CN115896240A - Method for constructing mitochondrial genome sequencing library - Google Patents
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Abstract
The invention discloses a method for constructing a mitochondrial genome sequencing library. The method comprises the steps of obtaining purified mtDNA through nuclear matter separation, adding exonuclease Exo V to remove nuclear genes, and adding Tn5 transposase to fragment the mtDNA so as to construct a mitochondrial genome sequencing library. The invention develops a method for efficiently separating and purifying mitochondrial DNA, which can effectively avoid the interference of homologous sequences of nuclear genome; PCR amplification of the initial mtDNA is not needed before the Tn5 transposase fragments the mtDNA, so that the problems of preference and introduction of wrong bases caused by PCR can be avoided; meanwhile, the method is short in time consumption, and the construction efficiency of the mitochondrial genome sequencing library is improved.
Description
Technical Field
The invention belongs to the field of biology, and relates to a method for constructing a mitochondrial genome sequencing library.
Background
Mitochondria are important organelles providing energy in cells, and are in a central position in energy conversion and metabolism of organisms. The mitochondria carry a set of mitochondrial genomes (i.e., mtDNA) that are independent of the nuclear genome. The human mitochondrial genome is a double-stranded closed circular DNA molecule with the total length of 16569bp, and is divided into a coding region and a non-coding D-loop region, wherein the coding region can code 13 polypeptide chains, 22 tRNA (tRNA) s and 2 rRNA (rRNA). mtDNA lacks nucleotide binding proteins on the molecule, lacks histone protection, has few gaps between genes, and has no DNA damage repair system within mitochondria, so mtDNA is prone to mutation and mutation is difficult to repair and inherit to progeny cells. In addition, since hundreds to thousands of mtdnas exist in each cell, there is often a phenomenon in which mutant type mtDNA coexists with wild type mtDNA, which is called heterogeneity. These characteristics of mitochondrial DNA present great difficulties for sequencing of the whole mitochondrial genome.
The following methods are currently available for mitochondrial genome sequencing:
1. mutation sites with heterogeneity more than 10% can be detected by one-generation Sanger sequencing, and the mutation sites are gold standards for single homozygous sites. However, one generation Sanger sequencing cannot be used for whole mitochondrial genome sequencing and typically cannot accurately detect heterogeneity below 10% based on the low sensitivity of this technique.
2. Pyrosequencing uses a technology of synthesizing and sequencing at the same time, firstly obtains a target DNA fragment of 300-800bp, then adds adaptors at two ends to form a sample library of the target DNA, and then fixes the target DNA fragment on magnetic beads to form a water-in-oil structure for independent amplification, thereby realizing parallel PCR amplification and high-throughput sequencing of all the target DNA fragments and being suitable for quantitative detection of specific sites. However, because the sample loss is large, and the whole mitochondrial genome cannot be detected in one experiment, the cost is relatively high, and thus pyrosequencing is difficult to be used for deep sequencing of the whole mitochondrial genome.
3. The probe capture method comprises the steps of preparing an oligonucleotide probe which is labeled by biotin and is complementary to mtDNA, hybridizing the oligonucleotide probe with the mtDNA, capturing and enriching target fragments, and carrying out high-throughput sequencing [ He Y, wu J, dressman DC, iacobiuzio-Donahue C, markowitz SD, velculus VE, diaz LA Jr, kinzler KW, vogelstein B, papodopoulos N.Heteroplastic mitochondral DNA molecules in normal and turour cells.Nature.2010Mar 25;464 (7288) 610-4.doi; PMCID PMC3176451. Although the method can be used for sequencing the whole mitochondrial genome and has high accuracy, the probe capture method is long in time consumption and high in cost, and cannot effectively avoid the interference of homologous sequences in the nuclear genome.
