CN114015749A - Construction method of mitochondrial genome sequencing library based on high-throughput sequencing and amplification primer - Google Patents
Construction method of mitochondrial genome sequencing library based on high-throughput sequencing and amplification primer Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention discloses a construction method of a mitochondrial genome sequencing library based on high-throughput sequencing and an amplification primer. The amplification primers comprise a primer pair 1 and a primer pair 2; the nucleotide sequence of the primer pair 1 is shown as SEQ ID NO.1 and 2, and the nucleotide sequence of the primer pair 2 is shown as SEQ ID NO.3 and 4. According to the invention, only two pairs of amplification primers are needed, and a sufficient amount of mitochondrial DNA sample can be obtained through one-time PCR amplification, so that the complex primer amount and the cost investment are greatly reduced, and the operation is simplified. According to the invention, the integrity and accuracy of the mtDNA are improved and the effectiveness of data analysis is ensured by mixed database building of amplification products of two pairs of primers.
Description
Technical Field
The invention belongs to the technical field of high-throughput sequencing, and relates to a construction method of a mitochondrial genome sequencing library based on high-throughput sequencing and an amplification primer.
Background
Mitochondria are factories of cellular energy production and are important organelles of energy metabolism of organisms. The human mitochondrial genome is in a double-chain ring structure, the size is 16569bp, and 13 functional proteins participating in the ATP synthesis process, 22 tRNA and 2 rRNA are coded. Mitochondrial DNA is not protected by histone, is positioned in a highly-oxidized environment and has the characteristic of high mutation rate. These mutations cause changes in gene structure, affect mitochondrial function, cause abnormal oxidative phosphorylation and energy metabolism, and finally cause diseases, such as KSS syndrome, Leigh syndrome and the like.
To detect mutations in mitochondrial DNA, a number of methods are currently available for sequencing mitochondrial DNA. Mutation sites with heterogeneity greater than 10% can be detected by first-generation Sanger sequencing. The sequencing method for mitochondrial DNA based on the high-throughput sequencing technology mainly comprises the following steps: the method comprises the steps of firstly, carrying out mitochondrial DNA separation, separating a mitochondrial genome from cells by cesium chloride density gradient centrifugation, column chromatography, alkali denaturation and other methods, and sequencing; the other is a probe hybridization capture method, which is to synthesize an oligonucleotide probe complementary to mtDNA, capture and enrich target segments after hybridizing with mtDNA, and then carry out high-throughput sequencing; and the third is PCR amplification method, which includes specific amplification of mitochondrial DNA via one or several pairs of primer and subsequent high throughput sequencing.
Traditional generation sequencing technologies have been difficult to adapt to the requirements of mitochondrial genome sequencing due to low sensitivity and high cost. The development of a new generation sequencing technology provides good technical support for the detection of complex and low-frequency mtDNA heterogeneous mutation. The mitochondrial genome yield of cesium chloride gradient centrifugation is low, and the requirements on manpower, time and sample size are high. The probe hybridization capture method is expensive and complicated to operate. In the PCR amplification method, multiple pairs of primers are required to be designed for short-segment amplification, various primer sets are combined for use, the operation is complicated, and the cost is increased; in long fragment amplification, some methods aggregate complete mitochondrial DNA through fragment amplification, but have the defect that amplification cannot be performed when primer binding sites mutate, or have a fragment amplification method capable of mutual correction, but different amplification products need to be subjected to subsequent purification and library construction respectively, so that the workload and the reagent cost are multiplied.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a simple, economic and rapid mitochondrial DNA PCR amplification primer combination and a sequencing method based on a high-throughput sequencing technology aiming at the limitations of the existing methods in operation or cost. The primer combination only needs two pairs of amplification primers, and a sufficient amount of mitochondrial DNA samples which can be mutually corrected can be obtained through one-time PCR amplification, namely when one pair of primers fails to amplify due to mutation, the other pair of primers can still amplify to obtain a complete DNA sample, and fragments obtained by amplifying the two pairs of primers do not need purification, can be directly merged for subsequent library construction, so that the investment of complex primer amount and cost is greatly reduced, and the operation is simplified.
