Single-cell mitochondria sequencing method and application thereof

Abstract

The invention discloses a high-throughput single-cell mitochondria sequencing method and application thereof. The method has the advantages of high mitochondrial DNA capture rate, high mutation detection rate, low pollution rate, low cost and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of sequencing, and in particular relates to a method for single-cell mitochondrial sequencing and its application. Background Art

[0002] The current conventional scATAC-seq (single-cell assay for transposase-accessible chromatin with sequencing) experimental method relies on the treatment of the cell nucleus, which consumes and damages mitochondria. Ultimately, only about 1% of the sequencing data can be mapped to mitochondrial DNA. This sequencing depth is not enough to explain the high copy number (100-1000 per cell) and high mutation rate (2-10 times that of nuclear genomic DNA) of mitochondrial DNA (mtDNA) variations in each cell. It is far from enough for the evaluation of mtDNA mutations in single cells and the inference of clonal origin. Recently, a study reported the mtscATAC-seq (mitochondrial single-cell assay for transposase-accessible chromatin with sequencing) technology based on the 10x Genomics Chromium platform, which can combine high-confidence mtDNA mutation detection technology with high-quality chromatin accessibility analysis technology to establish a high-throughput, droplet-based (Droplet-based) mtDNA single-cell transposase chromatin accessibility sequencing technology, namely mtscATAC-seq (Lareau CA, et al. Nature Biotechnology. 2021; 39(4): 451-461. doi: 10.1038 / s41587-020-0645-6.). This technology mainly uses special cell lysis buffer to slightly lyse or permeabilize cells, then integrates adapters into open chromatin and mtDNA, uses formaldehyde (FA) to cross-link cells during lysis or permeabilization to fix mitochondria in cells, then uses mild lysis buffer to punch holes in cells so that transposase can enter cells, adds adapter sequences to open regions of nuclear chromatin and mitochondrial DNA, and finally uses scATAC-seq technology in the 10x Genomics Chromium platform to achieve simultaneous sequencing of open regions of nuclear chromatin and mitochondrial DNA at the single-cell level. However, this method cannot be applied to the C4 platform.

[0003] The disadvantages of this method include: (1) the mtscATAC-seq system and method based on 10x Genomics Chromium is not suitable for use on the C4 platform; (2) the proportion of mtDNA fragments captured by this method is not high; (3) the method is lengthy and economically expensive (expensive commercial instruments and reagents are used); (4) the sequencing throughput of this method is relatively low, the contamination rate is high, and the detection rate of mtDNA with low mutation rate is low; (5) this method is only used for tumors and PBMC (peripheral blood monoculearcell), and its universality in other tissues and organs is generally poor.

[0004] Therefore, there is an urgent need in the art for a high-throughput single-cell mitochondrial sequencing method suitable for the C4 platform. Summary of the invention

[0005] As mentioned above, there is an urgent need in the art for a high-throughput single-cell mitochondrial sequencing method suitable for the C4 platform.

[0006] The present invention utilizes the domestic DNBelab C4 platform to perform single-cell mitochondrial ATAC-seq to detect mitochondrial DNA variation at the single-cell level. Thus, the present invention is realized.

[0007] In a first aspect, the present invention provides a method for single-cell mitochondrial sequencing, the method comprising the following steps:

[0008] (1) Treat cells with methanol fixation;

[0009] (2) performing cell membrane perforation treatment on the cells; for example, treating the cells with a mild cell lysis solution; preferably, the mild cell lysis solution is NP-40;

[0010] (3) adding transposase to carry out transposition reaction;

[0011] (4) Perform single-cell library construction and sequencing.

[0012] In a second aspect, the present invention provides an application of the method described in the first aspect, including but not limited to detecting SNPs of the mitochondrial genome, mutations of the mitochondrial genome, and cell typing.

[0013] The beneficial effects of the present invention are: (1) the method of the present invention is portable and has low economic cost; (2) the sequencing throughput is relatively high; (3) the capture rate of mtDNA fragments is high and the contamination rate is low; (4) the detection rate of mtDNA with a low mutation rate is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solution of the present invention, the accompanying drawings will be briefly introduced below.

