CN115896240B - Method for constructing mitochondrial genome sequencing library - Google Patents

Abstract

The invention discloses a method for constructing a mitochondrial genome sequencing library. The method comprises the steps of obtaining purified mtDNA through nuclear mass separation, adding exonuclease Exo V to remove nuclear genes, adding Tn5 transposase to fragment the mtDNA, and thus constructing a mitochondrial genome sequencing library. The invention develops a method for efficiently separating and purifying mitochondrial DNA, which can effectively avoid the interference of nuclear genome homologous sequences; the initial mtDNA is not required to be subjected to PCR amplification 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 has short time consumption and improves the efficiency of constructing the mitochondrial genome sequencing library.

Description

A method for constructing a mitochondrial genome sequencing library

Technical Field

The invention belongs to the biological field and relates to a method for constructing a mitochondrial genome sequencing library.

Background technique

Mitochondria are important organelles that provide energy in cells and play a core role in energy conversion and metabolism of organisms. Mitochondria carry a mitochondrial genome (mtDNA) that is independent of the nuclear genome. The human mitochondrial genome is a double-stranded closed circular DNA molecule with a total length of 16569bp, which is divided into a coding region and a non-coding D-loop region. The coding region can encode 13 polypeptide chains, 22 tRNAs and 2 rRNAs. There are no nucleotide binding proteins on the mtDNA molecule, lack the protection of histones, there are few intervals between genes, and there is no DNA damage repair system in mitochondria. Therefore, mtDNA is prone to mutation and mutations are difficult to repair and inherit to daughter cells. In addition, since there are hundreds or thousands of mtDNAs in each cell, there is often a phenomenon of coexistence of mutant mtDNA and wild-type mtDNA, which is called heteroplasmy. These characteristics of mitochondrial DNA bring great difficulties to the sequencing of the whole mitochondrial genome.

There are several methods currently available for mitochondrial genome sequencing:

First-generation Sanger sequencing can detect mutation sites with heteroplasmy greater than 10%, and is the gold standard for detecting single homozygous sites. However, first-generation Sanger sequencing cannot be used for whole mitochondrial genome sequencing, and based on the low sensitivity of this technology, it usually cannot accurately detect heteroplasmy below 10%.

Second, pyrosequencing uses sequencing-by-synthesis technology to first obtain a 300-800bp target DNA fragment, then add adapters at both ends to form a sample library of the target DNA, and then fix the target DNA fragment to the magnetic beads to form an oil-in-water structure for independent amplification, thereby achieving parallel PCR amplification and high-throughput sequencing of all target DNA fragments, which is suitable for quantitative detection of specific sites. However, due to the large amount of sample loss and the inability to detect the entire mitochondrial genome in one experiment, the relatively high cost makes pyrosequencing difficult to use for deep sequencing of the entire mitochondrial genome.

3. Probe capture method: Prepare biotin-labeled oligonucleotide probes complementary to mtDNA, then hybridize with mtDNA to capture and enrich the target fragments, and then perform high-throughput sequencing [He Y, Wu J, Dressman DC, Iacobuzio-Donahue C, Markowitz SD, Velculescu VE, Diaz LA Jr, Kinzler KW, Vogelstein B, Papadopoulos N. Heteroplasmic mitochondrial DNA mutations in normal and tumour cells. Nature. 2010 Mar 25; 464(7288): 610-4. doi: 10.1038/nature08802. Epub 2010 Mar 3. PMID: 20200521; PMCID: PMC3176451.]. Although this method can be used for whole mitochondrial genome sequencing with high accuracy, the probe capture method is time-consuming, costly, and cannot effectively avoid interference from homologous sequences in the nuclear genome.

Fourth, the long-fragment PCR method generally divides mitochondrial DNA into two segments, and uses two pairs of primers to specifically amplify mtDNA, followed by high-throughput sequencing, which can be used for whole mitochondrial genome sequencing. However, this method still has certain limitations, including: library construction is time-consuming and inefficient; and because long-fragment PCR does not have a good correction function, it is easy to incorporate incorrect bases and produce bias, which may affect the final sequencing results. But even so, this method is still the most commonly used method for whole mitochondrial genome sequencing [Emonet S, Grard G, Brisbarre N, Moureau G, Temmam S, Charrel R, deLamballerie X. LoPPS: a long PCR product sequencing method for rapid characterization of long amplicons. Biochem Biophys Res Commun. 2006 Jun 16; 344(4): 1080-5. doi: 10.1016/j.bbrc.2006.04.015. Epub 2006 Apr 19. PMID: 16643852.].

