WO2021114713A1 - Cell lysis solution, kit, and application - Google Patents

Abstract

A cell lysis solution, a kit, and an application, comprising a cell lysis method, a library construction method, and an application thereof. The cell lysis solution is composed of proteinase K and NP-40, has a simple composition, and can effectively lyse cells and remove proteins on chromosomes. The nucleic acid solution after lysis can be directly used for subsequent reactions such as DNA library construction, without being subjected to any treatment. The cell lysis method and the library preparation method of the cell lysis solution can be used to quickly prepare a DNA library. The library preparation consumes short time, the prepared library can be used for different sequencing platforms, and GC bias of the obtained sequencing data is not significant. In addition, the kit can also be applied to sequencing-based preimplantation genetic screening (PGS).

Description

Cell lysate, kit and application

Priority information

This application requests the priority and rights of the patent application number CN201911247526.9 filed with the State Intellectual Property Office of China on December 9, 2019, and the full text is incorporated herein by reference.

Technical field

The present invention relates to the field of molecular biology. Specifically, the present invention relates to a cell lysis solution, a cell lysis method, a method for building a library, and applications.

Background introduction

At present, the construction of a sequencing library is a necessary step for sequencing to achieve abnormal detection of a small amount of cell chromosome data. Among them, the gDNA obtained by cell lysis is a key factor that affects the success of the sequencing library.

Commonly used cell lysis methods include alkaline lysis (the main component is NaOH), proteinase K lysis and so on.

The alkaline lysis method mainly uses NaOH to lyse cells, and the obtained gDNA has high requirements for the buffer in the subsequent library construction system. If the subsequent addition of reagents that can neutralize alkalinity, it is easy to increase the risk of contamination.

Proteinase K lysis method uses proteinase K to degrade proteins to achieve cell lysis. Proteinase K is active in a wide pH range (pH 4-12.5), temperature range (37-60°C), higher salt concentration (such as 3M GuHCl) and denaturant (such as 4M urea); some detergents Agents such as Tween20 (5%), Triton X-100 (1%) or SDS (1%) and metal chelating agent EDTA etc. cannot inactivate proteinase K. Because Ca ²⁺ can protect proteinase K from autolysis and increase its thermal stability, proteinase K is often stored in a ^{buffer containing calcium ions (Ca 2+} ) or reacted in a buffer ^{containing Ca 2+.} Based on the above-mentioned various characteristics of proteinase K, the inventors’ known reagent formulations that utilize proteinase K to digest proteins to achieve cell lysis, usually, also include other protease K activity or stability, and/or can act on cells including It is used as a component in the cell membrane or cell content to improve the effect of cell lysis. For example, the common proteinase K cell lysate also contains 0.5% SDS and 10mmol/L EDTA to enhance or stabilize the proteinase K enzyme activity. SDS is a strong denaturant. Even a small amount of SDS will affect the activity of many proteases. For example, DNA fragmentation enzymes, transposases, etc. will decrease or even lose their activity if affected by SDS even a small amount of SDS. For proteinase K lysate containing SDS, in order to avoid the impact on subsequent experiments, it is generally necessary to purify or separate the lysed system to remove SDS. Using purification steps to remove SDS on the one hand increases the steps and time of the experimental operation, on the other hand, it will cause DNA loss. For samples with single cells or a small number of cells, these effects are even more obvious. For example, in the pre-implantation screening in assisted reproductive technology, the amount of detection cells is small, and the time and requirements for library construction and sequencing are relatively strict. Requires fast, simple, and effective cell lysis methods that are compatible with library building methods and sequencing methods.

Summary of the invention

Based on the need for sequencing of single cells and a small number of cells (1 to 200 cells) and the use of known cell lysates to lyse cells, it is usually necessary to replace a reaction system, such as separating or purifying the lysate, before proceeding to the next reaction. Invented Man provides a cell lysis solution, a cell lysis method, and a library preparation method based on the cell lysis method.

The first aspect of the present invention provides a cell lysate, the active ingredients of the cell lysate are composed of proteinase K and NP-40. The effective ingredient referred to here includes any substance that helps cell lysis, for example, a substance that helps to increase the activity of digestive enzymes, for example, a substance that can interact with proteins, lipids and other substances on the cell membrane, nuclear membrane, or genome.

The cell lysate has a simple composition and can effectively remove the protein on the genome to obtain the genomic DNA in the cell. Moreover, the lysed solution system does not need to be replaced or processed, and can be directly used for further detection of cell genomic DNA, such as sequencing.

In some embodiments, the cell lysate may also have at least one of the following additional technical features.

According to an embodiment of the present invention, the above-mentioned cell lysate is composed of proteinase K, NP-40 and H ₂ O. The proteinase K and NP-40 in the cell lysate are commercially available conventional reagents. It is understood that, preferably, the proteinase K, NP-40 and H ₂ O used to prepare the cell lysate do not contain DNA polymerase and DNA. Fragmentation enzyme activity component.

According to an embodiment of the present invention, the concentration of proteinase K in the cell lysate is 2 ng/mL to 200 μg/mL, and/or the volume percentage concentration of NP-40 is 1% to 10%. The cell lysates with different concentrations of proteinase K and NP-40 were tested. When the cell lysates were combined with proteinase K and NP-40 concentrations of 2ng/mL and 1%, 2ng/mL and 10%, 200μg/ The cell lysate can effectively lyse the cells at the time of mL and 10%, 200μg/mL and 1%, 1μg/mL and 5%, 100ng/mL and 1%, 100μg/mL and 10%. Based on the tested combinations of multiple different concentrations of proteinase K and NP-40, it is reasonable to believe that any concentration of proteinase K in the range of 2ng/mL～200μg/mL and any concentration of NP in the range of 1%～10% The combination of -40 can effectively lyse cells.