[0015] Figure 1 The C4_mtscATAC-seq method and workflow are shown.

[0016] Figure 2 The figure shows the comparison of the mitochondrial sequence alignment results. Figure A is the result of direct mitochondrial sequence alignment, and Figure B is the result of alignment after adding the mitochondrial starting 50bp sequence at the end of the mitochondrial reference sequence.

[0017] Figure 3 The proportion of mitochondrial sequences detected in different experimental conditions of 3T3 cells is shown.

[0018] Figure 4 The proportion of mitochondrial sequences detected in different experimental conditions of 293T cells is shown.

[0019] Figure 5 Shown are the alignment rates of nuclear genomes after treatment with formaldehyde and methanol.

[0020] Figure 6 A flow chart showing the use of the C4_mtscATAC-seq system to detect mouse tissues (spleen, bone marrow, lung, PBMC).

[0021] Figure 7 The figure shows the results of detecting mtDNA mutations in lung tissue of aged mice using the C4_mtscATAC-seq system, where VMR is the variance mean ratio. DETAILED DESCRIPTION

[0022] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is only used to illustrate the present invention by way of example, and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims. In addition, those skilled in the art will understand that the technical solution of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0024] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can usually be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this article, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. At this time, the expression is equivalent to the expression "consisting of..."

[0025] In order to better understand the present teachings and not to limit the scope of the present teachings, unless otherwise indicated, all numbers and other numerical values ​​used in the specification and claims indicating quantities, percentages or ratios should be understood as being modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values, which may vary depending on the desired properties sought to be obtained. At a minimum, each numerical parameter should at least be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0026] In this article, the term "ATAC-seq" refers to a technology for studying chromatin accessibility in molecular biology, which consists of two parts: ATAC experiment and high-throughput sequencing. The key part of the ATAC-seq experiment is the action of Tn5 transposase on sample genomic DNA. Transposons preferentially incorporate into open chromatin DNA without nucleosome protection. Therefore, open chromatin DNA is in a loosely exposed state, providing information about the active state of transcription of chromatin segments. ATAC-seq uses a mutated multi-active transposase, which allows efficient cutting of exposed DNA and simultaneous connection of adapters of specific sequences. Separate the adapter-connected DNA fragments and use them for high-throughput sequencing after PCR amplification.

[0027] In this article, the term "SNP" is a single nucleotide polymorphism (single nucleotide polymorphism), refers to a single base position in DNA, at which there are different alleles or alternative nucleotides of a population. The SNP position is usually preceded and followed by a highly conserved sequence of the allele (e.g., a sequence different in a member less than 1 / 100 or 1 / 1000 in a population). For the allele at each SNP position, an individual can be homozygous or heterozygous.

[0028] As used herein, the term “alignment” refers to the correspondence between a sequencing read sequence and a reference sequence. A sequencing read sequence may have multiple alignment results at the same time.

[0029] As used herein, the term "transposase" refers to a polypeptide that catalyzes the excision of a transposon from a first nucleic acid (eg, a vector) and integration into a target site (eg, a second nucleic acid or genomic or extrachromosomal DNA in a cell).

[0030] As mentioned above, the present invention aims to provide a method for high-throughput single-cell mitochondrial sequencing suitable for the C4 platform.

[0031] Therefore, in a first aspect, the present invention provides a method for single-cell mitochondrial sequencing (also referred to herein as C4_mtscATAC-seq), the method comprising the following steps:

[0032] (1) Treat cells with methanol fixation;

[0033] (2) Performing cell membrane perforation treatment on cells;

[0034] (3) adding transposase to carry out transposition reaction;

[0035] (4) Perform single-cell library construction and sequencing.

[0036] In one embodiment, the viability of the cells in step (1) is ≥ 80%. The cells in step (1) can be a single cell suspension.

[0037] In one embodiment, the concentration of the methanol in step (1) is 100%. It should be understood that other suitable concentrations of methanol can also be used in the present invention. In a preferred embodiment, the methanol is precooled methanol.