4. The long fragment PCR method generally divides mitochondrial DNA into two segments, specifically amplifies mtDNA with two pairs of primers, followed by high throughput sequencing, which can be used for whole mitochondrial genome sequencing. However, there are still some limitations to this method, including: the time for building the library is long, and the efficiency is low; moreover, long-fragment PCR has no good correction function, is easy to incorporate wrong bases and generate preference, and may influence the final sequencing result. However, even so, this method is still the most commonly used method for sequencing the whole mitochondrial genome at present [ Emonet S, grard G, brisbarre N, moureau G, temmam S, charrel R, de Lambolere X.LoPPS: a long PCR product sequencing method for rapid characterization of long amplification. Biochem Biophys Res Commun.2006Jun 16;344 (4) 1080-5.doi.
5. Whole genome sequencing can obtain the sequence information of the mitochondrial genome while obtaining the nuclear genome sequence, but the method has high cost, and like a probe capture method, whole genome sequencing cannot avoid the interference of the nuclear genome homologous sequence [ Schon KR, ratnaike T, van den Ameele J, horvath R, chinnery PF.Mitochon drial Diseases: A Diagnostic Revolume.trends Genet.2020Sep;36 (9): 702-717.doi.
Disclosure of Invention
Aiming at the limitations of the operation or cost existing in the current method, the invention aims to provide a database building method and a high-throughput sequencing method based on a mitochondrial separation and purification technology, and can realize simple, economic and rapid deep sequencing of mitochondrial genomes.
A method for constructing a mitochondrial genome sequencing library comprises the steps of obtaining purified mtDNA through nuclear matter separation, adding exonuclease Exo V to remove nuclear genes, and adding Tn5 transposase to fragment the mtDNA so as to construct the mitochondrial genome sequencing library.
As a preference of the present invention, the method is characterized by comprising the steps of:
i: separating Peripheral Blood Mononuclear Cells (PBMCs) by differential centrifugation using a lymphocyte separation fluid;
II: mitochondrial DNA isolation and purification:
a. removing cell nucleus as far as possible by nucleoplasm separation to obtain purified mitochondria;
b. adding exonuclease Exo V to remove a small amount of residual nuclear genome DNA;
III: tn5 enzyme fragmentation library construction:
a. adding Tn5 enzyme to the purified mitochondria to fragment the circular mtDNA;
b. double-round magnetic bead sorting;
c. and (4) absorbing the eluent sorted by the magnetic beads for the determination of the concentration of the Qubit, and then mixing the samples.
As a further preferred aspect of the present invention, the specific steps of separating nucleoplasm and obtaining mtDNA are as follows:
(1) Adding RSB Buffer into the cell sediment obtained after differential centrifugation;
(2) Blowing and beating for several times, and transferring into a centrifugal tube;
(3) Centrifuging at 4 ℃;
(4) The supernatant was immediately aspirated.
As a further preferred mode of the present invention, the addition of exonuclease Exo V to remove residual nuclear genomic DNA is carried out by adding the following components:
| components | Sample addition amount |
| Purified mitochondrial supernatant | 9.5uL |
| NEBuffer 4 | 1.2uL |
| ATP | 1.2uL |
| Exonuclease Ⅴ | 1uL |
Reacting at 37 ℃ for 25-35 minutes to remove residual nuclear genome; then reacting for 25-35 minutes at 65-70 ℃.
As a further preferred aspect of the present invention, said adding Tn5 enzyme to purified mitochondria to fragment mtDNA comprises the steps of:
(1) Adding 2xTD and Tn5 into the purified mitochondria and uniformly mixing;
(2) Performing metal bath at 37 deg.C, and shaking for 20-40min;
(3) Purification was performed using the Zymo D4014 kit;
(4) And adding a joint primer in PCR.
