Loading method, analysis method and application of nucleic acid template before sequencing

Abstract

The invention provides a method for loading a nucleic acid template before sequencing, a method for carrying out visual imaging analysis on the loaded nucleic acid template before sequencing and related applications of the nucleic acid template and the method. Specifically, the method for loading the nucleic acid template comprises the following steps: 1) loading the nucleic acid template to the surface of a solid support; 2) combining a fluorescent dye with the nucleic acid template; and (3) removing the uncombined fluorescent dye by adopting a cleaning reagent. Furthermore, the method also comprises the following steps: removing the fluorescent dye combined to the nucleic acid template by using a rinsing reagent; and carrying out sequencing pretreatment on the nucleic acid template by using a plurality of reagents after cleaning and before rinsing. Meanwhile, the invention also provides a method for analyzing the loaded nucleic acid template before sequencing based on the loading method, namely photographing and observing after the steps. By means of the analysis method, multiple steps of the sequencing pretreatment stage can be effectively monitored, quality inspection is conducted on all the stages, faults are confirmed in time, and then the one-time off-machine success rate is increased.

Description

Technical Field

[0001] This invention belongs to the field of gene detection, and particularly relates to a method for loading nucleic acid templates before sequencing, a method for analyzing the loaded nucleic acid templates before sequencing, and related applications. Background Technology

[0002] High-throughput sequencing generates light signals by nucleotides complementary to the target nucleic acid template (e.g., deoxyribonucleotides and ribonucleotides), and then identifies the type of complementary nucleotides by detecting the light signals, further determining the sequence of the target nucleic acid template. Taking the target nucleic acid template as a deoxyribonucleotide chain (DNA template chain) as an example, the identification (e.g., taking a picture) process begins from the first complementary deoxyribonucleotide pairing with the DNA template chain. Before sequencing, multiple sample pretreatment steps are required, and the effectiveness of pretreatment is closely related to the sequencing quality. Typically, after preparing the DNA template chain to be sequenced, two important steps are required: (1) DNA template chain loading (i.e., placing the target DNA template onto the surface of a solid support such as a chip) and (2) DNA template chain post-loading (also known as sequencing pretreatment) before sequencing can begin. However, once sequencing is completed, if the quality of the sequencing data is found to be poor, due to the numerous steps and reagents in current sequencing methods, it is difficult to quickly and accurately locate the problematic step or reproduce the problem during pretreatment, resulting in researchers being unable to promptly identify the problem and adjust the research plan.

[0003] The technique of loading a target nucleic acid template (hereinafter, we will use a deoxyribonucleic acid sequence (DNA template) as an example) onto a sequencing slide involves mixing the target nucleic acid template and loading buffer, then flowing the mixture through a sequencing slide (solid support) and incubating it at a controlled temperature. The slide contains hundreds of millions of sites arranged in a regular or non-array pattern, with the surface of each site modified by chemical functional groups. After loading the DNA template, each DNA template should be anchored at a single site, and there should be no DNA template adhering between sites or on the chip's positioning lines. However, in current sequencing methods, after the DNA template is loaded onto the slide used for gene sequencing, the tester is unaware of the loading effect because existing sequencing equipment lacks a method for real-time qualitative and quantitative monitoring of the DNA template loading effect for quality control. Therefore, current sequencing methods may encounter the following problems: (1) Due to poor surface modification of the slide (e.g., a chip), (i) empty sites on the slide are not occupied by DNA templates, resulting in less DNA template loading on the slide; (ii) some sites on the slide are occupied by multiple DNA templates simultaneously, resulting in multiple signals at one site during sequencing; (2) the quality of the slide itself cannot be known in a timely manner, for example, DNA templates may be non-specifically adsorbed at sites where they should not be adsorbed. The problems that occur after loading will affect signal recognition during testing, and failure to detect them in time will greatly affect the success rate and quality of sequencing.

[0004] After loading, the target DNA template requires a series of biochemical reagent treatments to ensure it is stably and regularly anchored on the slide. Post-loading reagent processing uses chemical reagents to tighten, stabilize, and protect the target DNA template already loaded at the sites, ensuring that hundreds of millions of target DNA templates remain anchored in their regular shape and do not shift when subsequent sequencing reagents flow through the slide. However, problems often arise during the post-loading reagent processing. For example, air bubbles may prevent the reagents from fully contacting the target DNA template; or the reagent flow rate may be too fast, causing the target DNA template to become loose or shifted, failing to anchor firmly at the sites on the slide. Failure to detect these problems in a timely manner or to clearly define the scope of troubleshooting will reduce sequencing quality.

[0005] Therefore, this invention provides an effective and visual imaging analysis method in the aforementioned gene sequencing pretreatment stage to confirm whether the nucleic acid template was loaded successfully and efficiently, as well as the actual situation of reagent treatment during each sequencing pretreatment step. On the one hand, based on the overall image brightness, the sparseness of bright spots, and the regularity of bright spot arrangement, users can quickly determine the success or failure of DNA template loading before sequencing begins. On the other hand, the grayscale values ​​provided by image processing software can be used to estimate the effectiveness of DNA template loading. This invention will play an important and positive role in improving the success rate of sequencing and troubleshooting sequencing failure factors. Summary of the Invention

[0006] Based on the above, the present invention provides a method for loading nucleic acid templates before sequencing and a method for analyzing the loaded nucleic acid templates before sequencing based on the method. This analysis method can effectively monitor multiple steps in the sequencing preprocessing stage, perform quality checks on each stage, promptly identify faults, quickly assist in solving problems, and thus improve the success rate of sequencing on the first attempt.

[0007] In a first aspect, the present invention provides a method for loading a nucleic acid template before sequencing, the method comprising the following steps:

[0008] 1) Load the nucleic acid template onto the surface of a solid support;

[0009] 2) Use a fluorescent dye to bind to the above nucleic acid template;

[0010] 3) Use cleaning reagents to remove unbound fluorescent dyes.

[0011] Furthermore, the above method also includes step 4), which includes removing the fluorescent dye bound to the nucleic acid template with a rinsing reagent.

[0012] Furthermore, the above method also includes pre-sequencing treatment of the nucleic acid template using several reagents between steps 3) and 4).

[0013] In one specific embodiment of the present invention, the fluorescent dye is a non-specific fluorescent dye. The fluorescent dye includes, but is not limited to, Qubit fluorescent detection reagent, SYBR Gold dye, SYBR Green II nucleic acid dye, ethidium bromide, GelRed dye, SolarRed dye, YeaRed dye, NA-Red dye, NA-Green dye, acridine orange, Diamond nucleic acid dye, Hoechst 33258 dye, SYBR Green I dye, YeaGreen dye, GeneRed nucleic acid dye, or CPRNA dye, etc.