[0038] In one embodiment, in step (2), cells can be treated with a mild cell lysis solution. In a preferred embodiment, the mild cell lysis solution is NP-40. In a preferred embodiment, the concentration of NP-40 is 0.1% (V / V).

[0039] In one embodiment, the transposase in step (3) is Tn5 transposase. It should be understood that other suitable transposases can also be used in the present invention to achieve transposition reactions.

[0040] In one embodiment, step (4) further includes the steps of adding sequencing primers and barcodes (label sequences for distinguishing different samples during sequencing, also called barcodes) to the DNA fragments of individual cells, mixing and purifying the DNA fragments of all cells, performing an amplification reaction, and purifying the amplified products. In step (4), special magnetic beads can be used to add barcodes to the DNA sequences in each cell. In a further embodiment, the amplification reaction includes two cycles, with the cycle numbers being 13 and 10 respectively, which can reduce the content of repeated sequences in the library caused by amplification.

[0041] In one embodiment, the method further comprises step (5): analyzing the sequencing data. In the data analysis process, the mitochondrial sequence comparison method is: artificially construct a new mitochondrial sequence, add the sequence of the mitochondrial starting region at the end of the mitochondria, and the added sequence length can be 50bp, 100bp, 150bp, 200bp, etc. In a preferred embodiment, the mitochondrial starting 50bp sequence is added at the end of the mitochondria. In a further embodiment, the mitochondrial sequence data is removed from the analysis and the cells are clustered.

[0042] In the second aspect, the present invention provides an application of the method described in the first aspect, which includes but is not limited to detecting SNPs in the mitochondrial genome, mutations in the mitochondrial genome, and cell typing, such as studying the mitochondrial genome mutation status of each cell in a cell line or animal tissue.

[0043] Example

[0044] It should be noted that the terms used in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The above summary of the invention and the detailed description below are only for the purpose of specifically explaining the present invention, and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the attached claims.

[0045] Example 1: Sequencing of human HEK-293T and mouse 3T3 cell libraries

[0046] The flowchart of this embodiment is as follows Figure 1 The specific process of the experiment is as follows:

[0047] (1) Resuscitation of human HEK-293T and mouse 3T3 cells, check the cell viability, which should be above 80%, centrifuge and discard the supernatant;

[0048] (2) Resuspend human HEK-293T and mouse 3T3 cells in 1 mL of formaldehyde and pre-cooled 100% methanol, incubate on ice for 10 min, centrifuge at 300 g for 5 min at 4°C, and discard the supernatant;

[0049] (3) Add 1 mL of pre-cooled PBS, centrifuge at 300 g for 5 min at 4°C, and discard the supernatant;

[0050] (4) Resuspend the cells in 100 μL of 0.1% NP-40 Lysis Buffer, pipette gently to mix, and incubate on ice for 5 min;

[0051] (5) Add 1 mL of Wash Buffer A, pipette gently to mix, centrifuge at 500 g for 5 min at 4°C, discard the supernatant, and wash the cells twice with 400 μL of Wash Buffer A;

[0052] (6) Resuspend the cell pellet with 1% BSA (PBS), pipette to mix, and count the DAPI;

[0053] (7) Take 100,000 human HEK-293T cells and mouse 3T3 cells, respectively, and transpose them in a PCR instrument using 50 μL of transposition system and 4 μL of Tn5 transposase;

[0054] (8) After transposition, 5000 3T3 and HEK-293T cells were taken for droplet generation;

[0055] (9) Single-cell library construction was completed using the DNBelab C series high-throughput single-cell ATAC library preparation kit (MGI, #1000021878), with 13 and 10 PCR amplification cycles respectively;

[0056] (10) Shenzhen National Gene Bank was commissioned to complete the sequencing, and the data volume of a single library was greater than 500M reads; (11) Bioinformatics analysis: Since mitochondria are a ring structure, it is easy to cause misalignment at the beginning and end during alignment. Therefore, in this step, a new mitochondrial sequence was artificially constructed, and the mitochondrial starting 50bp sequence was added to the end of the mitochondria to achieve a complete alignment of the mitochondria ( Figure 2 ), chromap was used as the specific alignment software; after the alignment was completed, the mtDNA sequence alignment results were removed, and d2c software was used for cell division; after cell division, the sequenced mitochondrial reads were assigned to the corresponding cells according to the barcode, and the sequencing depth and proportion of each cell were counted.