According to an embodiment of the present invention, the concentration of proteinase K in the cell lysate is 0.002-1 μg/mL or the concentration of proteinase K is 0.002-0.1 μg/mL, which can also ensure that cells are effectively lysed, and can further reduce the cost of the cell lysate.

The cell lysate in any of the above embodiments has a simple composition, can effectively lyse cells, and is particularly suitable for the lysis of a small number of animal cells (1 to 200 cells), and the components in the lysate do not affect subsequent reactions, such as nucleic acid fragmentation reactions. , Amplification reaction. In an example of the present invention, after using the cell lysate in any of the above embodiments to lyse the cells, the genome coverage in the result of database construction and sequencing is 99.54%. It can be seen that the use of any of the above cell lysates can effectively remove chromosomes. The protein on the chromosome to remove the influence of the protein on the sequencing.

The second aspect of the present invention provides a cell lysis method, which comprises using the cell lysis solution in the first aspect or any of the embodiments to lyse cells. The above description of the technical features and advantages of the cell lysis solution in the first aspect or any of the embodiments of the present invention is also applicable to the cell lysis method.

According to an embodiment of the present invention, the cells in the cell lysis method are animal cells, the number of cells is 1-200, and the number of cells in an example is 1-10.

According to an embodiment of the present invention, the cell lysis method performs lysis at 55°C to 65°C, and/or the lysis time is 30min to 60min.

According to an embodiment of the present invention, the cell lysis method further includes adding a termination reaction solution to terminate the lysis. The types of the termination reaction solution include protease inhibitors, which are used to terminate the activity of proteinase K, such as PMSF (phenylmethyl sulfonyl fluoride), antipain (anti-protease peptide), etc. In the example of the present invention, the termination reaction solution added is In PMSF aqueous solution, in order to avoid the influence of protease inhibitors on subsequent experiments, the PMSF concentration as the termination reaction solution should not be too high. The final concentration of the termination reaction solution selected is 0.5 mM ~ 1 mM, and the final concentration is the termination reaction solution to terminate the lysis The reaction concentration during the reaction.

The above cell lysis method has simple reaction conditions and convenient operation, can effectively lyse a small amount of animal cells to obtain genomic DNA, and the components in the cell lysis solution and the termination reaction solution do not affect DNA fragmentation and amplification, and the lysed cell solution can be used directly For follow-up experiments.

The third aspect of the present invention provides a nucleic acid preparation method, which includes obtaining cell nucleic acid by using the cell lysis method in the second aspect or any of the examples of the present invention. The nucleic acid is DNA and/or RNA of the cell. In the example, the nucleic acid is DNA. The DNA prepared by this method has no protein on it, and can be directly used to prepare a DNA library without purification to remove the cell lysate and stop the reaction solution. The above description of the technical features and advantages of the cell lysis method in the second aspect of the present invention or any of the embodiments is also applicable to the nucleic acid preparation method.

The fourth aspect of the present invention provides a nucleic acid library preparation method, which includes the steps of: obtaining a nucleic acid using the nucleic acid preparation method in the third aspect or any example of the present invention; and fragmenting the nucleic acid.

In an example, the nucleic acid library preparation method further includes amplifying the fragmented nucleic acid to obtain the nucleic acid library.

According to an embodiment of the present invention, there is no purification or separation operation between the steps of the method, that is, there is no purification or separation operation after the steps of obtaining the nucleic acid and fragmenting the nucleic acid respectively, and adding the fragment reagent after obtaining the nucleic acid to perform the fragmentation treatment , The fragmented product is directly used for amplification. In the nucleic acid library preparation method, a fragmentation reagent is used to perform the fragmentation. When the nucleic acid is DNA, the fragmentation reagent is used to fragment the DNA. It can be understood that there are many methods for fragmenting DNA, such as ultrasonic interruption and DNA fragmentation reagent interruption. The DNA fragmentation reagent includes transposase complexes, DNA interruption enzymes, and the like. For DNA obtained from a small number of cells, it is preferable that a DNA fragmentation reagent fragments the DNA. DNA fragmentation enzymes can be selected from Tiangen DNA Fragmentation Kit NG305-02, Qiagen CM0162 Fragmentase Kit, NEB Fragmentase and other endonucleases or combinations of endonucleases capable of fragmenting DNA, etc.; transposition Enzyme complexes include Tn transposition complex, Mu transposition complex and other complexes with transposition function. In the example, the transposase complex is Tn transposition complex, specifically Tn5 transposase complex body. The Tn5 transposase complex is a complex formed by Tn5 transposase and a transposable linker. The transposable linker consists of a Tn5 transposase recognition sequence or a Tn5 transposase recognition sequence and a known PCR primer for recognition. The sequence composition.

The nucleic acid library preparation method described above is to obtain nucleic acid by using the nucleic acid preparation method provided in the third aspect of the present invention or any example. The above description of the technical features and advantages of the nucleic acid preparation method in the third aspect of the present invention or any example also applies The nucleic acid library preparation method.

In the example, after the cell lysate is lysed, the termination reaction solution is added to the original reaction tube to terminate the lysis reaction to obtain nucleic acid; in the same reaction tube, a fragmentation reagent is added to fragment the nucleic acid; in the same reaction tube, the expansion is added The amplification reagent is used for amplification to obtain a nucleic acid library. There is no purification operation or separation operation between the steps of the method, and the reaction in each step is carried out in the same reaction tube. It can be seen from the experimental results that the cell lysate and termination reaction solution do not affect the effect of the fragmentation reagent and the amplification effect. Utilizing this nucleic acid library preparation method, it takes about 3.5 hours from cell lysing to obtaining a DNA library; a nucleic acid library preparation method including two amplification and multiple purification steps is used to obtain a nucleic acid library (lysing cells to obtain nucleic acid; MDA (multiplex) Displacement amplification) technology to amplify the nucleic acid obtained by cell lysis; fragment nucleic acid; purify the fragmented product; PCR amplify the purified product; purify the PCR amplified product to obtain a nucleic acid library.), it takes about 16 hours, and the time used is about this The nucleic acid preparation method provided in the fourth aspect of the invention or any of the embodiments takes 5 times the time. The nucleic acid preparation method provided in the fourth aspect or any of the embodiments of the present invention is simple and rapid in operation, and the library prepared by this method can be used for sequencing.

