times) is selected for assembly analysis, all 37 genes can still be assembled and annotated to achieve a coverage depth of 461x.

[0136] The present application does not need to design PCR primers for enrichment from total DNA, which reduces the difficulty of data analysis of sequencing. The mitochondrial genome sequencing data generated by the mitochondrial DNA extracted by the present application is a high-fidelity data, which avoids the influence of PCR errors, biases and pseudo genes in nuclear DNA brought by PCR amplification enrichment, and greatly reduces the sequencing error rate.

[0137] In the above embodiments, the components in the reagent A can be adjusted within a certain concentration range, specifically, it can include 3.25-4.75 M guanidine isothiocyanate, 0.65-0.85 M sodium citrate and 5-15 v / v% N-lauroylsarcosine sodium salt, and within the range, the mitochondrial DNA with high purity can be obtained by cooperating with reagents B and C, and the ratio of mitochondrial DNA can reach 50-99.9%, even more than 75%.

[0138] In the above embodiments, the concentration of sodium acetate in the reagent B can be 1.7-2.5 M, and within the range, the mitochondrial DNA with high purity can be obtained by cooperating with reagents A and C, and the ratio of mitochondrial DNA can reach 50-99.9%, even more than 75%.

[0139] In the above embodiments, the components in the reagent C can be adjusted within a certain range, specifically, it can include phenol, chloroform and isoamyl alcohol in a volume ratio of (120-127):(22-26):1, and within the range, the mitochondrial DNA with high purity can be obtained by cooperating with reagents A, B and C, and the ratio of mitochondrial DNA can reach 50-99.9%, even more than 75%.

[0140] Comparative Example 1

[0141] 1) The total DNA of Spodoptera litura was extracted by using the conventional mitochondrial genome kit.

[0142] 2) The library was prepared by using the illumina DNA library kit (TrueSeq DNA), and high-throughput sequencing was performed by using the illumina HiSeq2000. The obtained raw sequencing data was published in the NBCI-xx database and can be obtained by SRA number: SRR5132384 (Nat. Ecol. Ecol., 2017). We used the raw sequencing data obtained by SRR5132384, and the analysis process of step 4 in Example 1 was used for the preliminary resequencing analysis of the comparison data, the de novo assembly and annotation analysis. The resequencing analysis results are shown in Tables 7-8 and Figure 5, and the assembly and annotation analysis results are shown in Table 9 and Figure 6.

[0143] Table 7, Parameters of sequencing mitochondrial genome by total DNA sequencing method of Spodoptera litura

[0144] The results of Table 7 show that the ratio of mtDNA% in total DNA extracted by the present comparative example is only 0.5%, the mitochondrial DNA purity is low, and the total sequencing data amount is as high as 5305 Mb to produce 1332x mtDNA sequencing depth, which shows that the sequencing data amount of the existing mitochondrial DNA sequencing method is large, which greatly improves the sequencing cost and analysis difficulty.

[0145] The sequencing data of the mitochondrial genome of Spodoptera litura was compared with the reference genome (KF701043.1) at the variant sites and frequency results as shown in Figure 5 and Table 8.

[0146] Table 8, Variant sites and frequency of mitochondrial genome compared with reference genome (KF701043.1) by total DNA sequencing method of Spodoptera litura

[0147] The results of Figure 5 and Table 8 show that the mtDNA sequencing data based on total DNA of H. zea significantly reduces the sensitivity and specificity in detecting high frequency SNPs or low frequency mutation sites. For example, the mtDNA sequencing data of the comparative example detected up to 582 (233 + 349) low frequency mutation sites, including 349 mutation sites with a load higher than 1% and 233 sites with a load lower than 1%. The generation of these large numbers of low frequency mutation sites is not likely to be real variation but only the result of various sequencing errors and interference of nuclear DNA sequencing data (those skilled in the art know that the mtDNA low frequency somatic mutation rate is relatively low, and a sample can detect a few (0-10 variations), and theoretically cannot reach dozens or hundreds). Therefore, the mitochondrial DNA data based on total DNA sequencing faces great challenges in the accurate analysis of various mutation sites.

[0148] The mitochondrial assembly map using the sequencing data (1.3 Gb, i.e., about 1300 Mb) of part of H. zea in the comparative example is shown in Figure 6, and the sequencing, assembly and annotation analysis is shown in Table 9.

[0149] Table 9, sequencing, assembly and annotation analysis of total DNA samples of H. zea

[0150] The results of Figure 6 and Table 9 show that the comparative example using 1.3 Gb (i.e., about 1300 Mb) of total DNA sequencing data can only assemble a mitochondrial genome with a coverage depth of 196x, and only 36 genes are assembled and annotated (37 genes are annotated in Examples 1 and 2, and one mitochondrial gene is missing compared to Examples 1 and 2). This shows that total DNA sequencing data not only requires more data to assemble a complete mitochondrial genome, but also often results in low coverage depth and incomplete assembly due to insufficient sequencing data.