In order to solve the technical problem, the invention provides a human mitochondrial amplification primer on one hand, which comprises a primer pair 1 and a primer pair 2;
the upstream primer MT1-F of the primer pair 1 is shown as SEQ ID NO.1, the downstream primer MT1-R is shown as SEQ ID NO.2, the upstream primer MT2-F of the primer pair 2 is shown as SEQ ID NO.3, and the downstream primer MT2-R is shown as SEQ ID NO. 4.
The invention also provides a kit for constructing a mitochondrial genome sequencing library, which comprises the human mitochondrial amplification primer.
In another aspect, the present invention provides a method for amplifying a human mitochondrial genome, comprising the steps of:
and (3) carrying out PCR amplification by using the human mitochondrial amplification primer by using the whole blood DNA as a template.
Preferably, the PCR reaction system for PCR amplification comprises: 5 XPrimeSTAR GXL Buffer 10. mu.L, dNTP mix (2.5mM each) 4. mu.L, PrimeSTAR GXL DNApolymerase 1. mu.L, template DNA 1. mu.L, 10. mu.M MT1-F/MT 2-F1. mu.L, 10. mu.M MT1-R/MT 2-R1. mu.L,NF·H2O32μL。
preferably, the PCR reaction procedure of the PCR amplification is: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing/extension at 68 ℃ for 10min, and 32 cycles; prolonging at 68 deg.C for 15 min; then stored at 4 ℃.
The invention also provides a construction method of the mitochondrial genome sequencing library based on high-throughput sequencing, which comprises the following steps:
1) using whole blood DNA as a template, and adopting the human mitochondrial genome amplification method to carry out PCR amplification;
2) and (3) equivalently and uniformly mixing mtDNA obtained by amplifying the two pairs of primers, and constructing a library.
Preferably, the step 2) comprises:
2.1) equally and uniformly mixing mtDNA obtained by amplifying two pairs of primers, and fragmenting by using an ultrasonic disruptor;
2.2) repairing the fragmentation product and adding a single base A at the tail end;
2.3) carrying out a joint connection reaction;
2.4) magnetic bead purification and fragment screening are carried out, and quality control detection is carried out to complete library construction.
As a preferred embodiment of the present invention, the step 2.1): respectively taking 500ng of MT1 product and MT2 product of the same sample, mixing uniformly, and adding nucleic-free ddH2O make up volume to 55 μ L and fragment with an ultrasonic disruptor for 80 s.
The invention further provides a kit for constructing a mitochondrial genome sequencing library, which comprises the human mitochondrial amplification primer. The beneficial effects of the invention include:
(1) through simple PCR amplification of two pairs of primers, a complete and sufficient mtDNA sample can be obtained, complex primer combination pairing is not needed, the operation of personnel is simplified, and the method is economical and rapid;
(2) the integrity and accuracy of the mtDNA are improved and the effectiveness of data analysis is ensured by the mixed database construction of the amplification products of the two pairs of primers.
Drawings
FIG. 1 is a schematic diagram showing the positions of the primer pair MT1 and MT2 and the amplification directions thereof;
FIG. 2 is a gel electrophoresis identification chart of amplification products of the primer pair MT1 and MT 2.
FIG. 3 is mitochondrial sequencing data coverage.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to specific examples, but the present invention is not limited to the following examples.
Taking 4 samples, verifying the invention of the samples, and the specific steps of the implementation process are as follows:
1. extraction and preservation of mtDNA template
And (3) carrying out DNA extraction on the collected peripheral venous blood according to the conventional whole blood DNA extraction process.
2. Primer pair design
Mitochondrial amplification primer pair 1 and primer pair 2 were designed using PrimerPremier 5, with the primer sequences shown in Table 1.