The fifth aspect of the present invention provides a sequencing method, which includes using the nucleic acid library prepared in the fourth aspect or any example of the present invention for sequencing to obtain a sequencing result. The nucleic acid obtained by the cell lysis method in any of the above examples can be used to construct libraries of different sequencing platforms, such as Illumia's MiSeq and HiSeq sequencing platforms (HiSeq-2500, Hiseq-2000, NextSeq 500, HiseqX10 and other sequencers), PacBio sequencing Platforms (PacBio RS series, Sequel series sequencers), BGI gene sequencing platforms (MGISEQ-2000, MGISEQ-200, BGISEQ-500, DNBSEQ-T7 and other sequencers), etc.

In some embodiments, the nucleic acid is obtained by the cell lysis method in any of the above embodiments, or the nucleic acid library constructed in any example is sequenced using the sequencing platform built by the inventor, and CNV mutations of not less than 1M can be detected, and the sequencing result The GC bias and MAD (Median Absolute Deviation) in GC are lower than those in the sequencing results obtained by using a nucleic acid library preparation method including two amplification and multiple purification steps to obtain a nucleic acid library and sequencing using a high-throughput sequencing platform. Among them, a nucleic acid library preparation method including two amplification and multiple purification steps includes: cell lysis to obtain nucleic acid; MDA technology to amplify nucleic acid obtained from cell lysis; fragmentation of nucleic acid; purification of fragmented products; PCR amplification and purification of purified products; The PCR product was amplified to obtain a nucleic acid library. In the embodiment, the IlluminaHiSeqX10 high-throughput sequencing platform is used for high-throughput sequencing. The sequencing platform built by the inventor refers to CN201610209150.2, CN201710607295.2, CN201610958047.8, CN201710607306.7, CN201620278363.6, CN201520614342.2, CN201520614143.1, CN201520614143.1, CN201711105069.0 and/or CN201621419140.3. For one or more of the schemes, the operation process can be performed by referring to https://doi.org/10.1371/journal.pone.0188181(Single molecule sequencing of the M13 virus genome without amplification), for example.

The sixth aspect of the present invention provides a kit, which contains the cell lysate in the first aspect or any of the examples of the present invention. The active ingredients of the cell lysate in the kit can be stored in different forms, such as proteinase K and NP-40 separately stored in high concentration form, or stored in high concentration form after the two ingredients are mixed, or after mixing Store in a suitable concentration form. According to the different storage forms of the two reagents in the kit, different treatment methods are selected when using the cell lysate, and finally the two substances are configured into a mixture of appropriate concentrations, where the appropriate concentration refers to the concentration of proteinase K at 2ng/mL ~200μg/mL, the volume percentage concentration of NP-40 is 1%-10%; high concentration means that the concentration of one or both of the two substances is higher than the highest value of the respective suitable concentration.

According to an embodiment of the present invention, the above kit further includes a termination reaction solution, and the termination reaction solution is used to terminate the cell lysis reaction. The termination reaction solution can be a protease inhibitor, such as PMSF (phenylmethylsulfonyl fluoride), antipain (anti-protease peptide), etc., to terminate the activity of proteinase K. In the example, the termination reaction solution provided by the kit is PMSF. The concentration of PMSF provided in the kit is not less than 5mM, or the PMSF provided in the kit is solid, and when used, it is configured as an aqueous solution of PMSF with a concentration of not less than 5mM.

According to an embodiment of the present invention, the above kit further includes a nucleic acid fragmentation reagent, which is used to fragment the nucleic acid or to fragment the nucleic acid while adding a known sequence to both ends or ends of the fragmented nucleic acid. The types of nucleic acid fragmentation reagents include transposase complexes, DNA disrupting enzymes and so on. For small amounts of DNA (<50ng), it is preferred that DNA fragmentation reagents fragment the DNA. DNA fragmentation enzymes can be selected from Tiangen DNA Fragmentation Kit NG305-02, Qiagen CM0162 Fragmentase Kit, NEB Fragmentase and other endonucleases or combinations of endonucleases capable of fragmenting DNA, etc.; transposition The enzyme complex is selected from Tn transposition complex, Mu transposition complex and other complexes with transposition function. In the example, the DNA fragmentation reagent is the Tn5 transposase complex, the Tn5 transposase complex is a complex formed by the Tn5 transposase and the transposable linker, and the transposable linker is composed of the Tn5 transposase recognition sequence or Tn5 transposase recognition sequence and a known sequence for PCR primer recognition.

When using the above kit to construct a nucleic acid library, the cell lysis solution and the termination reaction solution do not affect the effect of the fragmentation reagent and the amplification effect, and the nucleic acid obtained after cell lysis does not need to be purified and can be directly used for nucleic acid library construction. Using this kit to construct a library can be done from cell lysis to amplification. The reaction is carried out in a reaction tube without the need to replace the reaction tube. The use of this kit to construct a nucleic acid library on the one hand simplifies the operation steps and reduces the cost, on the other hand, reduces the loss of DNA, and is suitable for library construction with a small number of samples. The description of the advantages of the cell lysis solution, cell lysis method, nucleic acid preparation method, and nucleic acid library preparation method in any of the above embodiments is also applicable to the kit.