5. Whole genome sequencing can obtain the sequence information of the mitochondrial genome while obtaining the nuclear genome sequence, but this method is too expensive, and like the probe capture method, whole genome sequencing cannot avoid the interference of homologous sequences of the nuclear genome [Schon KR, Ratnaike T, van den Ameele J, Horvath R, Chinnery PF. Mitochondrial Diseases: A Diagnostic Revolution. Trends Genet. 2020 Sep; 36 (9): 702-717. doi: 10.1016/j.tig.2020.06.009. Epub 2020 Jul 13. PMID: 32674947.].

Summary of the invention

In view of the operational or cost limitations of current methods, the present invention intends to provide a library construction method and a high-throughput sequencing method based on mitochondrial separation and purification technology, which can achieve simple, economical and rapid deep sequencing of the mitochondrial genome.

A method for constructing a mitochondrial genome sequencing library comprises obtaining purified mtDNA by nuclear-cytoplasmic separation, adding exonuclease Exo V to remove nuclear genes, and adding Tn5 transposase to fragment the mtDNA to construct a mitochondrial genome sequencing library.

As a preferred embodiment of the present invention, the method is characterized in that it comprises the following steps:

I: Use lymphocyte separation medium to separate peripheral blood mononuclear cells (PBMC) by differential centrifugation;

II: Mitochondrial DNA isolation and purification:

a. Through nuclear-cytoplasmic separation, remove the cell nucleus as much as possible to obtain purified mitochondria;

b. Add exonuclease Exo V to remove a small amount of residual nuclear genomic DNA;

III: Tn5 enzyme fragmentation library construction:

a. Add Tn5 enzyme to purified mitochondria to fragment circular mtDNA;

b. Double-wheel magnetic bead sorting;

c. Pipette the eluate from the magnetic bead separation for Qubit concentration determination, and then mix the samples.

As a further preferred embodiment of the present invention, the specific steps of separating nucleocytoplasm and obtaining mtDNA are as follows:

(1) Add RSB Buffer to the cell pellet obtained after differential centrifugation;

(2) Pipet and swirl several times and transfer to a centrifuge tube;

(3) Centrifugation at 4°C;

(4) Immediately aspirate the supernatant.

As a further preferred embodiment of the present invention, the exonuclease Exo V is added to remove the residual nuclear genomic DNA, and each component is added according to the following table:

Components Sample volume Purified mitochondrial supernatant 9.5uL NEBuffer 4 1.2uL ATP 1.2uL Exonuclease V 1uL

Incubate at 37°C for 25-35 minutes to remove residual nuclear genome; then incubate at 65-70°C for 25-35 minutes.

As a further preferred embodiment of the present invention, the step of adding Tn5 enzyme to the purified mitochondria to fragment the mtDNA comprises the following steps:

(1) Add 2xTD and Tn5 to the purified mitochondria and mix well;

(2) 37°C metal bath, shake for 20-40 min;

(3) Purification using the Zymo D4014 kit;

(4) PCR with adapter primers.

As a further preference of the present invention, the PCR procedure is:

Pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 63°C for 30 seconds, extension at 72°C for 1 minute, cycle 16-18 times; maintain at 4°C;

As a further preferred embodiment of the present invention, the double-wheel magnetic bead sorting comprises the following steps:

1) Pipette 0.5× magnetic beads into the PCR product, vortex to mix, centrifuge briefly and let stand at room temperature;

2): Place the EP tube on the magnetic rack and let it stand until the magnetic beads are completely adsorbed;

3): Keep the EP tube on the magnetic stand and carefully pipette the supernatant into a new EP tube to avoid contact with the magnetic beads;

4): Add 0.3× magnetic beads, vortex to mix, centrifuge briefly, and let stand at room temperature;

5): Place the EP tube on the magnetic rack and let it stand until the magnetic beads are completely adsorbed;

6) Keep the EP tube on the magnetic rack and carefully discard the supernatant;

7) Add freshly prepared 80% ethanol to the EP tube and let it stand for 3-5 minutes. After the suspended magnetic beads are completely adsorbed, discard the ethanol;

8): Repeat washing once with 80% ethanol, then aspirate clean;

9): Keep the EP tube on the magnetic rack and let it stand until the ethanol evaporates completely;

10): Add H ₂ 0 to the EP tube, vortex to mix, and let stand at room temperature;

11): Place the EP tube on the magnetic rack and let it stand until the magnetic beads are completely adsorbed;

12): Pipette the eluate into a new EP tube and discard the magnetic beads.

