Pigeon mitochondrial whole genome amplification and assembly method

Abstract

The invention discloses a pigeon mitochondrial whole genome amplification and assembly method. The method comprises the following steps: (1) extracting genome DNA of a to-be-detected pigeon; (2) carrying out PCR (Polymerase Chain Reaction) amplification through the primers to obtain two sections of pigeon mitochondrial DNA (Deoxyribose Nucleic Acid) fragments, and (3) carrying out high-throughput sequencing; and (4) assembling the mitochondrial genome to obtain the mitochondrial DNA full length.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for amplifying and assembling a pigeon mitochondrial whole genome. Background Art

[0002] As an important component of germplasm resources and even biodiversity, the protection and utilization of livestock and poultry genetic resources are of great significance for maintaining ecological balance and promoting sustainable agricultural development. Therefore, analyzing and evaluating the genetic diversity of local pigeon breeds is of great significance.

[0003] Animal cells contain two genomic systems: the nuclear genome and the mitochondrial genome. Mitochondrial DNA has an independent genetic coding system that does not follow Mendel's laws of inheritance. Its genome is simple, lacks homologous recombination, undergoes rapid sequence evolution, and is strictly maternally inherited. It is widely used in studies of animal origins and evolution, population genetic structure, species identification, and economic traits. Only a small sample of individuals is needed to reveal the genetic structure of a population. Pigeon mitochondria contain a D-loop region, 13 protein-coding genes, two rRNA genes (12S rRNA and 16S rRNA), and 22 tRNA genes.

[0004] The traditional method for obtaining the pigeon mitochondrial genome combines a primer walking strategy, designing multiple pairs of overlapping primers for PCR amplification and Sanger sequencing. Finally, the sequenced fragments are spliced together to obtain the complete mitochondrial genome. The pigeon mitochondrial genome is approximately 17,239 base pairs, and the effective read length of Sanger sequencing is typically around 700 base pairs. Including overlapping regions, more than 30 sequencing reactions are required to complete the sequence. This cumbersome process can also result in poor sequencing accuracy in some regions with high GC content.

[0005] With the development of second-generation sequencing technology and the rapid decline in sequencing costs, more and more researchers have begun using second-generation sequencing methods to obtain pigeon mitochondrial genomes. This method directly and randomly fragments the total DNA of the sample into small DNA fragments. The two ends of the small DNA fragments are then connected and sequenced with sequencing adapter sequences. Finally, the sequencing data is spliced using bioinformatics methods to obtain the sequence information of the entire mitochondrial genome. Because nuclear genomic DNA accounts for the absolute majority of the total DNA in the sample, the mitochondrial genome accounts for a relatively small proportion of the data generated by second-generation sequencing. To solve the problem of insufficient sequencing data, the total sequencing data volume can only be increased, which leads to increased costs. At the same time, the data processing and splicing process is cumbersome and requires professional bioinformatics knowledge and a large amount of computing resources, which is not conducive to its promotion and application in ordinary laboratories.

[0006] Therefore, there is an urgent need to develop a method for obtaining pigeon mitochondrial genomes that is simple, accurate, and low-cost. Summary of the Invention

[0007] The technical problem that the present invention aims to solve is to address the deficiencies of the above-mentioned prior art. The present invention provides a method for amplifying and assembling the whole mitochondrial genome of pigeons, comprising: (1) sample collection; (2) PCR amplification; (3) high-throughput sequencing; and (4) mitochondrial genome assembly. The method is easy to operate and has a high accuracy rate.

[0008] The present invention achieves the purpose of the present invention through the following technical solutions:

[0009] One of the purposes of the present invention is to provide a PCR primer for amplifying and assembling a pigeon mitochondrial genome, wherein the nucleotide sequence of the primer is:

[0010] The first pair of forward primers: 5'-AACCCTTGTACCTCTTGCATCAT-3' (SEQ ID NO. 1);

[0011] The first pair of reverse primers: 5'-AATGACGTTGCTTGGTTTTCGAT-3' (SEQ ID NO. 2);

[0012] The second pair of forward primers: 5'-TTCGACTCAACAGACCATAGCTC-3' (SEQ ID NO. 3);

[0013] The second pair of reverse primers: 5'-TCGATTGGCTTTTCACCTCTACT-3' (SEQ ID NO. 4). A second object of the present invention is to provide an application in a method for amplifying and assembling a pigeon mitochondrial genome, comprising: (1) extracting genomic DNA from a pigeon to be tested; (2) performing PCR amplification using the primers of the present invention to obtain two pigeon mitochondrial DNA fragments; (3) performing high-throughput sequencing; and (4) assembling the mitochondrial genome to obtain the full-length mitochondrial DNA.