[0057] The results of one of the above experiments are as follows: After analysis, the number of HEK-293T cells and 3T3 cells were 2565 and 1869, respectively, the median number of sequencing fragments per cell were 10546 and 7088, respectively, the median percentage of fragments with overlapping peaks were 20% and 21.3%, respectively, and the median percentage of fragments with overlapping TSS (Transcription Start Site) were 25.59% and 33.26%, respectively.

[0058] Figure 3 and Figure 4 The proportions of mitochondrial sequences detected under different experimental conditions for 3T3 cells and 293T cells are shown respectively. The results show that the proportion of mitochondrial sequences in the obtained sequences using the methanol fixation method for C4_mtscATAC-seq library construction is higher than that of scATAC-seq and C4_mtscATAC-seq using the formaldehyde fixation method. Figure 5 The alignment rates of nuclear genomes after treatment with formaldehyde and methanol are shown. The results show that the methanol fixation method obtained a higher sequence alignment rate than the formaldehyde fixation method.

[0059] Example 2: Sequencing of mouse tissue library

[0060] In this example, three 23-month-old old mice (C57BL / 6J) were used as the experimental group, and three 2-month-old young mice were used as the control group. The spleen, lung, PBMC and bone marrow of the mice were used as experimental objects, and single cell suspensions were prepared respectively, and then C4_mtscATAC-seq was performed. The flow chart of this example is shown in the figure. Figure 6 shown.

[0061] The preparation process of mouse spleen single cell suspension is as follows:

[0062] (1) After killing the mouse by cervical dislocation, the capsule and excess tissue of the freshly isolated mouse spleen were quickly removed and washed 1-2 times with tissue washing buffer / DMDM;

[0063] (2) Use a surgical blade to mince the tissue into a semi-homogeneous state (no more than 5 min) and wash the minced tissue with 1 mL of tissue washing solution;

[0064] (3) Use a 1 mL wide-mouth pipette tip to collect the chopped tissue into a 5 mL tube, add 2-3 mL of enzymatic solution, and seal with a sealing film;

[0065] (4) Digest spleen tissue in a 37°C water bath for 10 min, mixing gently during digestion;

[0066] (5) Place a 40 μm cell strainer in a 35 mm cell culture dish, rinse the cell strainer with 1 mL of tissue cleaning solution, filter the cell suspension, and then rinse the cell strainer with 4 mL of tissue cleaning solution to fully collect the cells. Finally, transfer the filtered liquid to a 15 mL centrifuge tube;

[0067] (6) Centrifuge at 300 g for 10 min at 4°C and discard the supernatant;

[0068] (7) Lyse red blood cells. Add 1 mL of pre-cooled cell washing solution to the obtained cell pellet, resuspend the cells, and then add 3 mL of red blood cell lysis solution. Mix gently without vortexing.

[0069] (8) Incubate on ice for 5 min;

[0070] (9) Add 5 mL of pre-cooled cell washing solution;

[0071] (10) Centrifuge at 300 g for 10 min at 4°C and discard the supernatant;

[0072] (11) Add 5 mL of pre-cooled cell washing solution to the obtained cell pellet, resuspend the cells, centrifuge at 300 g for 10 min at 4°C, and discard the supernatant;

[0073] (12) Repeat the previous step;

[0074] (13) Resuspend the cells using cell washing solution;

[0075] (14) Trypan blue staining was used to observe cell morphology, survival rate, aggregation rate, and count under a microscope.