TABLE 1 mtDNA amplification primers
| Numbering | Primer and method for producing the same | Sequence of |
| SEQ ID NO.1 | MT1-F | GGTAAAGGTCGGTTTATC |
| SEQ ID NO.2 | MT1-R | TATGCCTCTTCACG |
| SEQ ID NO.3 | MT2-F | CGATACTCGGACACCC |
| SEQ ID NO.4 | MT2-R | AGGGGCGTTTGGTATGG |
3. Two pairs of amplification primers are respectively used, whole blood DNA is taken as a template, mtDNA is amplified, and a PCR reaction system is configured on ice as follows:
and (3) PCR reaction conditions:
the amplification products of MT1 and MT2 were identified by electrophoresis on a 1% agarose gel, and the results are shown in FIG. 2. The product concentration was determined using Qubit.
4. Mitochondrial DNA library construction
Respectively taking 500ng of MT1 product and MT2 product of the same sample, mixing uniformly, and adding nucleic-free ddH2O make up volume to 55 μ L and fragment with an ultrasonic disruptor for 80 s.
Subsequently, the following reaction system was configured:
| components | Volume/amount |
| Breaking of the product | 50μL |
| End Prep Mix4 | 15μL |
| Total | 65μL |
The reaction procedure was as follows:
| step (ii) of | Temperature of |
| 1 | 105℃ |
| 2 | 20℃ |
| 3 | 65℃ |
| 4 | 4℃ |
The reaction repairs the broken product and adds A at the tail end, and then carries out the joint connection reaction, and the reaction system is as follows:
| components | Volume of |
| Repair product | 65μL |
| Rapid Ligation Buffer3 | 25μL |
| Rapid DNA Ligase | 5μL |
| DNAAdapter X | 5μL |
| Total | 100μL |
The mixture was placed in a PCR apparatus at 20 ℃ for 15 min.
5. Magnetic bead purification and fragment screening
Pipette 60. mu. L VAHTS DNA Clean Beads into 100. mu.L of adaptor ligation and vortex to mix well.
Incubate at room temperature for 5 min.
The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads and liquid, and the supernatant carefully removed after the solution cleared (about 5 min).
The PCR tube was kept on the magnetic rack, 200. mu.L of freshly prepared 80% ethanol was added to rinse the beads, and the supernatant carefully removed after incubation at room temperature for 30 s.
The rinsing was repeated once for a total of two rinses.
Keeping the PCR tube on the magnetic frame all the time, opening the cover and air-drying the magnetic beads for 5-10min until no ethanol remains.
Add 105. mu.L of nucleic-free water and vortex at room temperature for 2 min.
The PCR tube was briefly centrifuged and placed in a magnetic stand to stand, and after the solution cleared (about 5min), 100. mu.L of the supernatant was carefully removed to a new EP tube.
Pipette 59. mu. L VAHTS DNA Clean Beads into 100. mu.L of product and vortex well to mix.
Incubate at room temperature for 5 min.
The PCR tube was briefly centrifuged and placed in a magnetic stand to separate the beads from the liquid, after the solution was clarified (about 5min), the supernatant was carefully transferred to a new PCR tube and the beads discarded.
Aspirate 15. mu. L VAHTS DNA CLEAN Beads into the supernatant and vortex.
Incubate at room temperature for 5 min.
The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, and after the solution was clarified (about 5min), the supernatant was carefully removed to avoid contact with the beads.
The PCR tube was kept on the magnetic rack, 200. mu.L of freshly prepared 80% ethanol was added to rinse the beads, and after incubation at room temperature for 30sec the supernatant was carefully removed.
The rinsing was repeated once for a total of two rinses.
Keeping the PCR tube on the magnetic frame all the time, opening the cover and air-drying the magnetic beads for 5-10min until no ethanol remains.
The PCR tube was removed from the magnetic stand, 22.5. mu.L of nucleic-free water was added thereto, and the mixture was gently pipetted and mixed, followed by standing at room temperature for 2 min.
The PCR tube was briefly centrifuged and placed in a magnetic rack and left to stand, after the solution cleared (about 5min) 20. mu.L of supernatant was carefully pipetted into a new EP tube, and the beads were removed.
Taking 1 muL library to carry out the Qubit concentration determination and the QIAXcel fragment size determination respectively, wherein the size of the main peak of the library is between 400 and 500bp and is consistent with the size of a theoretical value, and the library construction quality is indicated to be in accordance with the sequencing requirement.
6. DNA library sequencing
The 4 sample libraries were sequenced using Illumina NOVA _ S4 and the data quality analysis is shown in table 2.