As a further preferred embodiment of the present invention, the PCR procedure is:
pre-denaturation at 98 ℃ for 30 seconds; denaturation at 98 deg.C for 10 s, annealing at 63 deg.C for 30 s, extension at 72 deg.C for 1 min, and circulation for 16-18 times; keeping at 4 ℃;
as a further preferred aspect of the present invention, the two-round magnetic bead sorting comprises the following steps:
1): sucking 0.5 times of magnetic beads into the PCR product, carrying out vortex oscillation and uniform mixing, carrying out instantaneous centrifugation, and then standing at room temperature;
2): placing the EP tube on a magnetic frame, and standing until the magnetic beads are completely adsorbed;
3): keeping the EP tube on the magnetic frame, carefully sucking the supernatant to a new EP tube, and avoiding contacting magnetic beads;
4): adding 0.3 Xmagnetic beads, vortex, shaking, mixing, centrifuging, and standing at room temperature;
5): placing the EP tube on a magnetic frame, and standing until the magnetic beads are completely adsorbed;
6): keeping the EP tube on the magnetic frame, and carefully absorbing and discarding the supernatant;
7): adding freshly prepared 80% ethanol into an EP tube, standing for 3-5min, and absorbing and removing the ethanol after the suspended magnetic beads are completely adsorbed;
8): washing with 80% ethanol once, and then completely sucking;
9): keeping the EP tube on a magnetic frame, and standing until the ethanol is completely volatilized;
10): addition of H to EP tubes 2 0, vortex, shake and mix evenly, and stand at room temperature;
11): placing the EP tube on a magnetic frame, and standing until the magnetic beads are completely adsorbed;
12): the eluate was pipetted into a new EP tube and the magnetic beads discarded.
A mitochondrial genome deep sequencing method is characterized in that a mitochondrial genome sequencing library is created by the method, and then an Illumina platform double-end sequencing is utilized to obtain a mitochondrial genome deep sequencing result.
Has the advantages that:
compared with the existing method, the technology innovatively realizes a database building method for mitochondrial genome sequencing based on mitochondrial purification and high-throughput sequencing. Mitochondrial DNA is purified by nucleoplasm separation, high-purity mtDNA (figure 1) without nuclear genome DNA can be obtained, PCR amplification of initial mtDNA is not needed before Tn5 transposase fragments the mtDNA, and the problems of sequence preference and introduction of additional mutation caused by PCR can be avoided. In addition, the library building method is short in time consumption: mitochondrial genome sequencing library construction needs 1.5h, exonuclease removes nuclear genes for 1h, tn5 fragmentation for 1h, PCR (polymerase chain reaction) joint primer and agarose gel electrophoresis for 1h, and magnetic bead sorting for 1h, so that library construction can be completed within 6h, the method is superior to a library construction method based on long-fragment PCR, and the mitochondrial genome sequencing library construction efficiency is improved.
Drawings
Fig. 1 is a graph of mitochondrial purification efficiency for 6 samples: s1-6 are from different blood samples, and 4 lanes for each sample are primers designed for nuclear genome 18S, 28S and mitochondrial genome m.8363, m.13513 sites: S1-S6 only amplify the target band of the mitochondrial genome, which shows that the method effectively separates and purifies the mitochondrial DNA and does not contain nuclear genome DNA.
FIG. 2 is a gel electrophoresis chart of products obtained by using the method of the present invention to separate and purify mitochondrial DNA and building Tn5 enzyme fragment libraries from A, B, C, D samples, wherein the size is 185-1000 bp.
FIG. 3 is a graph of sequencing library coverage of sample 1 by the method of the present invention and the long fragment PCR method.