[0014] In the method of the present invention, step (1) extracting the genomic DNA of the pigeon to be tested can be performed according to conventional methods in the art. For example, the genomic DNA of the pigeon to be tested can be extracted from materials including but not limited to feathers, blood, tissues, organs, etc.

[0015] In the method of the present invention, the PCR reaction system in step (2) can be a conventional system in the art. In a specific embodiment of the present invention, the PCR reaction system can be, for example, 25 μL of 2×Vazyme LAmp Master Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 2 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 19 μL of ultrapure water.

[0016] In the method described in the present invention, the PCR reaction procedure in step (2) can be a conventional system in the art. In a specific embodiment of the present invention, it is: 95°C for 5 min, 35 cycles of (95°C for 30 s, 66°C for 9 min), and 72°C for 10 min.

[0017] The method of the present invention, step (3) Chinese library construction can be a conventional operation in the art. In a specific embodiment of the present invention,

[0018] (1) Preparation of enzyme digestion system

[0019] a) Prepare sterile, enzyme-free PCR tubes and set up the enzyme digestion reaction system on ice; input 50 ng of amplified product, 1 μL of Tn5 Enzyme, and add Working Buffer to 20 μL;

[0020] b) After the enzyme digestion system is prepared, place the PCR tube on a PCR instrument with a pre-heated cover at 70°C and a temperature of 55°C, and perform the enzyme digestion reaction for 7 minutes.

[0021] (2) Termination of enzyme digestion reaction

[0022] a) After the enzyme digestion reaction is complete, place the tube on ice immediately and prepare the enzyme digestion stop reaction system: 20 μL of the enzyme digestion system from the previous step and 1 μL of Stop Buffer.

[0023] b) Place the PCR tube on a PCR instrument pre-heated at 70°C and 55°C, and perform the end-enzyme digestion program for 5 minutes.

[0024] (3) Purification and recovery of enzyme digestion products

[0025] a) Preparation: Remove DNA Selection Beads magnetic beads from the refrigerator and equilibrate them at room temperature for at least 30 minutes. Meanwhile, prepare 80% ethanol.

[0026] b) Vortex or invert the beads thoroughly to ensure thorough mixing.

[0027] c) Pipette 20 μl of DNA Selection Beads (0.9X, Beads:DNA=0.9:1) into the product after enzyme digestion, vortex or pipette to mix, and incubate at room temperature for 5 minutes.

[0028] d) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid. After the solution has clarified (approximately 5 minutes), carefully remove the supernatant.

[0029] e) Keeping the PCR tube in the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant.

[0030] f) Repeat step e and rinse once more.

[0031] g) Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads until they just begin to crack (no more than 5 minutes).

[0032] h) Remove the PCR tube from the magnetic rack and add 15 μl of ddH2O. Vortex or gently pipette to mix thoroughly. Let stand at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on the magnetic rack. Once the solution has cleared (approximately 3 minutes), carefully transfer 15 μl of the supernatant to a new PCR tube for the next amplification step.

[0033] (4) Library amplification

[0034] a) Prepare the amplification system: 10 μL of purified product, 15 μL of 2X High-Fidelity Mix, 2 μL of UDI, and add HO to 30 μL.

[0035] b) Amplification program: 72°C for 3 min, 98°C for 3 min, 7-15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 30 s), 72°C for 3 min.

[0036] (5) Library quality control

[0037] Typically, the quality of the constructed library can be evaluated by concentration detection and length distribution detection.

[0038] (6) High-throughput sequencing

[0039] The constructed NGS library was sequenced using an Illumina MiSeq sequencer in SE150 or PE150 mode.