[0014] Preferably, the concentration of the fluorescent dye used is between 59X and 82X.

[0015] In one specific embodiment of the present invention, the fluorescent dye described above is a Qubit fluorescent detection reagent, and a concentration of 59X to 82X is recommended.

[0016] In one specific embodiment of the present invention, the above-mentioned nucleic acid template is a deoxyribonucleotide sequence, preferably, the above-mentioned nucleic acid template is a single-stranded deoxyribonucleotide sequence, and preferably, the above-mentioned nucleic acid template is a DNA nanosphere.

[0017] In one specific embodiment of the present invention, the cleaning reagent is elution reagent 2 and / or Qubit dilution buffer from the MGI sequencing reagent tank.

[0018] In one specific embodiment of the present invention, the rinsing agent is an alcoholic substance or an organic solvent with polarity similar to alcohols, with a concentration of 50%-100%. Preferably, the rinsing agent is an alcoholic substance with a concentration of 75%, which includes, but is not limited to, methanol, ethanol, propylene glycol, propanol, or n-butanol.

[0019] In one specific embodiment of the present invention, the above-mentioned organic solvents include, but are not limited to, formamide, acetonitrile, acetone, dioxane, tetrahydrofuran, methyl ethyl ketone, ethyl acetate, diethyl ether, isopropyl ether, dichloromethane, chloroform, bromoethane, or toluene.

[0020] In one specific embodiment of this application, in the steps of binding the fluorescent dye to the nucleic acid template and cleaning the fluorescent dye with a cleaning reagent, the amount of fluorescent dye and cleaning reagent loaded onto the surface of the solid support is proportional to the number of flow channels on the surface of the solid support.

[0021] In one specific embodiment of this application, in the steps of binding the fluorescent dye to the nucleic acid template and cleaning the fluorescent dye with a cleaning reagent, the flow rate of the fluorescent dye and the cleaning reagent across the surface of the solid support is 80-120 μL / min; preferably, the flow rate is 100 μL / min.

[0022] In one specific embodiment of this application, the incubation temperature for binding the fluorescent dye to the nucleic acid template is 15-35°C, and the incubation time is 10-30 minutes. Preferably, the incubation temperature is 25°C, and the incubation time is 15 minutes.

[0023] In one specific embodiment of this application, in the step of removing fluorescent dye with rinsing reagent, the amount of rinsing reagent loaded onto the surface of the solid support is proportional to the number of flow channels on the surface of the solid support.

[0024] In one specific embodiment of this application, in the step of removing fluorescent dye with rinsing reagent, the incubation temperature for loading the rinsing reagent onto the surface of the solid support is 15-35°C, and the incubation time is 10-30 minutes. Preferably, the incubation temperature is 25°C, and the incubation time is 15 minutes.

[0025] In one specific embodiment of this application, in the step of removing fluorescent dye with rinsing reagent, the flow rate of the rinsing reagent across the surface of the solid support is 80-120 μL / min. Preferably, the flow rate is 100 μL / min.

[0026] Secondly, the present invention provides a method for analyzing nucleic acid templates loaded before sequencing. The method includes the following steps: (1) after the nucleic acid template to be tested is loaded onto the surface of a solid support, a fluorescent dye is used to bind the nucleic acid template loaded onto the surface of the solid support; (2) the fluorescent dye that has not bound to the nucleic acid template is cleaned with a cleaning reagent, and then photographed and analyzed.

[0027] Preferably, step (1) above further includes using a fluorescent dye to bind to the nucleic acid template and then taking a picture for analysis.

[0028] Furthermore, the above method also includes step (3): after step (2), the nucleic acid template is subjected to multiple reagent treatments before sequencing and then photographed and analyzed.

[0029] Furthermore, the above method also includes step (4): removing the fluorescent dye bound to the nucleic acid template with a rinsing reagent before sequencing. Preferably, step (4) further includes taking a picture for analysis after removing the fluorescent dye bound to the nucleic acid template with a rinsing reagent.

[0030] In one specific embodiment of the present invention, the fluorescent dye is a non-specific fluorescent dye. The fluorescent dye includes at least one of the following: Qubit fluorescent detection reagent, SYBR Gold dye, SYBR Green II nucleic acid dye, ethidium bromide, GelRed dye, SolarRed dye, YeaRed dye, NA-Red dye, NA-Green dye, acridine orange, Diamond nucleic acid dye, Hoechst 33258 dye, SYBR Green I dye, YeaGreen dye, GeneRed nucleic acid dye, or CP RNA dye.

[0031] In one specific embodiment of the present invention, the amount of fluorescent dye used is based on the average grayscale value of the image taken after cleaning with the cleaning reagent, and the average grayscale value ranges from 7713 to 12691. Further, if Qubit dilution buffer is used, the average grayscale value is from 7713 to 10606; if elution reagent 2 is used, the average grayscale value is from 9229 to 12691.

[0032] In one specific embodiment of the present invention, the amount of fluorescent dye used is based on the average gray value of the image taken after the fluorescent dye has been combined with the nucleic acid template without being cleaned with a cleaning reagent, and the average gray value is between 5577 and 7700.

[0033] In one specific embodiment of the present invention, the amount of the rinsing reagent used is based on the average gray value of the image taken after rinsing with the rinsing reagent, wherein the average gray value is 2369 to 3259.

[0034] In one specific embodiment of this application, the above-mentioned nucleic acid template is a deoxyribonucleotide sequence, preferably a single-stranded deoxyribonucleotide sequence, and preferably a DNA nanosphere.

[0035] In one specific embodiment of the present invention, the cleaning reagent is elution reagent 2 and / or Qubit dilution buffer from the MGI sequencing reagent tank.

[0036] In one specific embodiment of this application, in the steps of binding the fluorescent dye to the nucleic acid template and cleaning the fluorescent dye with a cleaning reagent, the amount of fluorescent dye and cleaning reagent loaded onto the surface of the solid support is proportional to the number of flow channels on the surface of the solid support.

[0037] In one specific embodiment of this application, in the steps of binding the fluorescent dye to the nucleic acid template and cleaning the fluorescent dye with a cleaning reagent, the flow rate of the fluorescent dye and the cleaning reagent across the surface of the solid support is 80-120 μL / min; preferably, the flow rate is 100 μL / min.

[0038] In one specific embodiment of this application, the incubation temperature for binding the fluorescent dye to the nucleic acid template is 15-35°C, and the incubation time is 10-30 minutes. Preferably, the incubation temperature is 25°C, and the incubation time is 15 minutes.