The seventh aspect of the present invention provides the application of the kit in a method or device involving cell lysis, including application to preimplantation genetic screening of embryos. The results of preimplantation genetic screening using this kit are consistent with the results of preimplantation genetic screening using high-throughput sequencing, which can detect chromosomal mutations of CNV (copy number variation) not less than 1M .

The cell lysate provided in any aspect or embodiment of the present invention has a simple composition and can effectively remove proteins on the genome, and the lysed cell solution is directly used for DNA library construction without purification.

The cell lysis method using the cell lysate can quickly obtain genomic DNA.

The library preparation method using the cell lysate can quickly prepare a DNA library, and the prepared library can meet the requirements of different sequencing platforms.

The cell lysis method, library construction method, sequencing method, etc., using the cell lysate provided in any of the foregoing embodiments are particularly suitable for clinical use due to simple operations.

The additional aspects and advantages of the embodiments of the present application will be partly given in the following description, and part of them will become obvious from the following description, or be understood through the practice of the embodiments of the present application.

Description of the drawings

Figure 1 shows the detection result of the Chinese library of Example 2 by LabChip GX Touch (PerkinElmer) microfluidic capillary electrophoresis;

Figure 2 shows the detection result of the Chinese library of Example 3 by LabChip GX Touch (PerkinElmer) microfluidic capillary electrophoresis;

Figure 3 shows the detection result of the Chinese library of the comparative example by LabChip GX Touch (PerkinElmer) microfluidic capillary electrophoresis;

Figure 4 shows the agarose electrophoresis detection diagram of the amplified product 1 in the comparative example;

Figure 5 shows the sequencing and analysis results of chES497 samples on the single-molecule sequencing platform built by Illumina HiseqX10 and the inventor;

Figure 6 shows the sequencing analysis results of chES497 samples on the single-molecule sequencing platform built by Illumina HiseqX10 and the inventor.

Detailed ways

The present invention will be described below with reference to specific embodiments and drawings. It should be noted that these embodiments are only illustrative, and cannot be understood as a limitation of the present invention. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. Those reagents or instruments that do not indicate the manufacturer are all conventional products that can be purchased on the market. The cell samples used in the examples are used to verify the feasibility of the cell lysis method and the library preparation method. The cells used are not limited to the cell lines selected in the examples. The cell lines used in Examples 2, 3 and 4 are 90-P50 And chES497(46,XX,del(1p36.33-1p36.32)1.29M, dup(16p13.3-16.13.11)16.72M) were all from Hunan Guangxiu Hospital.

The "high-throughput sequencing" referred to in this article refers to the ability to sequence hundreds of thousands to millions of nucleic acid molecules in parallel at a time. The GC bias (GC preference) value in the sequencing data is an important indicator for judging the effect of sequencing. The lower the GC bias, the lower the GC bias introduced during library construction and sequencing. The difference between DNA molecules with different GC composition and rich GC or less The bias of chromosome sequencing data containing GC is reduced. The MAD (Median Absolute Deviation) in sequencing data is the same as the conventional definition of MAD. For sequencing data, it refers to the median of the absolute value of the new data obtained by subtracting the median from the original data, which is used to measure the data The smaller the MAD value, the lower the degree of dispersion of the measured data, the more concentrated the distribution of data without anomalies, and the easier to distinguish discrete data.

In the following examples, the concentration of NP-40 refers to the volume percentage concentration of NP-40 in the cell lysate.

In the following examples, the concentration of proteinase K refers to the concentration of proteinase K in the cell lysate.

Example 1

The following is an example of how to prepare the kit:

The kit contains tube 1, which is a cell lysate. The active ingredients of the cell lysate are composed of proteinase K and NP-40. The concentrations of the two are 2ng/mL～200μg/mL and 1%～10%, respectively. 1%～10% is the volume percentage concentration. The cell lysates with different concentrations of proteinase K and NP-40 were tested. When the cell lysates were combined with proteinase K and NP-40 concentrations of 2ng/mL and 1%, 2ng/mL and 10%, 200μg/ The cell lysate can effectively lyse the cells at the time of mL and 10%, 200μg/mL and 1%, 1μg/mL and 5%, 100ng/mL and 1%, 100μg/mL and 10%. Proteinase K and NP-40 in the kit are conventional commercially available reagents, such as NP-40 with Sigma's article number 74385, and Merck Millipore's article number 539480-100mg Proteinase K. The two reagents do not contain DNase activity and have no other influence. Enzymatically active substances exist, such as SDS and so on.

In one case, the two active ingredients proteinase K and NP-40 contained in the cell lysate are stored in high concentrations respectively. When using the cell lysate, mix the two substances into a mixture of appropriate concentration; in another case Next, the cell lysate is stored in the form of a high-concentration mixture. When using the cell lysate, the high-concentration mixture is diluted with deionized water to a suitable concentration mixture. The appropriate concentration means that the concentration of proteinase K is 2ng～200μg/mL, and the volume percentage concentration of NP-40 is 1%～10%. A high-concentration mixture means that the concentration of one or both of the two substances is higher than the appropriate concentration.

In one case, the kit further includes a tube 2, which is a termination reaction solution, and the termination reaction solution is used to terminate the activity of proteinase K. The terminating reaction solution can be a protease inhibitor, such as PMSF (phenylmethylsulfonyl fluoride), antipain (anti-protease peptide) and so on. The concentration of the PMSF aqueous solution in tube 2 is not less than 5 mM, and the final concentration when used is 0.5 mM to 1 mM, that is, the concentration of the PMSF aqueous solution is 0.5 mM to 1 mM when the PMSF aqueous solution is added to the cell lysis solution to terminate the cell lysis reaction.

Use the kit containing tube 1 and tube 2 to lyse animal cells to obtain DNA in the cells. The kit has simple components and convenient operation for understanding cells, and can effectively lyse cells to obtain DNA. The obtained DNA is directly used for library construction without purification. This operation avoids the problem that the amount of nucleic acid is reduced after the nucleic acid of a small number of cells is purified. This kit is suitable for DNA extraction from single cell/micro cell samples or preimplantation genetic screening samples.