[0040] In the method of the present invention, the mitochondrial genome assembly in step (4) can be a conventional operation in the art. In a specific embodiment of the present invention,

[0041] (1) Use cutadapt (v 1.2.1) software to remove any partial sequence containing sequencing adapter sequences;

[0042] (2) PRINSEQ-lite (v 0.20.3) software was used to perform quality control on the remaining sequences. Bases with a quality threshold lower than 20 were deleted from the 3' end to the 5' end of the sequence. The remaining sequences were considered to have passed the quality control.

[0043] (3) SPAdes (v3.15.0) software was used to assemble the sequences that passed quality control, and the default parameters were selected to output the spliced sequences.

[0044] Compared with the prior art, the present invention has the following advantages:

[0045] (1) Conventional sequencing requires the design of more than 30 amplification or sequencing reactions to complete the test. The present invention only requires two pairs of primers to cover the entire length of the pigeon mitochondrial genome through the designed PCR primers, which greatly reduces the workload.

[0046] (2) Directly construct a library of two long-fragment PCR products for high-throughput sequencing, which has high accuracy and is priced at only one-fifth of conventional sequencing, one-tenth of the cost of second-generation sequencing, and one-twenty-fifth of the cost of third-generation sequencing. The specific comparison is shown in Table 1.

[0047] Table 1 Comparison of different methods for obtaining full-length mitochondrial genomes

[0048]

[0049] (3) The primers of the present invention have little restriction on pigeon breeds, so there is no restriction on the specific breed of the pigeons described in the present invention.

[0050] (4) The method for obtaining the pigeon mitochondrial genome of the present invention obtains two fragments of the full-length mitochondrial DNA by one-step PCR amplification from the total DNA, which can ensure the accurate amplification of the pigeon mitochondrial genome and is not contaminated by homologous sequences in the pigeon genome, making mutation detection more sensitive and accurate and saving time.

[0051] (5) The mitochondrial DNA library constructed by the present invention is subjected to high-throughput sequencing, and bioinformatics analysis is performed to obtain the mitochondrial genome DNA sequence of the test sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The structure of the assembled pigeon mitochondrial genome. DETAILED DESCRIPTION

[0053] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0054] Example 1

[0055] 1 Establishment of pigeon mitochondrial genome method

[0056] 1.1 Primer design

[0057] With reference to the standard sequence of pigeon mitochondrial genome (NCBI database ID number: NC013978), two pairs of primers were designed to cover the full length of the pigeon mitochondrial genome. The length of the amplified product of the first pair was 8819 bp, and the length of the amplified product of the second pair was 8799 bp, with an overlapping region of 379 bp.

[0058] The first pair of forward primers: 5′-AACCCTTGTACCTCTTGCATCAT-3′;

[0059] The first pair of reverse primers: 5′-AATGACGTTGCTTGGTTTTCGAT-3′;

[0060] The second pair of forward primers: 5′-TTCGACTCAACAGACCATAGCTC-3′;

[0061] The second pair of reverse primers: 5'-TCGATTGGCTTTTCACCTCTACT-3'.

[0062] 1.2 PCR amplification

[0063] The primers designed by the present invention were used to amplify the Tarim pigeon DNA preserved by the research group.

[0064] The PCR reaction system is:

[0065] 25 μL of 2×Vazyme LAmp Master Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 2 μL of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 19 μL of ultrapure water.

[0066] The PCR reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 66°C for 9 min), and 72°C for 10 min.

[0067] 1.3 High-throughput sequencing

[0068] (1) Preparation of enzyme digestion system

[0069] a) Prepare sterile, enzyme-free PCR tubes and prepare the enzyme digestion reaction system on ice. Input 50 ng of amplified product, 1 μL of Tn5 Enzyme, and add 20 μL of Working Buffer.

[0070] b) After the enzyme digestion system is prepared, place the PCR tube on a PCR instrument with a pre-heated cover at 70°C and a temperature of 55°C, and perform the enzyme digestion reaction for 7 minutes.

[0071] (2) Termination of enzyme digestion reaction

[0072] a) After the enzyme digestion reaction is complete, place the tube on ice immediately and prepare the enzyme digestion stop reaction system: 20 μL of the enzyme digestion system from the previous step and 1 μL of Stop Buffer.

[0073] b) Place the PCR tube on a PCR instrument pre-heated at 70°C and 55°C, and perform the end-enzyme digestion program for 5 minutes.