[0151] The results of Comparative Example 1 and the prior art show that the mitochondrial DNA ratio is only about 0.05-1%, and the enrichment efficiency is low, resulting in higher sequencing cost and difficulty in subsequent sequencing analysis.

[0152] Using the kit or method of the present application, only 10-500 Mb of sequencing data is required to assemble a complete mitochondrial genome in de novo assembly analysis, which is 10-500 times less than the amount of existing total DNA sequencing data; in resequencing analysis, 0.36 Mb of sequencing data can produce a mitochondrial genome sequencing depth of 1400x or more, which is at least 10,000 times less than the amount of data required to achieve the same sequencing depth by the existing total DNA sequencing method, greatly reducing the sequencing cost and analysis difficulty.

[0153] The application can extract high-purity mitochondrial DNA from insect tissues with low mitochondrial DNA content, and the proportion of extracted mitochondrial DNA in total DNA sequencing can reach more than 75%, which can effectively increase the sequencing ratio of mitochondrial DNA in total DNA, indicating that the enrichment efficiency of the application is thousands of times higher than that of the prior art, and far exceeds that of commonly used enrichment extraction kits on the market. Combined with the Tn5 transposase scheme, the small amount of circular mitochondrial DNA extracted from insect tissues can be more effectively converted into a DNA sequencing library. This technical progress overcomes the difficulty of using a single small insect for mitochondrial genome sequencing analysis, and has important practical significance in the identification of numerous small insects and biodiversity surveys.

[0154] The application is a universal method for rapid preparation and library construction of mitochondrial genome DNA of small arthropods (such as insects), and is used for mitochondrial whole genome sequencing, which overcomes various limitations of existing methods:

[0155] 1) The application does not require prior acquisition of mitochondrial genome information of the species to be tested, and is a species-independent method, providing an effective mitochondrial DNA extraction and library preparation method for various insects with a large number of species and lacking mitochondrial genome information.

[0156] 2) The Tn5 transposase scheme of the application can more effectively convert the small amount of circular mitochondrial DNA extracted from insect tissues into a DNA sequencing library. This technical progress overcomes the difficulty of using a single small insect for mitochondrial genome sequencing analysis, and has important practical significance in the identification of numerous small insects and biodiversity surveys.

[0157] 3) The application is a method for directly extracting high-purity mitochondrial DNA from small arthropod (such as insect) tissues and organs and using it for genome sequencing, without the need to first prepare total DNA of insect tissues, and then secondarily enrich mitochondrial DNA through PCR amplification or probe capture. Therefore, the method of the application is not only simple and fast, but also avoids the cumbersome technical operations and high experimental costs of PCR amplification or probe capture.

[0158] 4) Compared with insect total DNA sequencing data, the content or ratio of mitochondrial DNA in the sequencing product is increased from about 0.1% in total DNA sequencing of the prior art to 50-99% in the sequencing of the application, and the enrichment efficiency is increased by thousands of times. Since the application greatly improves the sequencing efficiency of mitochondrial DNA, it not only overcomes the limitation of total DNA sequencing method that cannot use a single small insect individual for sequencing, but also can simultaneously determine dozens or more mitochondrial DNA samples through a total DNA sample equivalent sequencing data amount. This not only reduces the waste of rare insect samples, but also greatly reduces the sequencing cost of individual samples.

[0159] 5) The mitochondrial genome sequencing data generated by the present application is a high-fidelity and high-sequencing-depth sequencing data. The high-fidelity refers to avoiding the PCR errors and biases brought by PCR amplification enrichment or the influence of pseudogenes in nuclear DNA, and the high-sequencing-depth overcomes the problem that the complete mitochondrial genome cannot be assembled due to insufficient target sequencing data. In summary, the present application provides a general solution for species-independent mitochondrial genome sequencing of the most diverse small insects, and especially solves the technical problem of the feasibility of species identification and biodiversity investigation of individual small insects.

[0160] The present application can use the trace amount of mitochondrial DNA extracted from individual small insects for library preparation and high-throughput sequencing, and provides a simple and efficient general technology for mitochondrial whole genome sequencing analysis of the most diverse small insects.

[0161] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. Industrial applicability

[0162] The present application provides a kit for small arthropod mitochondrial genome DNA extraction and sequencing, a sequencing method and its application, wherein the kit comprises the following components: 1) reagent A comprises cell lysis and balancing reagent; 2) reagent B comprises sodium acetate; 3) reagent C comprises phenol, chloroform and isoamyl alcohol with a volume ratio of (120-130):(19-29):1; wherein reagents B and C are used to mix with the lysis solution containing total DNA and obtain the aqueous phase containing mitochondrial DNA by layering; 4) Tn5 transposase system. The present application can use the trace amount of mitochondrial DNA extracted from individual small insects for library preparation and high-throughput sequencing, and provides a simple and efficient general technology for mitochondrial whole genome sequencing analysis of the most diverse small insects. The amount of sequencing data required for mitochondrial genome assembly using the kit or method of the present application can be reduced by tens of times or even thousands of times compared to the amount of data of the existing total DNA method, greatly reducing the sequencing cost and analysis difficulty.