[0039] In one specific embodiment of the present invention, the rinsing agent is an alcoholic substance or an organic solvent with polarity similar to alcohols, with a concentration of 50%-100%. Preferably, the rinsing agent is an alcoholic substance with a concentration of 75%, which includes, but is not limited to, methanol, ethanol, propylene glycol, propanol, or n-butanol.

[0040] In one specific embodiment of the present invention, the above-mentioned organic solvents include, but are not limited to, formamide, acetonitrile, acetone, dioxane, tetrahydrofuran, methyl ethyl ketone, ethyl acetate, diethyl ether, isopropyl ether, dichloromethane, chloroform, bromoethane, or toluene.

[0041] In one specific embodiment of this application, in the step of removing fluorescent dye with rinsing reagent, the amount of rinsing reagent loaded onto the surface of the solid support is proportional to the number of flow channels on the surface of the solid support.

[0042] In one specific embodiment of this application, in the step of removing fluorescent dye with rinsing reagent, the incubation temperature for loading the rinsing reagent onto the surface of the solid support is 15-35°C, and the incubation time is 10-30 minutes. Preferably, the incubation temperature is 25°C, and the incubation time is 15 minutes.

[0043] In one specific embodiment of this application, in the step of removing fluorescent dye with rinsing reagent, the flow rate of the rinsing reagent across the surface of the solid support is 80-120 μL / min. Preferably, the flow rate is 100 μL / min.

[0044] Thirdly, the present invention provides a kit comprising the reagents used in the methods mentioned in the first aspect and the methods mentioned in the second aspect, specifically including fluorescent dyes and cleaning reagents.

[0045] Furthermore, it also includes rinsing agents.

[0046] In one specific embodiment of the present invention, the fluorescent dye in the kit includes at least one of the following: Qubit fluorescent detection reagent, SYBR Gold dye, SYBR Green II nucleic acid dye, ethidium bromide, GelRed dye, SolarRed dye, YeaRed dye, NA-Red dye, NA-Green dye, acridine orange, Diamond nucleic acid dye, Hoechst 33258 dye, SYBR Green I dye, YeaGreen dye, GeneRed nucleic acid dye, or CP RNA dye.

[0047] In one specific embodiment of the present invention, the cleaning reagent includes elution reagent 2 from the MGI sequencing reagent tank and / or Qubit dilution buffer.

[0048] In one specific embodiment of the present invention, the rinsing agent includes an alcoholic substance or an organic solvent with polarity similar to that of alcohols at a concentration of 50-100%.

[0049] In one specific embodiment of the invention, the rinsing agent comprises an alcohol at a concentration of 75%. Preferably, the alcohol comprises at least one selected from methanol, ethanol, propylene glycol, propanol, or n-butanol.

[0050] In one specific embodiment of the present invention, the rinsing agent uses an organic solvent including at least one of formamide, acetonitrile, acetone, dioxane, tetrahydrofuran, methyl ethyl ketone, ethyl acetate, diethyl ether, isopropyl ether, dichloromethane, chloroform, bromoethane, or toluene.

[0051] Fourthly, the present invention provides a sequencing method, which uses the method described in the first aspect above to load nucleic acid templates before sequencing or uses the method described in the second aspect above to analyze nucleic acid templates loaded before gene sequencing, especially nucleic acid templates at each stage of preprocessing.

[0052] The beneficial effects of the present invention are as follows: The present invention provides a method for loading nucleic acid templates before sequencing and a method based on the method for analyzing nucleic acid templates loaded before sequencing. The above method can be used as a visual quality inspection scheme in the sequencing pretreatment stage and deployed in the sequencing process to improve the sequencing success rate. (1) The analysis method is a quality control method. It can monitor the quality of nucleic acid templates loaded onto solid supports (e.g., chips) and the morphology of nucleic acid templates after various reagent treatments before sequencing, so as to promptly investigate and accurately locate occasional problems such as chip quality problems, fluid inhomogeneity, reagent impurities, and abnormal laser intensity. Common quality problems that can be detected in time include, but are not limited to: low loading rate of nucleic acid template samples on chip surface; non-specific adsorption of nucleic acid templates by the chip; laser not turned on; low laser power; impurity precipitation in reagents; abnormal fluid reagents causing cavities or bubbles when passing through the chip. (2) The analysis method can be directly added to the existing sequencing process, which can ensure the quality monitoring of each step in the sequencing pretreatment stage, detect problems from images, and handle them in time, thereby improving the first-time sequencing success rate. (3) The analytical method is simple to use and easy to operate. The reagents are readily available and easy to prepare. It can be widely used in gene sequencers with single-stranded or double-stranded nucleic acid samples without the need for additional instruments and components. Attached Figure Description

[0053] Figure 1 The images are photographs taken before cleaning with a cleaning reagent after DNA nanoballs (DNB) were bound (stained) with fluorescent dye (software grayscale display: mean=6972.065, Min=6400, Max=11392);

[0054] Figure 2 The images show the background after DNB binding (staining) with fluorescent dyes and subsequent cleaning with different cleaning reagents. Figure A shows the background cleaned with Qubit dilution buffer (software grayscale display: mean = 9641.006, Min = 7552, Max = 15680), and Figure B shows the background cleaned with elution reagent 2 in well 18 of the MGI sequencing reagent slot (software grayscale display: mean = 11536.675, Min = 9216, Max = 17152).

[0055] Figure 3The images are photographs taken after staining with qubit fluorescent dye at a concentration reduced to 9.8X in the dye-compatible buffer (software grayscale display: mean = 3117.791, Min = 2880, Max = 3328).

[0056] Figure 4 For example, in step 8 of test 1, after DNB loading, the local image and fluorescence intensity of the elution reagent 1 in well 17 of the finished sequencing reagent slot after passing through the chip are shown (software grayscale display: mean = 3459.162, Min = 3136, Max = 4928).