[0074] (3) Purification and recovery of enzyme digestion products

[0075] a) Preparation: Remove DNA Selection Beads magnetic beads from the refrigerator and equilibrate them at room temperature for at least 30 minutes. Meanwhile, prepare 80% ethanol.

[0076] b) Vortex or invert the beads thoroughly to ensure thorough mixing.

[0077] c) Pipette 20 μl of DNA Selection Beads (0.9X, Beads:DNA=0.9:1) into the product after enzyme digestion, vortex or pipette to mix, and incubate at room temperature for 5 minutes.

[0078] d) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid. After the solution has clarified (approximately 5 minutes), carefully remove the supernatant.

[0079] e) Keeping the PCR tube in the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant.

[0080] f) Repeat step e and rinse once more.

[0081] g) Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads until they just begin to crack (no more than 5 minutes).

[0082] h) Remove the PCR tube from the magnetic rack and add 15 μl of ddH2O. Vortex or gently pipette to mix thoroughly. Let stand at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on the magnetic rack. Once the solution has cleared (approximately 3 minutes), carefully transfer 15 μl of the supernatant to a new PCR tube for the next amplification step.

[0083] (4) Library amplification

[0084] a) Prepare the amplification system: 10 μL of purified product, 15 μL of 2X High-Fidelity Mix, 2 μL of UDI, and add HO to 30 μL.

[0085] b) Amplification program: 72°C for 3 min, 98°C for 3 min, 7-15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 30 s), 72°C for 3 min.

[0086] (5) Library quality control

[0087] Typically, the quality of the constructed library can be evaluated by concentration detection and length distribution detection.

[0088] (6) High-throughput sequencing

[0089] The constructed NGS library was sequenced using an Illumina MiSeq sequencer in SE150 or PE150 mode.

[0090] 1.4 Mitochondrial genome assembly

[0091] (1) Use cutadapt (v 1.2.1) software to remove any partial sequence containing sequencing adapter sequences;

[0092] (2) PRINSEQ-lite (v 0.20.3) software was used to perform quality control on the remaining sequences. Bases with a quality threshold lower than 20 were deleted from the 3' end to the 5' end of the sequence. The remaining sequences were considered to have passed the quality control.

[0093] (3) SPAdes (v3.15.0) software was used to assemble the sequences that passed the quality control. The default parameters were selected and the spliced sequences were output. The assembled Tarim pigeon mitochondrial genome was 17279 bp long. After gene annotation, it contained 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA), 13 protein-coding genes and 1 D-loop region ( Figure 1 ).

[0094] 1.5 Comparison with third-generation sequencing results

[0095] The Tarim pigeon DNA sample was directly subjected to third-generation sequencing, and the sequence identity with the present invention was 100%, indicating that the method of the present invention can ensure the accurate amplification of the pigeon mitochondrial genome and is not contaminated by homologous sequences of the pigeon nuclear genome.

Claims

1. A method for amplifying and assembling a pigeon mitochondrial genome using PCR primers, wherein the two pairs of primers are as follows: The first pair of forward primers: 5'-AACCCTTGTACCTCTTGCATCAT-3' (SEQ ID NO. 1); The first pair of reverse primers: 5'-AATGACGTTGCTTGGTTTTCGAT-3' (SEQ ID NO. 2); The second pair of forward primers: 5'-TTCGACTCAACAGACCATAGCTC-3' (SEQ ID NO. 3); The second pair of reverse primers: 5'-TCGATTGGCTTTTCACCTCTACT-3' (SEQ ID NO. 4).

2. Application of the PCR primers according to claim 1 in obtaining pigeon mitochondrial genome.

3. A method for amplifying and assembling the whole mitochondrial genome of a pigeon, characterized in that The method comprises the following steps: (1) extracting genomic DNA from a pigeon to be tested; (2) performing PCR amplification using primers to obtain two pigeon mitochondrial DNA fragments; (3) performing high-throughput sequencing; and (4) assembling the mitochondrial genome to obtain the full-length mitochondrial DNA. The primer sequences used in step (2) are as follows: The first pair of forward primers: 5'-AACCCTTGTACCTCTTGCATCAT-3' (SEQ ID NO. 1); The first pair of reverse primers: 5'-AATGACGTTGCTTGGTTTTCGAT-3' (SEQ ID NO. 2); The second pair of forward primers: 5'-TTCGACTCAACAGACCATAGCTC-3' (SEQ ID NO. 3); The second pair of reverse primers: 5'-TCGATTGGCTTTTCACCTCTACT-3' (SEQ ID NO. 4).