Claims

1. A kit for small arthropod mitochondrial genome DNA extraction and sequencing, characterized by, Comprise the following components: 1) Reagent A: comprising cell lysis and balancing reagent, for lysing small arthropod tissue to obtain a lysate containing total DNA or preserving tissue lysate; 2) Reagent B: comprising 1.7-2.5 M sodium acetate; 3) Reagent C: comprising phenol, chloroform and isoamyl alcohol in a volume ratio of (120-130):(19-29):1; wherein, reagent B and C are used to mix with the lysate containing total DNA and obtain the water phase containing mitochondrial DNA by layering; 4) Tn5 transposase system, for sequencing library preparation.

2. The kit of claim 1, wherein In reagent A, the cell lysis and balancing reagent comprises 3.25-4.75 M guanidine isothiocyanate, 0.65-0.85 M sodium citrate and 5-15 v / v% N-lauroylsarcosine sodium salt; optionally comprising 3.85-4.25 M guanidine isothiocyanate, 0.7-0.8 M sodium citrate and 5-10 v / v% N-lauroylsarcosine sodium salt; optionally comprising 3.85-4.1 M guanidine isothiocyanate, 0.7-0.74 M sodium citrate and 5-10 v / v% N-lauroylsarcosine sodium salt; And / or, in reagent B, the concentration of sodium acetate is 1.8-2 M; And / or, in reagent C, the volume ratio of phenol, chloroform and isoamyl alcohol is (120-127):(22-26):

1.

3. A method for small arthropod mitochondrial genome DNA extraction and sequencing, characterized by, The kit of claim 1 or 2 comprises the following steps: I) Extracting mitochondrial DNA from small arthropod tissue organ samples, mixing reagent B and C with reagent A lysed tissue lysate, layering, precipitating, and obtaining mitochondrial DNA; II) Taking the mitochondrial DNA of step I) and mixing it with the Tn5 transposase system to perform cleavage reaction, and using the cleavage reaction product for sequencing.

4. The method of claim 3, wherein, In step I), the extraction method of mitochondrial DNA is: 1) Taking small arthropod tissue organs; 2) Mixing the tissue organ sample with reagent A to lyse the cells and release free nucleic acids to obtain a lysate; 3) Adding reagent B and reagent C to the lysate of step 2), mixing thoroughly, and waiting for the water phase and organic phase to layer; 4) Taking the water phase containing mitochondrial DNA in step 3), and precipitating and recovering small arthropod mitochondrial DNA by alcohol.

5. The method of claim 4, wherein, In step 1), the small arthropod tissue organs include one or more of the head, cephalothorax, compound eye, and arthropod part; And / or, in step 1), the sample amount is 1-20 mg; And / or, the tissue organs are processed into small pieces by cutting or crushing; And / or, in step 1), the sample is preserved with reagent A; And / or, the tissue organs are freshly collected, or frozen, or ethanol preserved, or reagent A preserved.

6. The method according to claim 4 or 5, characterized in that, In step 3), 7-13% reagent B is added according to the volume fraction of the lysate; And / or, in step 3), the amount of reagent C added is 1-1.5 times the volume of the lysate; And / or, in steps 2) and 3), the mixing method includes shaking; And / or, in step 3), the water phase and organic phase are layered by low-temperature high-speed centrifugation.

7. The method according to any one of claims 3 to 6, characterized in that, In step 4), anhydrous ethanol or isopropanol is used for alcohol precipitation; and / or, in step 4), the obtained mitochondrial DNA is dissolved in TE solution.

8. The method according to any one of claims 3 to 7, characterized in that, In step II), the sequencing method comprises: library preparation: the cleavage product of Tn5 transposase is added into universal sequencing adapters by PCR amplification, purification, multiplex sequencing and analysis by multiple mitochondrial DNA library mixing; the optional purification method is magnetic bead purification.

9. Use of the kit of claim 1 or 2 or the method of any one of claims 3 to 8 in the manufacture of a de novo assembly analysis product, or a re-sequencing analysis product, or a mutation analysis product, of a mitochondrial genome of a small arthropod, optionally the mutation analysis product comprises a base and structural mutation analysis product.

10. Use of the kit of claim 1 or 2 or the method of any one of claims 3 to 8 in the manufacture of a small arthropod species identification and / or diversity analysis product.

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