[0057] Figure 5 Example of a partial image of the chip with only the camera background and fluorescence intensity after dye rinsing in step 9 of Test 1 (software grayscale display: mean = 2962.012, Min = 2752, Max = 3392);

[0058] Figure 6 As an example of a typical anomaly in the embodiment: an example image with bubbles (software grayscale display: mean = 9257.133, Min = 7616, Max = 12160);

[0059] Figure 7 Here is a typical abnormal situation as an example in the embodiment: an image example of the DNB being deflected by the rapid scouring of fluid after loading (software grayscale display: mean=4081.632, Min=3200, Max=63488);

[0060] Figure 8 The third typical anomaly in the example is an image example of impurity precipitation in biochemical reagents (software grayscale display: mean=8447.718, Min=5695, Max=17152);

[0061] Figure 9 The example shown is a normal loading result: when the chip is in the presence of reagents, a magnified local image shows a clear and regularly arranged DNB on the chip positioning line. Detailed Implementation

[0062] As mentioned in the background section, in existing sequencing technologies, there is no method to promptly and qualitatively reflect the DNA template loading efficiency and the morphology of the loaded DNA template after loading the nucleic acid template (for ease of explanation, a DNA template will be used as an example below). Sometimes, substandard chip quality or batch-to-batch quality variations can significantly reduce sequencing success and quality. Due to the numerous types and steps involved in the biochemical reactions and reagents (e.g., reagent processing after DNA template loading typically includes six steps), it is difficult to quickly and accurately trace the source of the problem—whether it is a chip quality issue, instrument malfunction, or a problem with the chemical reagents, enzyme reagents, or the DNA sample—after sequencing is completed. Furthermore, there is currently no method to monitor the macroscopic morphology and regular arrangement of the deoxyribonucleic acid sequence on the slide in real time during and after reagent processing for the aforementioned six steps. The problems that these six steps may cause include, but are not limited to: 1) Bubbles generated when switching reagents or the reagents themselves contain bubbles, which affect the full contact between the reagents and the DNA template, resulting in the DNA template not being firmly loaded onto the chip; 2) Due to the blockage of the reagent tubing or the excessive flow rate, the regular arrangement of the DNA template is disrupted, and adjacent molecules are too close, which interferes with the recognition of fluorescence signals during sequencing; 3) Impurities in the chemical processing reagents after loading may block part of the deoxyribonucleic acid, affecting the imaging during the sequencing stage, causing blockage of the fluid tubing, affecting the reagent pump solution, and hindering the sufficiency of the biochemical reaction.

[0063] It should be understood that, apart from the DNA templates in the following examples, the present invention is also applicable to RNA templates, and single-stranded or double-stranded nucleic acid templates do not constitute a limitation of the present invention.

[0064] Based on repeated research and experimentation with the aforementioned problems, this invention proposes a solution: a non-specific fluorescent dye is intercalated with the DNA template to be tested. This fluorescent dye can be excited by a laser of a specific excitation wavelength in the sequencer. Imaging (using the sequencer's built-in imaging equipment) allows visualization of the DNA template's morphology on a solid support, such as a chip surface, showing either a regular or non-array arrangement within the reagents. This provides a visual representation of the DNA template's macroscopic morphology. Based on this DNA template loading method and the aforementioned visualization analysis, the loading of DNA template samples onto the chip and each step of reagent processing before sequencing can be effectively monitored. By deploying this analytical method into the sequencing workflow, quality control can be achieved at each step of the pre-sequencing stage, further improving the first-run success rate.

[0065] It should be noted that, to avoid redundancy, the following description will only use the method of analyzing DNA templates loaded before sequencing as an example. It should be understood that the reagents and methods of use used in each step of the DNA template loading method and the DNA template analysis method in this invention are consistent. The reagents, specific dosages, and methods used in the method for analyzing DNA templates loaded before sequencing are also applicable to the method for loading nucleic acid templates before sequencing.

[0066] 1. The main steps of the method (analysis method) for analyzing DNA templates loaded before sequencing in this invention include:

[0067] (1) After the DNA template is loaded onto a solid support (e.g., a chip; for ease of explanation, a chip will be used as an example in the following description), a fluorescent dye capable of staining DNA is used to bind to the DNA template. Preferably, photographic analysis can be performed after binding to detect the phenomenon of insufficient DNA template loading earlier.

[0068] (2) Use a cleaning reagent to clean off excess fluorescent dye that has not bound to the DNA template, and then take pictures for analysis.

[0069] After cleaning, the DNA template bound to the fluorescent dye can be photographed by emitting a laser using a sequencer. The image can reflect the immediate morphology of the DNA template after it is loaded onto the chip, and the DNA template loading status at the current step can be analyzed by the photographic results to determine whether it meets the requirements.

[0070] In one specific embodiment of the present invention, the fluorescence intensity of the captured image can be quantified using ImageJ software. The fluorescence intensity is positively correlated with the size or length of the DNA template and the density loaded onto the chip surface. This is an imaging method that does not rely on the complementary pairing of fluorescent deoxyribonucleotides with the DNA template.

[0071] Therefore, one inventive aspect of this invention lies in: using embedded fluorescent dye labeling for precise DNA template localization, obtaining the location of the DNA template through imaging, establishing sequencing-as-synthesis sites, and observing the presence of fluorescent signals at the template sites to determine the saturation of the DNA template loaded onto the chip. This step provides intuitive feedback and timely evaluation, solving the problem of unknown DNA loading quality onto the chip. The image can reflect whether the DNA sequence loading is uniform, clearly showing whether there is non-specific adsorption of deoxyribonucleic acid on the chip surface, and quantitatively determining whether the number of loaded DNA templates is normal, too high, or too low through fluorescence intensity and image grayscale values.

[0072] For example, by scoring the image results, if the score is unsatisfactory, a prompt will be made to replace the sequencing slide before sequencing; or the target DNA template will be unloaded and reloaded to improve the sequencing success rate and ensure sequencing quality.

[0073] It should be noted that after the fluorescent dye binds to the DNA template loaded onto the chip, the image needs to be washed with a cleaning reagent. Taking images directly without washing after staining will result in blurry images. Figure 1 As shown, the chip positioning lines are faintly visible, but they are dark and the edges are unclear. The column of bright spots after the DNA template is loaded in the middle of the positioning lines is also not visible, making it impossible to see the regular arrangement of the DNA template (e.g., DNB dots) on the chip. However, after dye binding and washing, a clear image is obtained. The grayscale values ​​of each figure are indicated in the accompanying drawings of this invention. These grayscale values ​​reflect the brightness after the corresponding step, such as the brightness of the fluorescence after dye binding and the brightness after washing.

[0074] (3) After the DNA template is loaded onto the chip, images are taken during the multiple reagent processing steps before sequencing to reflect the real-time impact of each reagent flowing through the chip on the DNA template. This step can solve the problem of quality monitoring of reagent processing steps after DNA template loading is missing.

[0075] The present invention provides a method for visually monitoring the DNA template morphology during and after reagent processing, enabling step-by-step confirmation of the proper completion of reagent pretreatment before sequencing. 1) It clearly and intuitively shows whether air bubbles are introduced when reagents flow through the chip, thus affecting the sufficient contact between the reagents and the DNA template. 2) It allows for timely and intuitive observation of the overall morphology of the DNA template after reagent flow through the chip, ensuring its regularity. Extensive experimental data has shown that excessively fast reagent flow rates can lead to irregular arrangement of the DNA template to be sequenced, reducing sequencing quality. This invention takes photos during the six steps of reagent flow through the slide and during reagent switching, enabling real-time detection and localization of fluid problems. 3) Impurities in the chemically treated reagents after loading can affect sequencing photography or clog the tubing. Serious fluid problems and reagent impurities are clearly displayed in the captured images. Furthermore, based on the method of this invention, software can help users determine whether the image quality is acceptable. If acceptable, the next step of processing can be continued or sequencing can proceed directly; if unacceptable, the user is prompted to replace the chip and reload the DNA template and / or process the reagents.