[0076] The preparation process of mouse bone marrow single cell suspension is as follows:

[0077] (1) Using sterile techniques, 2-month-old and 23-month-old mice were killed, their tibiae and femurs were removed, and the muscles were stripped to expose the bone marrow cavity of the mice;

[0078] (2) Collect bone marrow and place the bone marrow sample into a sterile cell culture dish and wash with cold phosphate-buffered saline (PBS) to obtain bone marrow cells;

[0079] (3) The cell suspension was filtered using a 70 μm cell strainer (Falcon cell strainer, Franklin Lakes, NJ, USA) and centrifuged at 4°C, 125 g for 10 min;

[0080] (4) Resuspend the cell pellet in PBS;

[0081] (5) Add ammonium chloride solution to the harvested bone marrow cells, incubate at room temperature for 5 min, remove red blood cells, and wash with PBS three times.

[0082] In addition, single cell suspensions of lungs and PBMCs from 2-month-old and 23-month-old mice were prepared in this example.

[0083] Single cell suspensions of spleen, lung, PBMC and bone marrow of the above mice were taken to prepare libraries for sequencing and analysis, and the steps were the same as the experimental process in Example 1.

[0084] Figure 7 The results of detecting mutations in mtDNA in lung tissue of aged mice using the C4_mtscATAC-seq system are shown, including 13023T>C and 12174G>A.

[0085] In summary, the present invention uses a droplet-based method to partition and barcode single cells in a nanoliter reactor, uses magnetic beads coated with barcode oligonucleotides to capture DNA molecules from each cell, analyzes more cells and reduces the negative impact of technical and intrinsic noise, uses methanol (100%) to fix cells, fixes mitochondria in cells, and then uses 0.1% concentration of NP-40 to perforate cells, effectively ensuring that mitochondria in cells will not flow out; at the same time, the number of PCR amplification cycles is reduced to reduce redundant data in sequencing data, thereby establishing an experimental system of C4_mtscATAC-seq with independent intellectual property rights in my country. The proportion of mtDNA in the data obtained by C4_mtscATAC-seq can reach 55%, and the double cell rate is reduced to 1.89%, which can effectively analyze the mutation of mtDNA in single cells.

[0086] The single-cell mitochondrial sequencing method and its application provided by the present invention are introduced in detail above. The principles and implementation methods of the present invention are explained in this article using specific embodiments. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for single-cell mitochondrial sequencing, It is characterized in that The method comprises the following steps: (1) Treat cells with methanol fixation; (2) performing cell membrane perforation treatment on the cells; for example, treating the cells with a mild cell lysis solution; preferably, the mild cell lysis solution is NP-40; (3) adding transposase to carry out transposition reaction; (4) Perform single-cell library construction and sequencing.

2. The method according to claim 1, It is characterized in that The concentration of the methanol in step (1) is 100%; preferably, the methanol is pre-cooled methanol.

3. The method according to claim 1 or 2, It is characterized in that The concentration of NP-40 in step (2) is 0.1% (V / V).

4. The method according to any one of claims 1 to 3, It is characterized in that The transposase in step (3) is Tn5 transposase.

5. The method according to any one of claims 1 to 4, It is characterized in that Step (4) further includes the steps of adding sequencing primers and barcodes to the DNA fragments of individual cells, mixing and purifying the DNA fragments of all cells, performing an amplification reaction, and purifying the amplified products.

6. The method according to claim 5, It is characterized in that The amplification reaction includes two cycles, and the cycle numbers are 13 and 10 respectively.

7. The method according to any one of claims 1 to 6, It is characterized in that The method also includes step (5): analyzing the sequencing data.

8. The method according to claim 7, It is characterized in that The mitochondrial sequence alignment method in the analysis is: adding the sequence of the mitochondrial starting region at the end of the mitochondria as a reference mitochondrial sequence for alignment; preferably, adding the mitochondrial starting 50bp sequence at the end of the mitochondria.

9. The method according to any one of claims 1 to 8, It is characterized in that The sequencing was performed on the C4 platform.

10. Use of the method according to any one of claims 1 to 9, It is characterized in that The applications include but are not limited to detecting SNPs of the mitochondrial genome, mutations of the mitochondrial genome, and cell typing.

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