In one case, the kit further includes tube 3, which is a DNA fragmentation reagent. The types of fragmentation reagents can include transposase complexes, DNA interrupting enzymes, etc., and the DNA interrupting enzymes can be selected from Tiangen DNA Fragmentation kit NG305-02, Qiagen CM0162 fragmentation enzyme kit or NEB fragmentation enzyme; transposase complex can choose Tn transposition complex, Mu transposition complex, etc. In one case, the DNA fragmentation reagent of tube 3 is a Tn5 transposase complex. The Tn5 transposase complex is a complex formed by connecting Tn5 transposase and a transposable linker. The sequence of the transposable linker is determined by Tn5. The recognition sequence is composed of a Tn5 recognition sequence and a known sequence for PCR primer recognition.

When using the kit containing tubes 1, 2 and 3 to construct a sequencing library, the DNA obtained after cell lysis can be constructed without purification. The cell lysate and termination reaction solution have no effect on fragmentation reagents, especially for Tn5 transposase complexes. influences. Using this kit to construct a DNA library, from cell lysis to amplification, the reaction is carried out in a reaction tube, which can simplify the operation steps and reduce the cost on the one hand, and reduce the loss of DNA on the other hand.

In one case, the kit also includes amplification reagents and purification reagents.

Example 2

This example provides the application of the kit in Example 1 in library construction and sequencing on the Illumina company's high-throughput sequencing platform.

1. Cell Lysis

1) Separation of a small number of cell samples

A small amount of cells (1 to 200 cells) can be separated by techniques such as micromanipulators and mouth pipettes, and a small amount of 90-P50 (chHES90, 46, XY, source: CITIC Xiangya Reproductive and Genetic Hospital) A sample of normal cells (1 to 200 cells), the sample contains PBS (pH 7.2 to 7.4), and the total volume does not exceed 4 μL.

2) Cell lysis

Take 6 μL of the cell lysate prepared in Example 1 and add it to the cell sample to mix, and incubate at 55°C to 65°C for 30 to 60 minutes. The proteinase K and NP-40 concentrations of the cell lysate used in this example are 1 μg/ mL and 5%, 100ng/mL and 1%, 2ng/mL and 1%, the selected three combinations of cell lysates can effectively lyse cells. In order to ensure the uniformity of experimental conditions, Example 2 and Examples 3 The proteinase K and NP-40 concentrations of the cell lysate used were 100ng/mL and 1%, respectively.

3) Stop cell lysis

Add the termination reaction solution PMSF (phenylmethylsulfonyl fluoride) to the solution in step 2), the final concentration of PMSF is 0.5mM-1mM, and the reaction is at room temperature for 15min～35min to obtain the cleavage product.

2. Library construction

The library was constructed with the reagents of Nanjing Novozan kit (Cat NO.S111-C3, Vazyme), and the specific steps are as follows:

1) Fragmentation of genomic DNA

Use Nanjing Novozan kit (Cat NO.S111-C3, Vazyme) reagents to fragment the lysate in step 3) of cell lysis, and add 4μL of 5× fragmentation buffer to the lysate to fragment the complex. C3 1μL, add water to 20μL, mix well, place in a PCR machine, incubate at 37℃～55℃ for 10～30min, add 5μL of stop reaction buffer after the reaction, mix well, and react at room temperature for 5～7min.

2) PCR amplification

Add the PCR reaction system to the fragmented product in the library construction step 1). The PCR reagents are the reagents in the Nanjing Novozan kit (Cat NO.S111-C3, Vazyme), and the PCR reaction system contains 5×TAB amplification buffer Solution 10μL, TAE 1μL, primer 12μL, primer 22μL, make up to 50μL with water, mix well, and place in a preheated PCR machine. The PCR amplification process settings are shown in Table 1.

Table 1

3) Purification

The PCR product was purified using Nanjing Novozan's Purification Magnetic Bead Kit (Cat NO.N411-03, Vazyme). If the PCR product system is less than 50μL, add water to make up to 50μL, and then perform two rounds of purification. The first round of purification uses 35μL 0.7×VAHTS DNA Clean Beads (0.7×purified magnetic beads), and the second round uses 7.5μL 0.15×VAHTS DNA Clean Beads (0.15x purified magnetic beads). Purification was carried out in accordance with the instruction manual or the conventional purification steps of magnetic bead purification, and the final elution volume was 20 μL to obtain the DNA library.

3. Sequencing

1) Library detection

^{Use Qubit TM} ssDNA Assay Kit for concentration detection (Cat No. Q10212, Invitrogen Biotech.) and LabChip GX Touch (PerkinElmer) for the concentration detection of the obtained DNA library. ~300bp or so. Figure 1 shows the detection result of the library by the LabChip GX Touch bioanalyzer. In the figure, the abscissa is the fragment length, the unit is BP, the ordinate is the fluorescence intensity, and the concentration of the library is reflected.

2) IlluminaHiseqX10 sequencing

Sequencing results show that the total amount of unqiue data obtained (the total amount of uniquely compared data) is about 614.98M, and the coverage of the genome is as high as 99.54% when the sequencing depth of the DNA library constructed by this method is 30.75×. GC bias is 0.06. From the sequencing results, the coverage of the genome is as high as 99.54%. It can be judged that using the cell lysate in the example to lyse cells can effectively remove the proteins on the genome, thereby avoiding the influence of the proteins on the genome on fragmentation and the sequencing results. For the impact of coverage, the specific sequencing results are shown in Table 2.