[0076] (4) Remove the fluorescent dye that has bound to the DNA template with a rinsing agent and begin sequencing.

[0077] It is important to note that in routine sequencing procedures, it is generally recommended to remove fluorescent dyes using rinsing reagents before sequencing. However, rinsing may not be necessary in some special cases. For example, in case 1), troubleshooting and locating problems such as the quality of the chip itself or chip surface modification; whether the chip is overloaded with DNA template or underloaded; or whether the DNA template is non-specifically adsorbed outside the chip sites. If it is determined after investigation that sequencing will not be performed, then rinsing is not required. In case 2), if there is a significant difference between the fluorescence emission wavelength of the fluorescent group used in sequencing and the fluorescence emission wavelength of the dyes used, the dye signal can be filtered out using filters or the dye signal value can be subtracted using an algorithm during sequencing.

[0078] 2. The fluorescent dye reagents used in this invention are described as follows:

[0079] (1) The fluorescent dye used in this invention is used to non-specifically bind and stain DNA. In one specific embodiment of this invention, the fluorescent dye used is Qubit fluorescent detection reagent (brand: Thermo Fisher Scientific, or Yisheng Bio).

[0080] It should be understood that the fluorescent dyes suitable for this invention are not limited to Qubit reagents, but can also be fluorescent reagents that can bind to single-stranded or double-stranded DNA, such as SYBR Gold dye, SYBR Green II nucleic acid dye, ethidium bromide, GelRed dye, SolarRed dye, YeaRed dye, Beyotime Biotechnology NA-Red dye, NA-Green dye, Promega Diamond nucleic acid dye, acridine orange, and Hoechst series dyes such as Hoechst 33258, or SYBR Green I dye, YeaGreen dye, GeneRed nucleic acid dye, CP RNA dye, etc., that can bind to RNA. The fluorescent dyes suitable for this invention can be excited by the sequencing system to emit fluorescent signals and generate grayscale images. When the DNA template is loaded onto the chip surface, the fluorescent molecules can bind to the DNA template, and the sequencer can take pictures to see the clear macroscopic morphology of the DNA template on the chip surface.

[0081] (2) The cleaning reagent of this invention serves to remove excess dye molecules. Suitable cleaning reagents for this invention include, but are not limited to, elution reagent 2 or Qubit dilution buffer from the MGI sequencing reagent tank. Both reagents can remove excess dye molecules, preventing fluorescent dye adsorption on chip areas without loaded DNA templates, thus enabling the acquisition of clear images during photography, such as... Figure 2As shown, Figure A is a photograph taken after washing the background with Qubit dilution buffer, and Figure B is a photograph taken after washing the background with elution reagent 2 in well 18 of the MGI sequencing reagent slot in a specific embodiment of this application.

[0082] It should be understood that the cleaning reagents applicable to this invention should, as far as possible, use reagents already available in sequencing laboratories to achieve a new function that allows for timely and visual analysis of nucleic acid templates loaded before sequencing, rather than simply eliminating pre-sequencing quality control. This not only controls reagent costs but also ensures the ease of operation of the new function.

[0083] (3) The function and effect of the rinsing reagent of the present invention is to detach the fluorescent dye from the DNA molecule, enabling the DNA to undergo subsequent gene sequencing steps. Suitable rinsing reagents can be high-concentration alcohols. For example, 50%-100% alcohols, specifically including but not limited to 50%, 53%, 55%, 57%, 60%, 63%, 65%, 67%, 70%, 73%, 75%, 77%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, and 100%. Specific alcohols that can be used include, but are not limited to, methanol, ethanol, propylene glycol, propanol, and n-butanol. In one specific embodiment of this application, 75% ethanol is used.

[0084] Meanwhile, the rinsing agent suitable for this invention can also be other organic solvents with similar polarity to alcohols. Specifically, the organic solvents that can be used include, but are not limited to, formamide, acetonitrile, acetone, dioxane, tetrahydrofuran, methyl ethyl ketone, ethyl acetate, diethyl ether, isopropyl ether, dichloromethane, chloroform, bromoethane, toluene, etc.

[0085] By combining the above-mentioned fluorescent dyes, cleaning reagents, and rinsing reagents, a kit for the analysis method applicable to the gene sequencing pretreatment stage of this invention can be obtained.

[0086] 3. The dosage and usage instructions for fluorescent dyes, cleaning reagents, and rinsing reagents are as follows:

[0087] (1) Regarding the required concentration of the fluorescent dye applicable to this invention, the following experimental results from this invention can be used for selection. It should be particularly noted that, in this invention, the principle for the appropriate amount of fluorescent dye used is to ensure that the fluorescent dye fully binds to the DNA template and produces sufficient brightness for clear image capture. However, excessive amounts of fluorescent dye should be avoided to prevent waste or insufficient cleaning. In a specific embodiment of this invention, the Qubit fluorescent detection reagent is used, with a final concentration of 74X in the dye-compatible buffer.

[0088] Experiments revealed that when the concentration of the Qubit fluorescent dye was reduced to 9.8X in the dye-compatible buffer, the image became darker (e.g., ...). Figure 3As shown in the image, the grayscale value is slightly higher than the camera's background signal. The image clarity is average. If a lower concentration of fluorescent dye is used, the image will be too dark, and registration will fail during photography due to the small difference in brightness between the positioning lines and the DNA signal points, resulting in image acquisition failure. Therefore, when using Qubit fluorescent dye, the preferred concentration range is 59X to 82X.

[0089] In addition, the grayscale value of the photographed image can also be used as a reference for the amount of fluorescent dye used. The following grayscale values ​​are provided for reference by those skilled in the art. It should be understood that when abnormal conditions occur during loading and preprocessing, the grayscale values ​​will change significantly due to interference from abnormal factors. Therefore, the following description uses grayscale values ​​under normal conditions for reference only.

[0090] In one specific embodiment of the present invention, when the amount of fluorescent dye used is based on the average grayscale value of the images taken after cleaning with the cleaning reagent, the average grayscale value is between 7713 and 12691. Specifically, if Qubit dilution buffer is used, the average grayscale value is between 7713 and 10606, that is, the above-mentioned average grayscale value includes, but is not limited to, 7713, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, and 8950. 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 9980, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10606. If elution reagent 2 is used, the average gray value will be between 9229 and 12691. That is, the average gray value includes, but is not limited to, 9229, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 9980, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 108... 00, 10850, 10900, 10950, 11000, 11050, 11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450, 12500, 12550, 12600, 12650, 12691.