Detailed Description
Example 1
1. Differential centrifugation, peripheral blood PBMC isolation:
(1) Taking 1ml of fresh EDTA anticoagulation, mixing with 1:1 physiological saline for injection, and gently blowing;
(2) Diluted blood was carefully added to the surface of 2ml of lymphocyte separation medium (STEMCELL);
(3) 400g, centrifuging for 20 minutes;
(4) At this time, the cells in the centrifuge tube are layered from top to bottom: the first layer is a plasma layer, the second layer is a ring-shaped milky white cell layer (mononuclear cells including lymphocytes and monocytes), the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer. Collecting the second layer of cells, putting the second layer of cells into a test tube containing 5-10 ml of normal saline for injection, and fully and uniformly mixing;
(5) 250g, centrifuging for 10 minutes, and discarding the supernatant;
(6) Repeating the step 5, and sucking the supernatant;
2. mitochondrial DNA separation and purification:
2.1: nuclear-cytoplasmic separation to obtain mtDNA
(1) Adding 15uL RSB Buffer into the sediment obtained after differential centrifugation;
(2) Blowing and beating for five times, and transferring into a 1.5ML centrifugal tube;
(3) Centrifuging at 4 ℃ for 10000g and 5min;
(4) Immediately sucking the supernatant to a new 1.5ML centrifuge tube;
2.2, exonuclease Exo V removal of nuclear genes: (NEB # M0345L)
| Components | Sample addition amount |
| Purified mitochondrial supernatant | 9.5 |
| NEBuffer | |
| 4 | 9.5uL |
| ATP | 1.2uL |
| Exonuclease Ⅴ | 1uL |
Reacting at 37 ℃ for 25-35 minutes to remove residual nuclear genome; then reacting for 25-35 minutes at 65-70 ℃.
2.3 purification of the product, 9uL H, using the Zymo D4014 kit 2 0, eluting;
2.4, PCR run gel verification, using Vazyme P112-01 kit:
18S rRNA：For：CACGGACAGGATTGACAGATTGAT(SEQ ID NO.1)；
Rev：GCCAGAGTCTCGTTCGTTATCG(SEQ ID NO.2)；
28S rRNA：For：TGGAATGCGAGTGCCTAGTG(SEQ ID NO.3)；
Rev：ACCGTCCTGCTGTCTATATCAAC(SEQ ID NO.4)；
13513-For：ACCATTGGCAGCCTAGCATT(SEQ ID NO.5)；
13513-Rev：GTTGTTTGGAAGGGGGATGC(SEQ ID NO.6)；
8363-For：GCAAACCACAGTTTCATGCCCA(SEQ ID NO.7)；
8363-Rev：TTATGGTGGGCCATACGGTAGTA(SEQ ID NO.8)；
| components | Sample addition amount |
| Purified product | 2uL |
| H 2 0 | 6.4uL |
| 2X PCR Mix | 10uL |
| Primer For | 0.8uL |
| Primer Rev | 0.8uL |
The reaction system is as follows: pre-denaturation at 98 ℃ for 30 seconds; denaturation at 98 ℃ for 10 seconds, annealing at 63 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, and circulation for 35 times; keeping at 4 ℃;
the results of running 2.5% and 2% agarose gel electrophoresis at 180V and 180mA at 100W for 30 minutes are shown in FIG. 1.