Table 2

Sample name Total amount of unqiue data coverage GCbias 90-P50 614.98M 99.54% 0.06

Example 3

This example is the application of the kit in Example 1 in cell lysis, library construction and single molecule sequencing platforms. Refer to one or more of the solutions disclosed in CN201610209150.2, CN201710607295.2, CN201610958047.8, CN201710607306.7, CN201620278363.6, CN201520614342.2, CN201520614143.1, CN201520614143.1, CN201711105069.0 and/or CN201621419140.3 To build a single-molecule sequencing platform, for example, the operation flow can refer to https://doi.org/10.1371/journal.pone.0188181(Single molecule sequencing of the M13 virus genome without amplification).

1. Cell Lysis

1) Separation of a small number of cell samples

A small number of cell samples can be separated by techniques such as micromanipulators and mouth pipettes to obtain 5-10 CNV (copy number variation) cell lines chES497(46,XX,del(1p36.33-1p36.32)1.29M, dup(16p13.3-16.13.11)16.72M) (Source: CITIC Xiangya Reproductive and Genetic Specialist Hospital), and place the cells in 1-4μL PBS (pH7.2～7.4) solution to obtain cell samples.

2) Cell lysis

Take 6 μL of the cell lysate prepared in Example 1 and add it to the cell sample for mixing, and incubate at 55°C to 65°C for 30min to 60min. The cell lysate proteinase K and NP-40 concentrations used in this example are 100ng/mL and 1 %.

3) Stop cell lysis

Add the termination reaction solution (PMSF, phenylmethylsulfonyl fluoride) to the solution in step 2), the final concentration of PMSF is 0.5 mM to 1 mM, and the reaction is kept at room temperature for 15 to 35 minutes to obtain the cleavage product.

2. Library construction

GeneMind Biotech (full name: Shenzhen GeneMind Biotech Co., Ltd.) single-cell whole genome amplification kit (Cat NO.C0BD01-01AA, GeneMind Biotech) was used for library construction. The specific steps are as follows:

1) Fragmentation of genomic DNA

Use Zhenmai (full name: Shenzhen Zhenmai Biotechnology Co., Ltd.) single-cell whole genome amplification kit (Cat NO.C0BD01-01AA, GeneMind Biotech) to fragment the lysate in step 3) of cell lysis in this example, Add 4μL of 5× fragmentation buffer and 1μL of fragmentation complex to the lysate, make up to 20μL with water, mix well, place in a PCR machine, incubate at 37℃～55℃ for 10～30min, add 5×stop reaction buffer after the reaction is over 5μL of solution, mix well, and react at room temperature for 5-7 minutes.

2) PCR amplification

Add the PCR reaction system to the fragmented product in the library construction step 1) of this example. The PCR reaction system includes 5×PCR amplification buffer 10μL, 1U/μL and PCR amplification enzyme 1μL, primer 12μL, primer 20.2μL, supplemented with water To 50μL, mix well, and place in a pre-heated PCR machine. The PCR amplification process settings are shown in Table 3.

table 3

3) Purification

The PCR products were purified with Nanjing Novozan's Purification Magnetic Bead Kit (Cat NO.N411-03, Vazyme). If the PCR product system is less than 50μL, add water to make up to 50μL, and then perform two rounds of purification. The first round of purification uses 35μL 0.7×VAHTS DNA Clean Beads (0.7×purified magnetic beads), and the second round uses 7.5 μL 0.15×VAHTS. DNA Clean Beads (0.15x purified magnetic beads). Purification was carried out in accordance with the instructions in the manual or the conventional purification steps of magnetic bead purification. The final elution volume was 20 μL to obtain the DNA library.

3. Sequencing

1) Library detection

^{Use Qubit TM} ssDNA Assay Kit for concentration detection (Cat No. Q10212, Invitrogen Biotech.) and LabChip GX Touch (PerkinElmer) for the concentration detection of the obtained DNA library. ~300bp or so. Figure 2 shows the detection result of the library by the Bioanalyzer LabChip GX Touch. In the figure, the abscissa is the fragment length, the unit is BP, the ordinate is the fluorescence intensity, and the concentration of the library is reflected.

2) Sequencing by single molecule sequencer

Sequencing was performed using the single-molecule sequencing platform built by the inventor. The sequencing results showed that the total amount of unique data obtained by sequencing the DNA library constructed by this method (the total amount of uniquely compared data) was 5.5M, and the coverage (genome coverage) was 7.7% , GC bias is 0.05 and MAD (median absolute deviation) is 0.21. The specific results are shown in Table 4.

Table 4

DNA library name The total amount of unique data coverage GC bias MAD chES497-GC 5.5M 7.7% 0.05 0.21 chES497-NGS 4.9M 13.31% 0.15 0.34

Note: chES497-GC: DNA library constructed from cell samples in Example 3, using the single molecule sequencing platform built by the inventor for sequencing; chES497-NGS: DNA library constructed from cell samples in Example 3, using Illumina HiseqX10 sequencing platform Perform sequencing.

Comparative example

This comparative example is a comparative experiment of Example 3. This comparative example and Example 3 use the same cell sample, and use different cell lysis methods, library construction methods and sequencing platforms to perform library construction and sequencing on the cell samples.

This comparative example is a method commonly used by PGS at present. The current preimplantation screening method is to use 3 to 5 cells of the blastocyst trophoblast as the biopsy material, and then through various genetic screening methods, such as fluorescence in situ hybridization (Fish), polymerase chain reaction (PCR) Screening for chromosomal abnormalities. Based on the high-throughput sequencing platform PGS (preimplantation genetic screening), its data requires that the coverage of a single sample data is not less than 4%, and the data volume of a single sample is not less than 1M (according to the sequencing of each platform It depends on the length of reading). The detection performance needs to achieve the detection rate of chromosomal abnormalities corresponding to the positive reference product with an abnormal fragment size greater than 4M to reach 100%, and the detection rate of chromosome abnormalities corresponding to the positive reference product with the abnormal fragment size less than or equal to 4M to reach more than 30%.