[0091] In one specific embodiment of the present invention, when the amount of fluorescent dye used is based on the average gray value of the image taken after the fluorescent dye has been combined with the nucleic acid template without being cleaned with a cleaning reagent, the average gray value is between 5577 and 7700. Specifically, the average values ​​of the above grayscale values ​​include, but are not limited to, 5577, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, and 7700.

[0092] In one specific embodiment of the present invention, when the amount of rinsing reagent used is based on the average grayscale value of the images taken after rinsing with the rinsing reagent, the average grayscale value is between 2369 and 3259. Specifically, the aforementioned average grayscale value includes, but is not limited to, 2369, 2380, 2400, 2420, 2450, 2480, 2500, 2520, 2550, 2580, 2600, 2620, 2650, 2680, 2700, 2720, 2750, 2780, 2800, 2820, 2850, 2880, 2900, 2920, 2950, ​​2980, 3000, 3020, 3050, 3080, 3100, 3120, 3150, 3180, 3200, 3220, and 3259.

[0093] (2) General characteristics regarding the flow rate, temperature, time, and loading amount of fluorescent dyes, cleaning reagents, and rinsing reagents loaded onto the chip.

[0094] For clarity, a general example using a large four-channel chip is provided. It should be understood that for a two-channel chip, the reagent volume is approximately half the volume described below. For a single-channel chip, the reagent volume is approximately one-quarter of the chip volume flowing through all four channels. Since the internal channel volumes of chips vary depending on the sequencer, the reagent volume may need to be adjusted accordingly; the volumes in the example below are excess volumes.

[0095] (i) Using a large chip with four channels, the volume of fluorescent dye reagent drawn is 100 μL, and the dye flows through the chip at a rate of 80-120 μL / min, for example 80 μL / min, 83 μL / min, 85 μL / min, 87 μL / min, 90 μL / min, 93 μL / min, 95 μL / min, 97 μL / min, 100 μL / min, 103 μL / min, 105 μL / min, 107 μL / min, 110 μL / min, 113 μL / min, 115 μL / min, 117 μL / min, 120 μL / min, with a preferred rate of 100 μL / min. The dye is incubated at 15-35℃ (specifically including but not limited to 15℃, 17℃, 20℃, 23℃, 25℃, 27℃, 30℃, 33℃, 35℃) for 10-30 minutes (specifically including but not limited to 10 minutes, 13 minutes, 15 minutes, 17 minutes, 20 minutes, 23 minutes, 25 minutes, 27 minutes, 30 minutes). Preferably, the dye is incubated at 25℃ for 15 minutes.

[0096] (ii) The volume of the cleaning reagent is 160 μL, and the rate at which the cleaning reagent flows through the chip is 80-120 μL / min (specifically including but not limited to 80 μL / min, 83 μL / min, 85 μL / min, 87 μL / min, 90 μL / min, 93 μL / min, 95 μL / min, 97 μL / min, 100 μL / min, 103 μL / min, 105 μL / min, 107 μL / min, 110 μL / min, 113 μL / min, 115 μL / min, 117 μL / min, 120 μL / min), preferably 100 μL / min.

[0097] (iii) The rinsing reagent should flow through the chip at a rate of 80-120 μL / min (80 μL / min, 83 μL / min, 85 μL / min, 87 μL / min, 90 μL / min, 93 μL / min, 95 μL / min, 97 μL / min, 100 μL / min, 103 μL / min, 105 μL / min, 107 μL / min, 110 μL / min, 113 μL / min, 115 μL / min, 117 μL / min, 120 μL / min), preferably 100 μL / min. Rinse thoroughly multiple times and incubate at low temperature.

[0098] Step 1: Push the following volumes of rinsing reagent into the chip (four-channel chip): 250 μL, then 500 μL, 500 μL, and 300 μL sequentially. Incubate the rinse solution at 15-35°C (specifically including but not limited to 15°C, 17°C, 20°C, 23°C, 25°C, 27°C, 30°C, 33°C, and 35°C) for 10-30 minutes (specifically including but not limited to 10 minutes, 13 minutes, 15 minutes, 17 minutes, 20 minutes, 23 minutes, 25 minutes, 27 minutes, and 30 minutes). Preferably, incubate the rinse solution at 25°C for 15 minutes. (The maximum capacity of the syringe pump on this machine is 500 μL, so it is divided into multiple pumping operations.)

[0099] Step 2: Push the following volumes of rinsing reagent into the chip (four-channel chip): 500 μL, 500 μL, 500 μL, 300 μL. Rinse at 15-35℃ (specifically including but not limited to 15℃, 17℃, 20℃, 23℃, 25℃, 27℃, 30℃, 33℃, 35℃) for 10-30 minutes (specifically including but not limited to 10 minutes, 13 minutes, 15 minutes, 17 minutes, 20 minutes, 23 minutes, 25 minutes, 27 minutes, 30 minutes). Preferably, rinsing is incubated at 25℃ for 15 minutes.

[0100] Step 3: Push the following volumes of rinsing reagent into the chip (four-channel chip): 500μL, 500μL, 500μL, 300μL, 150μL, and take a picture.

[0101] Alternatively, if the above steps are performed manually, simply use a pipette to slowly push 4 mL of rinsing reagent into the chip through the inlet.

[0102] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0103] Example

[0104] 1. Key equipment used in this embodiment:

[0105] MGISEQ-2000 gene sequencer manufactured by BGI Genomics, MGISEQ-2000 sequencing chip, one set of pipettes, and MGISEQ-2000 sequencing reagent container.

[0106] 2: The key reagents used in this embodiment are shown in Table 1 below:

[0107] Table 1 Reagent Instructions

[0108] Reagent Name brand Qubit fluorescence detection reagent Thermo Fisher Scientific Qubit Quantitative Reagent Kit Yisheng Bio MGISEQ-2000 High-Throughput Sequencing Reagent Basket MGI E. coli DNA library MGI DNA template loading buffer II MGI Anhydrous ethanol Xilong Science

[0109] 3. Reagent preparation:

[0110] 1) Preparation of fluorescent dye: As shown in Table 2 below, dilute the Qubit concentrated assay reagent (concentration 200X, DMSO as solvent) 2.7 times with Qubit buffer.

[0111] Table 2: Preparation of Fluorescent Dyes

[0112] Reagent Name Final concentration effect Qubit dye 74X in dye-compatible buffer DNA is stained for photographing.