3. Tn5 enzyme fragmentation library construction:
3.1, tn5 enzyme fragmentation: (Vazyme TD501 kit)
(1) Adding 12.4ul 2xTD +0.5ul Tn5 into the purified mitochondria and mixing evenly;
(2) Performing metal bath at 37 ℃, and shaking for 20-40min;
(3) Purification was performed using Zymo D4014 kit;
(4) PCR plus adaptor primer (Vazyme TD501 kit):
| components | Sample addition amount |
| Purified DNA | 6uL |
| 5XTAB | 4uL |
| i5 | 2uL |
| i7 | 2uL |
| TAE | 0.5uL |
| H 2 0 | 5.5uL |
PCR conditions were as follows: pre-denaturation at 98 ℃ for 30 seconds; denaturation at 98 deg.C for 10 s, annealing at 63 deg.C for 30 s, extension at 72 deg.C for 1 min, and circulation for 16-18 times; maintaining at 4 deg.C
3.2, pipetting 1uL to run gel on 2% agarose gel electrophoresis, and the result is shown in FIG. 2;
3.3, double-round magnetic bead sorting:
1): sucking 0.5 Xmagnetic beads into the PCR product, mixing by vortex oscillation, instantly centrifuging, and standing at room temperature for 5min;
2): placing the EP tube on a magnetic frame, and standing for 5min until the magnetic beads are completely adsorbed;
3): keeping the EP tube on the magnetic frame, carefully sucking the supernatant to a new EP tube to avoid contacting the magnetic beads;
4): adding 0.3 Xmagnetic bead, vortex, shaking, mixing, centrifuging, and standing at room temperature for 5min;
5): placing the EP tube on a magnetic frame, and standing for 5min until the magnetic beads are completely adsorbed;
6): keeping the EP tube on the magnetic frame, and carefully absorbing and discarding the supernatant;
7): adding 250ul of freshly prepared 80% ethanol into an EP tube, standing for 3-5min, and absorbing and removing the ethanol after the suspended magnetic beads are completely adsorbed;
8): washing with 80% ethanol for one time, and then completely sucking;
9): keeping the EP tube on a magnetic frame, and standing for 5min until the ethanol is completely volatilized;
10): addition of H to EP tubes 2 0, vortex, shake and mix evenly, and stand for 5min at room temperature;
11): placing the EP tube on a magnetic frame, and standing for 5min until the magnetic beads are completely adsorbed;
12): absorbing the eluent into a new EP tube, and discarding the magnetic beads;
3.4, respectively absorbing 2uL to measure the concentration of the Qubit, and then mixing the samples;
4. the library was paired-end sequenced using the Illumina platform, 150bp each. The resulting sequences were then aligned with the human mitochondrial genome NC _012920 database using the GATK mitochondrial genome standardization analysis procedure, and all short sequences mapped to the mitochondrial genome were used for downstream mutation site analysis.
5. And (4) result finishing:
the average sequencing depth of the method was 35298 ×. The sequencing results are shown in table 1.
| Sample name | RAW_READS | RAW_BASES | CLEAN_READS | CLEAN_BASES | READ_LENGTH |
| Sample 1 | 5,760,551 | 1,728,165,300 | 5,759,832 | 1,727,949,600 | 150；150 |
TABLE 1
Example 2
The same samples of example 1 were pooled and sequenced using the long fragment PCR method. The long fragment PCR library building method uses 2 pairs of primers to divide mitochondrial DNA into two fragments, wherein the sequences of the primers are as follows:
F1-For：GCAAATCTTACCCCGCCTG(SEQ ID NO.9)；
F1-Rev：AATTAGGCTGTGGGTGGTTG(SEQ ID NO.10)；
F2-For：GCCATACTAGTCTTTGCCGC(SEQ ID NO.11)；
F2-Rev：GGCAGGTCAATTTCACTGG(SEQ ID NO.12)；
the average sequencing depth for the long fragment PCR method was 26030 x. The sequencing results are shown in table 2.
| Sample name | RAW_READS | RAW_BASES | CLEAN_READS | CLEAN_BASES | READ_LENGTH |
| Sample 1 | 2,433,774 | 730,132,200 | 2,433,774 | 730,132,200 | 150；150 |
TABLE 2
The methods of the invention compared to the long-fragment PCR method, both were found to have better library uniformity by analyzing library coverage (see FIG. 3). However, for the whole library construction process, the long-fragment PCR method takes 2 days, and the method takes 6 hours, so that the efficiency of the method is far higher than that of the long-fragment method. As shown by comparison of sequencing results (tables 1 and 2), for the sites with the heterogeneous mutation ratio of 10-100%, the points detected by the method and the long-fragment PCR method are completely consistent with the heterogeneity; when the degree of heterogeneity is less than 10%, the results obtained by the two methods are slightly different but substantially identical.