The steps of cell lysis, library construction and sequencing in the comparative example are as follows:

1 cell lysis

1) Separation of a small number of cell samples

A small number of cell samples can be separated by techniques such as micromanipulators and mouth pipettes to obtain 5-10 CNV (copy number variation) cell lines chES497(46,XX,del(1p36.33-1p36.32)1.29M, dup(16p13.3-16.13.11)16.72M) (Source: CITIC Xiangya Reproductive and Genetic Specialist Hospital), and place the cells in 1-4μL PBS (pH7.2～7.4) solution to obtain cell samples, Subsequently, Discover-sc single-cell kit (Cat no. N601-02, Vazyme) was used for cell lysis and library construction.

2) Cell lysis

Take 3μL buffer D2 (prepared by adding 4μL 1MDTT to 36μL buffer D) and add it to the cell sample, flick the tube wall to mix and centrifuge briefly, and incubate at 65°C for 10 minutes;

Add 3μL of buffer N, mix well to terminate the lysis reaction, and place the reaction tube on ice to obtain the cell lysate.

2. Library construction

1) MDA (Multiple Displacement Amplification) technology amplification

Add 40 μL of the reaction mixture (as shown in Table 5) prepared in advance to the 10 μL cell lysate of step 2), mix and centrifuge briefly, and incubate at 30°C for 6 hours.

table 5

ingredient volume H2O 8μL Discover-sc Reaction Buffer 30μL Discover-sc DNA Polymerase (DNA amplification enzyme) 2μL total capacity 40μL

After the amplification, incubate at 65°C for 3 minutes to inactivate the DNA amplification enzyme, and the amplification product 1 is obtained.

2) Detection of amplification product 1

The amplified product 1 was diluted 10-fold, and the Qubit ^TM dsDNA BR Assay Kit concentration detection (CatNo. Q32853, Invitrogen Biotech.) and 1% agarose electrophoresis detection were performed respectively. Figure 4 is the result of agarose electrophoresis detection of amplified product 1. Marker 1 in the figure is a 50bp Marker; 2 is amplified product 1 with a total load of about 150 ng; 3 is amplified product 1 with a total load of about 180 ng; 4 is the negative control group (H ₂ O). The results show that the amplification products can be successfully obtained by using MDA technology.

3) DNA fragmentation

Use the kit TruePrep DNA Library Prep Kit V2 for Illumina (Cat NO.TD501, Vazyme) to fragment the amplified product 1. The specific operation is: take 50ng of the amplified product 1 and add 4μL 5×TTBL, 1.5μL TTE MixV50, replenish water To 20μL, mix well, place in a PCR machine, and incubate at 55°C for 10 min to obtain a fragmented product.

4) Purification

Enter 20μLVAHTS DNA Clean Beads (Cat NO.N411-03, Vazyme) into the fragmented product for purification. The purification steps are carried out in accordance with the instructions in the manual or the conventional purification steps of magnetic beads. The final elution volume is 24 μL to obtain purified product 1.

5) PCR amplification

Add the PCR reaction system to the purified product 1. The PCR reaction system contains 10μL 5×TAB, 1μL TAE, 5μL N5-XX (XX means label position number), 5μL N7-XX (XX means label position number), 5μL PPM, replenishment To 50μL, mix well, and place in a pre-heated PCR machine. The PCR amplification process settings are shown in Table 6.

Table 6

6) Purification

The PCR product was purified using Nanjing Novozan's Purification Magnetic Bead Kit (Cat NO.N411-03, Vazyme). If step 5) PCR product system is less than 50μL, make up to 50μL, and then perform two rounds of purification. The first round of purification uses 0.7×VAHTS DNA Clean Beads (0.7×purified magnetic beads), and the second round of purification uses 7.5μL 0.15× VAHTS DNA Clean Beads (0.15x purified magnetic beads). Purification steps were carried out in accordance with the instruction manual or the conventional purification steps of magnetic beads, and the final elution volume was 20 μL to obtain the DNA library.

3. Sequencing

1) Library detection

The obtained DNA library was tested with Qubit dsDNA BR Assay Kit (Cat No. Q32853, Invitrogen Biotech.) for concentration detection and LabChip GX Touch (PerkinElmer) for microfluidic capillary electrophoresis detection. The main band size of the detected DNA library nucleic acid fragments was 250 ~450bp or so. Figure 3 shows the detection result of the library by the Bioanalyzer LabChip GX Touch. The abscissa is the fragment length, the unit is BP, the ordinate is the fluorescence intensity, and the library concentration is detected.

2) Illumina HiseqX10 sequencing

The sequencing results showed that the total amount of unique data (the total amount of uniquely aligned data) obtained by sequencing the DNA library constructed by this method was 4.9M, and the coverage (genome coverage) was 13.31%. The data quality and analysis results obtained by this method , The specific results are shown in Table 4.

The sequencing results were analyzed using hg19 (normal human chromosome, the complete sequence can be downloaded from ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/chromosomes/ website) as a reference genome for analysis. The chES497 samples were detected and analyzed according to different cell lysis methods, library construction methods, and sequencing methods. The two sequencing platforms used were Illumina HiseqX10 and the single-molecule sequencing platform built by the inventor. Figures 5 and 6 show the sequencing analysis results of chES497 samples on the single-molecule sequencing platform built by Illumina HiseqX10 and the inventor. The results show that the detection and analysis results of the two sequencing platforms are consistent. Figure 5 shows that both platforms can detect about 1M CNV (copy number variation), the chES497-NGS marked on the upper right of the figure is the sequencing analysis result of the Illumina HiseqX10 library, and the chES497-GC marked on the lower right is the sequencing result of the single molecule sequencing platform built by the inventor. The abscissa is the position of the chromosome, the ordinate is log2 (normalize bin depth), that is, the ordinate is log2 (standardized window depth), and the arrow mark is the position of the chromosome CNV in the chromosome; Figure 6 shows that both platforms can detect larger than 10M CNV (copy number variation), the chES497-NGS marked on the upper right of the figure is the sequencing analysis result of the Illumina HiseqX10 library, and the chES497-GC marked on the lower right is the sequencing result of the single molecule sequencing platform built by the inventor. The abscissa is the position of the chromosome, the ordinate is log2 (normalize bin depth), that is, the ordinate is log2 (normalized window depth), and the arrow mark is the position of the chromosome CNV in the chromosome.