[0113] 2) Cleaning reagent:

[0114] Table 3: Cleaning reagents (elution reagent 2 for the 18 wells in the finished sequencing reagent tank)

[0115] Reagent Name Final concentration effect Trizma base 0.05M Buffer pairs, stable system Sodium chloride 0.05M Provide monovalent ions EDTA 1mM Chelating metal ions Twain 20 0.05％ surfactants hydrochloric acid Adjust to pH 7.5 Adjusting pH

[0116] 3) Rinsing reagent: Use 75% ethanol.

[0117] Table 3: Rinsing reagents

[0118] Reagent Name Final concentration effect ethanol 75％ Dissolving and removing organic dyes

[0119] 4. Operating steps:

[0120] DNA nanospheres (DNBs) were prepared using E. coli circular DNA as a standard library and reagents from the BGI Genomics high-throughput sequencing reagent tank.

[0121] Test 1: Place the new chip on the MGISEQ-2000 sequencer stage. Add the fluorescent dye, cleaning reagent, and rinsing reagent to the replacement kit or the empty wells of the kit. This kit contains the four reagents commonly used with the MGI sequencer. Then, mix DNB with DNA loading buffer II and place the mixture into an MGISEQ-2000 sample tube. For instructions on using the fluorescent dye, cleaning reagent, and rinsing reagent, please refer to the previous instructions; they will not be repeated here.

[0122] Execute an integrated script on the sequencer. The script starts from loading DNBs onto the chip until the sequencing primers hybridize. During this process, photos are automatically taken after the following nine steps in sequence: Step 1: After the DNBs are loaded onto the chip and stained with a fluorescent dye; Step 2: After the excess fluorescent dye is washed away with a cleaning reagent; Step 3: After the DNB cleaning reagent 1 in well No. 11 of the finished sequencing reagent tank flows through the chip after the DNBs are loaded; Step 4: After the DNB loading reagent 1 in well No. 16 of the finished sequencing reagent tank flows through the chip; Step 5: After the DNB elution reagent 1 in well No. 17 of the finished sequencing reagent tank flows through the chip; Step 6: After the DNB loading reagent 2 in well No. 14 of the finished sequencing reagent tank flows through the chip; Step 7: After the DNB loading reagent 1 in well No. 16 of the finished sequencing reagent tank flows through the chip; Step 8: After the DNB elution reagent 1 in well No. 17 of the finished sequencing reagent tank flows through the chip; Step 9: After the rinsing reagent passes through the chip.

[0123] After the script runs, nine groups of pictures are obtained. Each group of pictures contains a thumbnail of the entire chip and local pictures. By viewing the thumbnails after each step, the loading quality of the DNBs and the macroscopic morphology of the DNBs on the chip after each reagent treatment can be known in a timely, accurate and intuitive manner. If the images are normal, the quality inspection is qualified. In addition, when the sequencing results are poor, the present invention can also be used to separately check and reproduce the problems of reagents, chips and instrument hardware during the pre-sequencing treatment.

[0124] Results: Figure 4 、 5 They are respectively the local images of the chip for testing Steps 8 and 9. As shown in the figure, the present invention can take clear images without relying on the complementary pairing of dNTPs, has a good staining effect of the fluorescent dye on DNA, and the reagent rinsing is sufficient. The present invention can clearly, intuitively and real-time reflect the quality levels of multiple steps of loading DNBs onto the chip and reagent treatment before sequencing.

[0125] Test Two: On the basis of the quality inspection of the Test One plan, after taking pictures during the process of loading DNBs and reagent treatment before sequencing, if the image quality is normal, further carry out subsequent gene sequencing. Sequencing is carried out according to the process of the supporting MGISEQ-2000 sequencing platform. After Test One is completed, immediately judge whether the DNBs on the chip meet the on-machine standards through nine groups of images. Use the MGISEQ-2000 high-throughput sequencing reagent tank and carry out sequencing according to the process of the MGISEQ-2000 sequencing platform. Only when it is determined that the loading and reagent treatment of single-stranded DNBs on the chip meet the on-machine standards, then sequencing is carried out, so as to ensure the sequencing quality and improve the one-time success rate of getting off the machine. At the same time, a comparative experiment (control) was carried out with the same samples as in Test Two. The experimental results show that the sequencing results after using the analysis method of the present invention are basically the same as the sequencing result data without analyzing the pre-sequencing treatment stage (control). As shown in Table 4.

[0126] Table 4 Comparison of test data and control data.

[0127] Category Test 2 Comparison CycleNumber 320 320 Total Reads (M) 491.99 502.94 Q30(％) 94.34 94.18 MappingRate (%) 99.98 99.98 AvgErrorRate(%) 0.21 0.22 AvgErrorRate! N(%) 0.21 0.21

[0128] It should be understood that the improvement in first-pass success rate is particularly significant for machines under development. For machines in development, various problems arise during the development process, especially during DNB loading and post-loading reagent processing. Based on extensive experimental testing, the analytical method of this invention can help quickly improve the efficiency of problem detection, thereby increasing the first-pass success rate by 50%. For already released sequencer products, the first-pass success rate is already very high, exceeding 90%. Multiple experiments have verified that the analytical method of this invention can further improve the existing success rate by 3% to 5%.

[0129] It is particularly important to note that the technical solution employed in this invention can visually represent the current step's problems through images, helping users determine whether DNA template loading or reagent processing needs to be repeated. High-throughput whole-genome sequencing often takes tens of hours; this invention avoids the problem of tracing the source after sequencing results become abnormal, shortening the time for troubleshooting and saving sequencing reagent costs. Furthermore, the method provided by this invention can be directly applied to commercially available sequencing instruments. Users already possess the skills to view and interpret conventional images taken after base synthesis during sequencing results; therefore, they can also easily interpret the images provided by the analysis method of this invention. This new function provides users with additional information.

[0130] This example provides a set of images as illustrations based on the various anomalies that occurred during the extensive testing, such as... Figure 6-8 As shown, where Figure 6 Here is an example image with bubbles; you can see the bubbles clearly in the picture. Figure 7 This is an example image showing the shift caused by the rapid scouring of fluid by the DNB after loading. Figure 7 The phenomenon of "growing grass" or umbrella-shaped objects was observed. Figure 8 This is an example image showing impurities precipitating in a biochemical reagent, with noticeable large bright spots. Additionally, an example after normal loading is also provided in this example. Figure 9 As shown, this is a clear image example of the regular arrangement of DNBs on the chip positioning line after magnification of a local image in the presence of reagents.

[0131] The above examples illustrate the present invention only to aid in understanding it and are not intended to limit the scope of the invention. Those skilled in the art can make various simple deductions, modifications, or substitutions based on the principles of this invention.