A method for deep sequencing of a whole mitochondrial genome, which uses the method described in the present invention to create a mitochondrial genome sequencing library, and then uses Illumina platform double-end sequencing to obtain the deep sequencing results of the whole mitochondrial genome.

Beneficial effects:

Compared with existing methods, this technology innovatively implements a library construction method for mitochondrial genome sequencing based on mitochondrial purification and high-throughput sequencing. By separating and purifying mitochondrial DNA by nucleoplasm, high-purity mtDNA that does not contain nuclear genome DNA can be obtained (Figure 1), and there is no need to perform PCR amplification on the initial mtDNA before the Tn5 transposase fragments it, which can avoid the sequence preference caused by PCR and the problem of introducing additional mutations. In addition, this library construction method is time-saving: mitochondrial separation and purification requires 1.5 hours, nuclease exonuclease removal of nuclear genes requires 1 hour, Tn5 fragmentation requires 1 hour, PCR plus adapter primers and agarose gel electrophoresis requires 1 hour, and magnetic bead sorting requires 1 hour, so the library construction can be completed within 6 hours, which is better than the library construction method based on long-fragment PCR and improves the efficiency of mitochondrial genome sequencing library construction.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph of the mitochondrial purification efficiency of six samples: S1-6 are from different blood samples, and the four lanes of each sample are primers designed for the nuclear genome 18S, 28S and the mitochondrial genome m.8363, m.13513 sites: S1-S6 only amplified the mitochondrial genome target bands, indicating that this method effectively separates and purifies mitochondrial DNA and does not contain nuclear genome DNA.

FIG2 is a gel electrophoresis diagram of the products obtained from mitochondrial DNA of four different samples A, B, C, and D using the method of the present invention, after Tn5 enzyme fragmentation and library construction, with a size ranging from 185 to 1000 bp.

FIG3 is a diagram showing the coverage of the sequencing library of sample 1 measured by the method of the present invention and the long-fragment PCR method.

Detailed ways

Example 1

1. Differential centrifugation to separate peripheral blood PBMC:

(1) Take 1 ml of fresh EDTA anticoagulated blood and mix it with normal saline for injection in a ratio of 1:1, then gently pipette and vortex;

(2) Carefully add the diluted blood to the surface of 2 ml of lymphocyte separation medium (STEMCELL);

(3) Centrifugation at 400 g for 20 min;

(4) At this time, the cells in the centrifuge tube are divided into four layers from top to bottom: the first layer is the plasma layer, the second layer is the ring-shaped milky white cell layer (mononuclear cells, including lymphocytes and monocytes), the third layer is the transparent separation liquid layer, and the fourth layer is the red blood cell layer. Collect the second layer of cells and put them into a test tube containing 5-10 ml of injection saline and mix them thoroughly;

(5) Centrifuge at 250 g for 10 min and discard the supernatant;

(6) Repeat step 5 and aspirate the supernatant;

2. Mitochondrial DNA separation and purification:

2.1: Nuclear and cytoplasmic separation to obtain mtDNA

(1) Add 15uL RSB Buffer to the precipitate obtained after differential centrifugation;

(2) Pipette five times and transfer to a 1.5 mL centrifuge tube;

(3) Centrifugation at 4°C, 10,000 g, 5 min;

(4) Immediately transfer the supernatant to a new 1.5 mL centrifuge tube;

2.2. Exonuclease Exo V to remove nuclear genes: (NEB#M0345L)

Components Sample volume Purified mitochondrial supernatant 9.5uL NEBuffer 4 9.5uL ATP 1.2uL Exonuclease V 1uL

Incubate at 37°C for 25-35 minutes to remove residual nuclear genome; then incubate at 65-70°C for 25-35 minutes.

2.3. Purify the product using the Zymo D4014 kit and elute with _9uL H20;

2.4. PCR 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: GCAACCAGTTTCATGCCCA (SEQ ID NO. 7);

8363-Rev: TTATGGTGGGCCATACGGTAGTA (SEQ ID NO. 8);

Components Sample volume Purified product 2uL H ₂ 0 6.4uL 2X PCR Mix 10uL Primer For 0.8uL Primer Rev 0.8uL

The reaction system was as follows: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 63°C for 30 seconds, extension at 72°C for 1 minute, 35 cycles; hold at 4°C;

2.5. 2% agarose gel electrophoresis was performed at 180 V, 180 mA, 100 W for 30 minutes. The results are shown in FIG1 .