According to the above sequencing results, using the kit in Example 1 to perform cell lysis and using the single molecule sequencing platform built by the inventor to lyse, build and sequence a small number of cells such as blastocyst stage cells, the sequencing results can be used to analyze the CNV of the cells. , The size of CNV detected is greater than 10M, and some are about 1M. The detection result is consistent with the result of HiseqX10 detection and meets the PGS detection standard. Therefore, using the kit in Example 1 of the present invention for cell lysis and the single molecule sequencing platform built by the inventor for sequencing can be used for PGS (preimplantation genetic screening). At the same time, when two different platforms are used for PGS, the traditional PGS library building process takes about 16 hours to build the library, while the use of the kit in Example 1 of the present invention for cell lysis and library building takes about 3.5 hours, so Using the method of lysing library construction in the embodiment of the invention can greatly shorten the detection time. It can be seen from Table 4 that the GC bias (GC preference) of the sequencing results of the two methods is different. The GC bias of the single-molecule sequencing platform built by the inventor is obvious when the kit in Example 1 of the present invention is used for cell lysis to build a database. Lower than the sequencing result of HiseqX10 library construction; meanwhile, the MAD (median absolute deviation) of the sequencing results of the two methods is different. The MAD of the cell lysis library construction sequencing using the kit in Example 1 of the present invention is significantly less than the sequencing result of HiseqX10 library construction , The smaller the value, the easier it is to analyze the missing chromosomes from the detection results. For example, in Figure 5, the position indicated by the arrow is the position of CNV. Compared with the sequencing result (chES497-NGS) in the example, the position of CNV is still around. There are other sites that affect the judgment of CNV. On the contrary, the position of CNV in the sequencing result (chES497-GC) in Example 3 is obvious and easy to judge.

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structures, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above-mentioned terms are not necessarily directed to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without contradicting each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. A person of ordinary skill in the art can comment on the above-mentioned embodiments within the scope of the present invention. The embodiment undergoes changes, modifications, substitutions, and modifications.

Claims (30)

A cell lysate, characterized in that the effective components of the cell lysate contain proteinase K and NP-40. The cell lysis solution according to claim 1, wherein the cell lysis solution is composed of proteinase K, NP-40 and H ₂ O. The cell lysate according to claim 1 or the cell lysate, wherein the concentration of the proteinase K is 0.002-200 μg/mL. The cell lysate according to claim 3, wherein the volume percentage concentration of the NP-40 is 1%-10%. The cell lysate according to claim 4, wherein the concentration of the proteinase K is 0.002-1 μg/mL. The cell lysate according to claim 5, wherein the concentration of the proteinase K is 0.002-0.1 μg/mL. A method for cell lysis, which is characterized in that it comprises using any cell lysis solution of claims 1 to 6 to lyse cells. The lysis method according to claim 7, wherein the cell is an animal cell. The lysis method according to claim 8, wherein the number of the cells is 1-200; Optionally, the number of the cells is 1-10. The cracking method according to any one of claims 7-9, wherein the cracking is performed at 55°C to 65°C, and/or the cracking time is 30min to 60min. The lysis method according to claim 10, wherein the lysis method further comprises adding a termination reaction solution to terminate the lysis. The lysis method according to claim 11, wherein the termination reaction solution is PMSF aqueous solution. The lysis method according to claim 12, wherein the final concentration of the termination reaction solution is 0.5 mM to 1 mM. A method for preparing nucleic acid, which is characterized in that it comprises using the lysis method of any one of claims 7-13 to lyse cells to obtain nucleic acid. The method for preparing nucleic acid according to claim 14, wherein the nucleic acid is DNA. A method for preparing a nucleic acid library, which is characterized in that it comprises: Using the nucleic acid preparation method of claim 14 to obtain nucleic acid; and Fragment the nucleic acid; Optionally, it also includes amplifying the fragmented nucleic acid to obtain the nucleic acid library. The method for preparing a nucleic acid library according to claim 16, wherein there is no purification and/or separation operation between the steps of the method for preparing a nucleic acid library. The method for preparing a nucleic acid library according to claim 16 or 17, wherein the fragmentation is performed using a fragmentation reagent. The method for preparing a nucleic acid library according to claim 18, wherein the fragmentation reagent comprises a transposase complex. The method for preparing a nucleic acid library according to claim 19, wherein the transposase complex is a Tn5 transposase complex. A sequencing method, characterized in that it comprises sequencing a nucleic acid library, which is prepared by the nucleic acid library preparation method according to any one of claims 16-20. A kit, characterized by comprising the cell lysate according to any one of claims 1-6. The kit according to claim 22, wherein the kit further comprises a termination reaction solution. The kit according to claim 23, wherein the termination reaction solution comprises an aqueous PMSF solution. The kit according to claim 24, wherein the concentration of the PMSF aqueous solution is not less than 5 mM. The kit according to any one of claims 22-25, wherein the kit further comprises a nucleic acid fragmentation reagent. The kit according to claim 26, wherein the fragmentation reagent comprises a transposase complex. The kit according to claim 27, wherein the transposase complex is a Tn5 transposase complex. Use of the kit of any one of claims 22-28 in a method or device involving cell lysis. The application according to claim 29, wherein the application is preimplantation genetic screening of embryos.

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