Claims

1. A method for loading a nucleic acid template before sequencing, characterized in that, Includes the following steps: 1) Load the nucleic acid template onto the surface of a solid support; 2) Use a fluorescent dye to bind to the nucleic acid template; 3) Use cleaning reagents to remove unbound fluorescent dyes.

2. The method as described in claim 1, characterized in that, The method further includes step 4): removing the fluorescent dye bound to the nucleic acid template with a rinsing reagent; Preferably, the method further includes performing a sequencing pretreatment of the nucleic acid template using several reagents between steps 3) and 4); Preferably, the fluorescent dye is a non-specific fluorescent dye; Preferably, the concentration of the fluorescent dye used is between 59X and 82X; Preferably, the cleaning reagent is elution reagent 2 and / or Qubit dilution buffer from the MGI sequencing reagent tank; Preferably, the nucleic acid template is a deoxyribonucleotide sequence; Preferably, the rinsing agent is an alcoholic substance or an organic solvent with polarity similar to alcohols at a concentration of 50%-100%. Preferably, in the steps of binding the fluorescent dye to the nucleic acid template and cleaning the fluorescent dye with the cleaning reagent, the amount of fluorescent dye and cleaning reagent loaded onto the surface of the solid support is proportional to the number of flow channels on the surface of the solid support. Preferably, in the steps of binding the fluorescent dye to the nucleic acid template and cleaning the fluorescent dye with the cleaning reagent, the flow rate of the fluorescent dye and the cleaning reagent through the surface of the solid support is 80-120 μL / min. Preferably, the incubation temperature for binding the fluorescent dye to the nucleic acid template is 15-35°C, and the incubation time is 10-30 minutes; Preferably, in the step of removing fluorescent dye with rinsing reagent, the amount of rinsing reagent loaded onto the surface of the solid support is proportional to the number of flow channels on the surface of the solid support; Preferably, in the step of removing fluorescent dye with rinsing reagent, the incubation temperature of the rinsing reagent loaded onto the surface of the solid support is 15-35°C, and the incubation time is 10-30 minutes. Preferably, in the step of removing fluorescent dye with rinsing reagent, the flow rate of the rinsing reagent over the surface of the solid support is 80-120 μL / min.

3. A method for analyzing loaded nucleic acid templates before sequencing, characterized in that, The method includes the following steps: (1) After the nucleic acid template is loaded onto the surface of the solid support, a fluorescent dye is used to bind the nucleic acid template; (2) Use cleaning reagents to remove unbound fluorescent dyes and take pictures for analysis; Preferably, the nucleic acid template is bound to a fluorescent dye before photographic analysis.

4. The method as described in claim 3, characterized in that, The analytical method further includes step (3): after step (2), the nucleic acid template is subjected to multiple reagent treatments before sequencing and then photographed and analyzed. Preferably, the analytical method further includes step (4): removing the fluorescent dye bound to the nucleic acid template with a rinsing reagent before sequencing; Preferably, the fluorescent dye bound to the nucleic acid template is removed by rinsing reagent before photographic analysis.

5. The method as described in claim 3 or 4, characterized in that, The fluorescent dye is a non-specific fluorescent dye; Preferably, the fluorescent dye includes at least one of the following: Qubit fluorescent detection reagent, SYBR Gold dye, SYBR Green II nucleic acid dye, ethidium bromide, GelRed dye, SolarRed dye, YeaRed dye, NA-Red dye, NA-Green dye, acridine orange, Diamond nucleic acid dye, Hoechst 33258 dye, SYBR Green I dye, YeaGreen dye, GeneRed nucleic acid dye, or CP RNA dye; Preferably, the amount of rinsing reagent used is based on the average gray value of the images taken after rinsing with the rinsing reagent, and the average gray value is between 2369 and 3259. Preferably, the amount of fluorescent dye used is based on the average gray value of the images taken after cleaning with the cleaning reagent, and the average gray value is between 7713 and 12691. Preferably, when the cleaning reagent is a Qubit dilution buffer, the average gray value is between 7713 and 10606; Preferably, when the cleaning agent is elution agent 2, the average gray value is between 9229 and 12691.

6. The method according to any one of claims 3-5, characterized in that, The nucleic acid template is a deoxyribonucleotide sequence; Optionally, the cleaning reagent includes elution reagent 2 from the MGI sequencing reagent tank and / or Qubit dilution buffer; Preferably, in the steps of binding the fluorescent dye to the nucleic acid template and cleaning the fluorescent dye with the cleaning reagent, the amount of fluorescent dye and cleaning reagent loaded onto the surface of the solid support is proportional to the number of flow channels on the surface of the solid support. Preferably, in the steps of binding the fluorescent dye to the nucleic acid template and cleaning the fluorescent dye with the cleaning reagent, the flow rate of the fluorescent dye and the cleaning reagent through the surface of the solid support is 80-120 μL / min. Preferably, the incubation temperature for binding the fluorescent dye to the nucleic acid template is 15-35°C, and the incubation time is 10-30 minutes.

7. The method as described in claim 4 or 5, characterized in that, The rinsing agent is an alcoholic substance or an organic solvent with similar polarity to alcohols, with a concentration of 50%-100%. Preferably, when the rinsing agent removes the fluorescent dye, the amount of rinsing agent loaded onto the surface of the solid support is proportional to the number of flow channels on the surface of the solid support; Preferably, in the step of removing fluorescent dye with rinsing reagent, the incubation temperature of the rinsing reagent loaded onto the surface of the solid support is 15-35°C, and the incubation time is 10-30 minutes. Preferably, in the step of removing fluorescent dye with rinsing reagent, the flow rate of the rinsing reagent over the surface of the solid support is 80-120 μL / min.

8. A kit for use in the method according to any one of claims 1-7, characterized in that, The kit includes a fluorescent dye and a cleaning reagent; Preferably, the fluorescent dye includes at least one of the following: Qubit fluorescent detection reagent, SYBR Gold dye, SYBR Green II nucleic acid dye, ethidium bromide, GelRed dye, SolarRed dye, YeaRed dye, NA-Red dye, NA-Green dye, acridine orange, Diamond nucleic acid dye, Hoechst 33258 dye, SYBR Green I dye, YeaGreen dye, GeneRed nucleic acid dye, or CP RNA dye; Preferably, the cleaning reagent includes elution reagent 2 from the MGI sequencing reagent tank and / or Qubit dilution buffer.

9. The reagent kit according to claim 8, characterized in that, The kit further includes a rinsing agent; Preferably, the rinsing agent comprises an alcoholic substance or an organic solvent with polarity similar to that of alcohols at a concentration of 50-100%.

10. A sequencing method, characterized in that, The sequencing method employs the method described in claim 1 or 2 to load a nucleic acid template, or the method described in any one of claims 3-7 to analyze the gene sequencing pretreatment stage.

Images