TABLE 2 sequencing data quality analysis
| Sample_ID | Clean_reads | Clean_Q30％ | Average_Depth | chrM％ |
| B2020111147 | 644024 | 92.19％ | 11100.04 | 96.47％ |
| B2020111342 | 557201 | 91.85％ | 9625.2 | 96.68％ |
| E21020074 | 515465 | 91.49％ | 8863 | 96.25％ |
| E21030317 | 659826 | 92.08％ | 11460.16 | 97.28％ |
The results show that the mitochondrial sequencing data accounts for more than 96%, and the Q30% reaches more than 91%, which indicates that the sequencing data quality is good, and the coverage of mitochondria is shown in figure 3, and the coverage of mtDNA reaches 100%. The detection rate of the positive mutation sites A1555G and A1557G reaches 100 percent, the sequencing depth is 5500 multiplied by more, and the method can be used for simply, quickly and effectively analyzing the human mitochondrial sequence.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> Zhejiang Bosheng Biotechnology Ltd
Hangzhou Bosheng medical laboratory Co., Ltd
<120> construction method of mitochondrial genome sequencing library based on high-throughput sequencing and amplification primer
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggtaaaggtc ggtttatc 18
<210> 2
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tatgcctctt cacg 14
<210> 3
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cgatactcgg acaccc 16
<210> 4
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aggggcgttt ggtatgg 17
Claims (8)
1. A human mitochondrial amplification primer is characterized by comprising a primer pair 1 and a primer pair 2;
the upstream primer MT1-F of the primer pair 1 is shown as SEQ ID NO.1, the downstream primer MT1-R is shown as SEQ ID NO.2, the upstream primer MT2-F of the primer pair 2 is shown as SEQ ID NO.3, and the downstream primer MT2-R is shown as SEQ ID NO. 4.
2. A human mitochondrial genome amplification method is characterized by comprising the following steps:
PCR amplification is carried out using the human mitochondrial amplification primer of claim 1 using whole blood DNA as a template.
3. The amplification method according to claim 2, wherein the PCR reaction system for PCR amplification comprises: 5 XPrimeSTAR GXL Buffer 10. mu.L, dNTP mix (2.5mM each) 4. mu.L, PrimeSTAR GXL DNA Polymerase 1. mu.L, template DNA 1. mu.L, 10. mu.M MT1-F/MT 2-F1. mu.L, 10. mu.M MT1-R/MT 2-R1. mu.L, NF. H2O 32μL。
4. The amplification method according to claim 2, wherein the PCR reaction procedure of the PCR amplification is: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing/extension at 68 ℃ for 10min, and 32 cycles; prolonging at 68 deg.C for 15 min; then stored at 4 ℃.
5. A construction method of a mitochondrial genome sequencing library based on high-throughput sequencing is characterized by comprising the following steps:
1) performing PCR amplification by using a whole blood DNA as a template and using the human mitochondrial genome amplification method according to any one of claims 2 to 4;
2) and (3) equivalently and uniformly mixing mtDNA obtained by amplifying the two pairs of primers, and constructing a library.
6. The building method according to claim 5, wherein the step 2) comprises:
2.1) equally and uniformly mixing mtDNA obtained by amplifying two pairs of primers, and fragmenting by using an ultrasonic disruptor;
2.2) repairing the fragmentation product and adding a single base A at the tail end;
2.3) carrying out a joint connection reaction;
2.4) magnetic bead purification and fragment screening are carried out, and quality control detection is carried out to complete library construction.
7. The construction method according to claim 6, wherein the step 2.1): respectively taking 500ng of MT1 product and MT2 product of the same sample, mixing uniformly, and adding nucleic-free ddH2O make up volume to 55 μ L and fragment with an ultrasonic disruptor for 80 s.
8. A kit for constructing a mitochondrial genome sequencing library, comprising the human mitochondrial amplification primer of claim 1.
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| CN117070511A (en) * | 2023-08-16 | 2023-11-17 | 广州卿泽生物科技有限公司 | Primer group for amplifying mitochondrial whole genome and application thereof |
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