Claims (8)
1. A method for constructing a mitochondrial genome sequencing library is characterized by comprising the steps of separating live nucleoplasm to obtain purified mtDNA, adding exonuclease Exo V to remove nuclear genes, and adding Tn5 transposase to fragment the mtDNA so as to construct the mitochondrial genome sequencing library.
2. The method according to claim 1, characterized by comprising the steps of:
i: separating peripheral blood PBMC by using lymphocyte separating medium and differential centrifugation;
II: mitochondrial DNA separation and purification:
a. removing cell nucleus as far as possible by nucleoplasm separation to obtain purified mitochondria;
b. adding exonuclease Exo V to remove a small amount of residual nuclear genome DNA;
III: tn5 enzyme fragmentation library construction:
a. adding Tn5 enzyme to the purified mitochondria to fragment the circular mtDNA;
b. double-round magnetic bead sorting;
c. and (5) absorbing the eluent sorted by the magnetic beads for Qubit concentration determination, and then mixing the samples.
3. The method of claim 2, wherein said nucleoplasm separation, obtaining mtDNA comprises the steps of:
(1) Adding RSB Buffer to the precipitate;
(2) Blowing and beating for several times, and transferring into a centrifugal tube;
(3) Centrifuging at 4 ℃;
(4) The supernatant was immediately aspirated.
4. The method of claim 2, wherein the addition of exonuclease Exo V removes residual nuclear genomic DNA by adding the components according to the following table:
Reacting at 37 ℃ for 25-35 minutes to remove residual nuclear genome; then reacting for 25-35 minutes at 65-70 ℃.
5. The method of claim 1, wherein said adding Tn5 enzyme to the purified mitochondria to fragment the mtDNA comprises the steps of:
(1) Adding 2xTD and Tn5 into the purified mitochondria and uniformly mixing;
(2) Performing metal bath at 37 deg.C, and shaking for 20-40min;
(3) Purification was performed using the Zymo D4014 kit;
(4) And adding a joint primer in PCR.
6. The method of claim 5, wherein the PCR process is: pre-denaturation at 98 ℃ for 30 seconds; denaturation at 98 deg.C for 10 s, annealing at 63 deg.C for 30 s, extension at 72 deg.C for 1 min, and circulation for 16-18 times; keeping at 4 ℃.
7. The method of claim 2, wherein said two-round magnetic bead sorting comprises the steps of:
1): sucking 0.5 Xmagnetic beads into the PCR product, mixing the mixture evenly by vortex oscillation, and standing the mixture at room temperature after instantaneous centrifugation;
2): placing the EP tube on a magnetic frame, and standing until the magnetic beads are completely adsorbed;
3): keeping the EP tube on the magnetic frame, carefully sucking the supernatant to a new EP tube, and avoiding contacting magnetic beads;
4): adding 0.3 Xmagnetic beads, vortex, shaking, mixing, centrifuging, and standing at room temperature;
5): placing the EP tube on a magnetic frame, and standing until the magnetic beads are completely adsorbed;
6): keeping the EP tube on the magnetic frame, and carefully absorbing and discarding the supernatant;
7): adding freshly prepared 80% ethanol into an EP tube, standing for 3-5min, and absorbing and removing the ethanol after the suspended magnetic beads are completely adsorbed;
8): washing with 80% ethanol for one time, and then completely sucking;
9): keeping the EP tube on a magnetic frame, and standing until the ethanol is completely volatilized;
10): addition of H to EP tubes 2 0, vortex, shake and mix evenly, and stand at room temperature;
11): placing the EP tube on a magnetic frame, and standing until the magnetic beads are completely adsorbed;
12): the eluate was pipetted into a new EP tube and the magnetic beads discarded.
8. A method for deep sequencing of a whole mitochondrial genome, which is characterized in that a mitochondrial genome sequencing library is created by the method of any one of claims 1 to 7, and then a result of deep sequencing of the whole mitochondrial genome is obtained by using Illumina platform double-ended sequencing.
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