3. Tn5 enzyme fragmentation library construction:

3.1. Tn5 enzyme fragmentation: (Vazyme TD501 kit)

(1) Add 12.4ul 2xTD + 0.5ul Tn5 to the purified mitochondria and mix well;

(2) Metal bath at 37°C, shake for 20-40 min;

(3) Purification using the Zymo D4014 kit;

(4) PCR plus adapter primer (Vazyme TD501 kit):

Components Sample volume Purified DNA 6uL 5XTAB 4uL i5 2uL i7 2uL TAE 0.5uL H ₂ 0 5.5uL

PCR conditions: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 10 seconds, 63℃ annealing for 30 seconds, 72℃ extension for 1 minute, 16-18 cycles; 4℃ hold

3.2. Take 1uL and run it on 2% agarose gel electrophoresis. The result is shown in Figure 2.

3.3. Double-wheel magnetic bead sorting:

1) Pipette 0.5× magnetic beads into the PCR product, vortex to mix, centrifuge briefly, and let stand at room temperature for 5 minutes;

2): Place the EP tube on the magnetic rack and let it stand for 5 minutes until the magnetic beads are completely adsorbed;

3): Keep the EP tube on the magnetic stand and carefully pipette the supernatant into a new EP tube to avoid contact with the magnetic beads;

4): Add 0.3× magnetic beads, vortex to mix, centrifuge briefly, and let stand at room temperature for 5 minutes;

5): Place the EP tube on the magnetic rack and let it stand for 5 minutes until the magnetic beads are completely adsorbed;

6) Keep the EP tube on the magnetic rack and carefully discard the supernatant;

7) Add 250ul of freshly prepared 80% ethanol to the EP tube, let it stand for 3-5min, and then discard the ethanol after the suspended magnetic beads are completely adsorbed;

8): Repeat washing once with 80% ethanol, then aspirate clean;

9) Keep the EP tube on the magnetic rack and let it stand for 5 minutes until the ethanol evaporates completely;

10): Add H ₂ 0 to the EP tube, vortex and mix, and let stand at room temperature for 5 minutes;

11): Place the EP tube on the magnetic rack and let it stand for 5 minutes until the magnetic beads are completely adsorbed;

12): Pipette the eluate into a new EP tube and discard the magnetic beads;

3.4. Take 2uL of each sample for Qubit concentration determination, and then mix the samples;

4. Use the Illumina platform to perform double-end sequencing of the constructed library, 150bp each. Then use the GATK mitochondrial genome standardized analysis process to compare the obtained sequence with the human mitochondrial genome NC_012920 database, and use all short sequences mapped to the mitochondrial genome for downstream variant site analysis.

5. Results summary:

The average sequencing depth of this 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

For the same sample in Example 1, a long-fragment PCR method was used to construct a library and sequence it. The long-fragment PCR library construction method uses two pairs of primers to divide the mitochondrial DNA into two segments, and the primer sequences are:

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 of the long-fragment PCR method was 26030×. 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

Compared with the long-fragment PCR method, the method of the present invention has good library uniformity by analyzing the library coverage (see Figure 3). However, for the entire library construction process, the long-fragment PCR method takes 2 days, and the present method takes 6 hours. It can be seen that the efficiency of the method of the present invention is much higher than that of the long-fragment method. By comparing the sequencing results (Table 1 and Table 2), it can be seen that for sites with heterogeneous mutation ratios between 10% and 100%, the points detected by the method of the present invention and the long-fragment PCR method and their heterogeneity are completely consistent; when the heterogeneity is less than 10%, the results obtained by the two methods are slightly different, but basically consistent.