4. The pigeon mitochondrial whole genome amplification and assembly method according to claim 3, characterized in that Step (1) extracting the genomic DNA of the pigeon to be tested from feathers, blood, tissues or organs.

5. The pigeon mitochondrial whole genome amplification and assembly method according to claim 3, characterized in that: The PCR reaction system in step (2) is 25 μL of 2×Vazyme LAmp Master Mix, 2 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 19 μL of ultrapure water.

6. The pigeon mitochondrial whole genome amplification and assembly method according to claim 3, wherein The PCR reaction program in step (2) was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 66°C for 9 min), and 72°C for 10 min.

7. The pigeon mitochondrial whole genome amplification and assembly method according to claim 3, characterized in that: Step (3) Chinese library construction includes the following steps: (1) Preparation of enzyme digestion system a) Prepare the enzyme digestion reaction system: input 50 ng of amplified product, 1 μL of Tn5 Enzyme, and add working buffer to 20 μL. b) After the enzyme digestion system is prepared, place the PCR tube on a PCR instrument pre-heated at 70°C and 55°C and perform the enzyme digestion reaction for 7 minutes; (2) Termination of enzyme digestion reaction a) After the enzyme digestion reaction is completed, immediately place on ice and prepare the enzyme digestion termination reaction system: 20 μL of the enzyme digestion system from the previous step and 1 μL of Stop Buffer; b) Place the PCR tube in a PCR instrument pre-heated at 70°C and 55°C and perform the end-enzyme digestion program for 5 minutes; (3) Purification and recovery of enzyme digestion products a) Preparation: Remove DNA Selection Beads from the refrigerator and equilibrate at room temperature for at least 30 minutes. Meanwhile, prepare 80% ethanol. b) Vortex or invert the beads thoroughly to ensure thorough mixing; c) Pipette 20 μl of DNA Selection Beads (0.9X, Beads:DNA = 0.9:1) into the product after enzyme termination, vortex or pipette to mix, and incubate at room temperature for 5 minutes; d) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid. Once the solution has clarified (approximately 5 minutes), carefully remove the supernatant. e) Keeping the PCR tube in the magnetic rack, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds, then carefully remove the supernatant. f) Repeat step e and rinse once more; g) Keep the PCR tube in the magnetic rack at all times and air-dry the magnetic beads with the lid open until they just begin to crack, no more than 5 minutes. h) Remove the PCR tube from the magnetic rack, add 15 μl of ddH2O, vortex or gently pipette to mix thoroughly, and let it stand at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on the magnetic rack to let it stand. Once the solution has clarified, carefully transfer 15 μl of the supernatant to a new PCR tube for the next amplification step. (4) Library amplification a) Prepare the amplification system: 10 μL of purified product, 15 μL of 2X High-Fidelity Mix, 2 μL of UDI, and add HO to 30 μL. b) Amplification program: 72°C for 3 min, 98°C for 3 min, 7-15 cycles of (98°C for 30 s, 60°C for 30 s, 72°C for 30 s), 72°C for 3 min; (5) Library quality control The constructed library is quality evaluated by concentration detection and length distribution detection; (6) High-throughput sequencing The constructed NGS library was sequenced using an Illumina MiSeq sequencer in SE150 or PE150 mode.

8. The pigeon mitochondrial whole genome amplification and assembly method according to claim 3, characterized in that: The mitochondrial genome assembly in step (4) includes the following steps: (1) Use cutadapt software to remove any partial sequence containing sequencing adapter sequences; (2) Use PRINSEQ-lite software to perform quality control on the remaining sequences. Delete bases with a quality threshold lower than 20 from the 3' end to the 5' end of the sequence. The remaining sequences are considered to have passed the quality control. (3) Use SPAdes software to assemble the sequences that have passed quality control, select the default parameters, and output the spliced sequences.

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