Claims (4)

1. A method of obtaining high purity mitochondrial genome mtDNA independent of PCR comprising the steps of:

I: differential centrifugation, peripheral blood PBMC separation:

(1) Mixing fresh EDTA anticoagulant 1ml with injectable normal saline at a ratio of 1:1, and gently beating;

(2) Carefully add diluted blood to the surface of 2ml lymphocyte separator STEMCELL;

(3) 400g, centrifuging for 20 minutes;

(4) At this time, the centrifuge tube is divided into four layers from top to bottom: the first layer is a plasma layer, the second layer is an annular milky white cell layer, the third layer is a transparent separation liquid layer, the fourth layer is a red cell layer, wherein the second layer is a mononuclear cell comprising lymphocytes and monocytes, and the second layer of cells is collected and put into a test tube containing 5-10 ml of physiological saline for injection and fully and uniformly mixed;

(5) 250g, centrifuging for 10 minutes, and discarding the supernatant;

(6) Repeating the step 5, and sucking the supernatant;

II: mitochondrial DNA isolation and purification:

2.1: separating nuclear mass to obtain mtDNA

(1) Adding 15uL RSB Buffer into the precipitate obtained after differential centrifugation;

(2) Blowing for five times, and transferring into a 1.5ML centrifuge tube;

(3) Centrifuging at 4 ℃ for 10000g and 5min;

(4) Immediately pipetting the supernatant to a new 1.5ML centrifuge tube;

2.2, exonuclease Exo V removal of nuclear genes:

Component (A) Sample addition amount Purified mitochondrial supernatant 9.5uL NEBuffer4 9.5uL ATP 1.2uL ExonucleaseⅤ 1uL

Reacting at 37 ℃ for 25-35 minutes, and removing residual nuclear genome; then reacting for 25-35 minutes at 65-70 ℃;

2.3, purifying the product by using a Zymo D4014 kit, eluting with 9uL H ₂, and obtaining purified mitochondrial genome mtDNA.

2. A method of constructing a mitochondrial genome sequencing library, characterized in that mitochondrial genome mtDNA is prepared according to the method of claim 1, and Tn5 transposase is added thereto to fragment mtDNA to construct a mitochondrial genome sequencing library.

3. The method according to claim 2, characterized by the steps of:

3.1, tn5 enzyme fragmentation:

(1) Adding 12.4ul 2xTD+0.5ul Tn5 to the purified mtDNA prepared according to the method of claim 1, mixing well;

(2) Metal bath is carried out at 37 ℃ and shaking is carried out for 20-40min;

(3) Purification was performed using the Zymo D4014 kit;

(4) PCR adaptor primer:

Component (A) Sample addition amount Purified DNA 6uL 5XTAB 4uL i5 2uL i7 2uL TAE 0.5uL H₂0 5.5uL

PCR conditions: pre-denaturation at 98 ℃ for 30 seconds; denaturation at 98℃for 10 seconds, annealing at 63℃for 30 seconds, elongation at 72℃for 1 minute, and circulation 16-18 times; keeping at 4 ℃;

3.2, sucking 1uL and performing 2% agarose gel electrophoresis gel running;

3.3, sorting the magnetic beads with two wheels:

1): sucking 0.5 Xmagnetic beads into the PCR product, mixing by vortex oscillation, and standing at room temperature for 5min after instantaneous centrifugation;

2): placing the EP tube on a magnetic rack, and standing for 5min until the magnetic beads are completely adsorbed;

3): holding the EP tube on a magnetic rack, carefully sucking the supernatant to a new EP tube, avoiding contacting the magnetic beads;

4): adding 0.3×magnetic beads, mixing by vortex oscillation, centrifuging instantaneously, and standing at room temperature for 5min;

5): placing the EP tube on a magnetic rack, and standing for 5min until the magnetic beads are completely adsorbed;

6): holding the EP tube on a magnetic rack, carefully pipetting the supernatant;

7): adding 250ul of freshly prepared 80% ethanol into an EP tube, standing for 3-5min, and absorbing and discarding the ethanol after the suspended magnetic beads are completely absorbed;

8): repeatedly washing with 80% ethanol once, and then sucking;

9): keep EP tube set on the magnetic force frame, the magnetic force frame is provided with a plurality of magnetic holes, standing for 5min until ethanol volatilizes completely;

10): adding H ₂ 0 into the EP pipe, mixing by vortex oscillation, and standing at room temperature for 5min;

11): placing the EP tube on a magnetic rack, 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;

And 3.4, respectively sucking 2uL to measure the Qubit concentration, and mixing the samples.

4. A full mitochondrial genome deep sequencing method, which is characterized in that a mitochondrial genome sequencing library is created by the method of claim 2 or 3, and then a full mitochondrial genome deep sequencing result is obtained by utilizing Illumina platform double-ended sequencing.