Nucleic acid loading reagent and method for detecting adsorption capacity of sequencing chip by using nucleic acid loading reagent

Abstract

The invention provides a nucleic acid loading reagent and a method for detecting the nucleic acid adsorption capacity of a sequencing chip by using the nucleic acid loading reagent. The method comprises at least one of the following conditions: (i) the pH value of the nucleic acid loading reagent is between 3 and 5; (ii) the nucleic acid loading reagent contains metal ions, and the metal ions are selected from one or more of the following solutions: MgCl2, CeCl3, ZnCl2, MnCl2, CaCl2, CuCl2 and NiCl2; and (iii) jointly incubating the chip and the nucleic acid loading reagent for more than 10 minutes. The invention provides a sequencing chip quality control scheme which is not limited by a platform, simple to operate, low in cost and high in speed; a scheme capable of rapidly optimizing the loading efficiency and loading bias of the nucleic acid library is provided; the invention provides a reaction reagent capable of effectively improving the loading efficiency and loading bias of a nucleic acid library.

Description

A nucleic acid loading reagent and a method for detecting the adsorption capacity of a sequencing chip using the same

[0001] The present invention relates to the field of biotechnology, and in particular to a nucleic acid loading reagent and a method for detecting the adsorption capacity of a sequencing chip using the same.

[0002] With the development of sequencing technology, the improvement of social awareness, and the continuous reduction of sequencing costs, high-throughput gene sequencing has been widely used in various fields such as scientific research, precision medicine, social health and public safety. The cost of human whole genome sequencing (30×, about 100Gb data) based on massively parallel sequencing technology has rapidly increased from US$1,000 to US$100, which is expected to further reduce the sequencing cost of the entire sequencing market, thereby promoting high-throughput sequencing to achieve more widespread application.

[0003] There are many factors that determine the quality and cost of high-throughput sequencing, such as biochemical protocols, sequencing materials or system integration capabilities. One of the key points is whether the sequencing chip used can fully support the high-density loading, amplification and signal detection of the samples to be tested. For large-scale parallel sequencing, the largest sequencing chip has a diameter of 8 inches and the nucleic acid loading density has reached 400nm spacing. Therefore, a single chip will be able to generate more than 50Gb of effective reads, corresponding to the whole genome of about 100 people, so its scientific and economic value are very considerable.

[0004] However, there are still many challenges in loading sequencing samples (such as Illumina's clusters or BGI's DNA nanoballs (DNBs)) onto sequencing chips. The mainstream high-throughput sequencers on the market, such as NovaSeq-6000 and DNBSEQ-T7, use sequencing chips prepared using semiconductor technology and bind to the nucleic acid sample to be tested through active sites. However, the former couples the probe sequence to the active site, and bridge PCR amplification begins after the nucleic acid sample to be tested binds to the probe; the latter does not require amplification at the active site. After the nucleic acid sample to be tested undergoes rolling circle amplification (RCA) in a test tube, it is adsorbed and loaded through interactions such as proteins, surface charges or ions.

[0005] Therefore, there is an urgent need to provide a solution that can quickly, simply and accurately control the quality of sequencing chips, improve the loading efficiency, bias and robustness of sequencing libraries, so as to effectively further reduce the cost of high-throughput sequencing, increase data output, and improve sequencing quality.

[0006] Summary of the invention

[0007] In order to solve the defects of low loading efficiency, high cost and complex quality control sequencing in the prior art sequencing, the present invention provides a nucleic acid loading reagent and a method for detecting the adsorption capacity of a sequencing chip using the same.

[0008] The first aspect of the present invention provides a method for detecting the ability of a sequencing chip to adsorb nucleic acids, comprising at least one of the following conditions:

[0009] (i) the pH value of the nucleic acid loading reagent is between 3 and 5;

[0010] (ii) the nucleic acid loading reagent contains metal ions, such as metal ions from one or more of the following solutions: MgCl2, CeCl3, ZnCl2, MnCl2, CaCl2, CuCl2 and NiCl2;

[0011] (iii) The chip is incubated with the nucleic acid loading reagent for more than 10 minutes, preferably 15 to 120 minutes.

[0012] In some embodiments, in (i), the pH value is 3 or the pH value is 5.

[0013] In some embodiments, in (ii), the metal ions are from a ZnCl2 or CaCl2 solution.

[0014] In some embodiments, the final concentration of the ZnCl2 and CaCl2 solutions is 15 mM-50 mM, for example, the ZnCl2 solution is 15 mM, 20 mM and 40 mM, preferably 15 mM; the CaCl2 solution is 20 mM and 30 mM, preferably 30 mM.

[0015] In some embodiments, the nucleic acid loading reagent is diluted with a buffer, such as one or more of a HEPES buffer, a citrate buffer, or a MES buffer.

[0016] In some embodiments, the pH of the buffer is 3-7.4.

[0017] In some embodiments, the buffer is a citrate buffer, and the concentration of the citrate is, for example, 20 mM.

[0018] In some embodiments, in (iii), the incubation time is ≥ 15 min.

[0019] The second aspect of the present invention provides a nucleic acid loading reagent, which comprises a buffer and metal ions as defined in the method described in the first aspect of the present invention.

[0020] The third aspect of the present invention provides a use of the nucleic acid loading reagent described in the second aspect of the present invention in sequencing or preparing sequencing reagents.

[0021] A fourth aspect of the present invention provides a method for rapidly detecting the ability of a chip to adsorb nucleic acid, comprising the following steps:

[0022] S1: aspirate the nucleic acid loading reagent described in the second aspect of the present invention, add the labeled DNA to be tested, drop it onto the surface of the sequencing chip or a small chip cut from the sequencing chip, and incubate;

[0023] S2: Pipetting the supernatant of the nucleic acid loading reagent into a detection container for detection;

[0024] S3: Calculate the adsorption ratio.

[0025] In some embodiments, in S1, the DNA to be tested is a single-stranded oligonucleotide.

[0026] In some embodiments, the label is a fluorescent label, preferably Cy3.

[0027] In some embodiments, the detection container is an ELISA plate or a 0.5 ml transparent centrifuge tube; the detection is performed in an ELISA reader or a Qubit fluorescence quantifier.

[0028] In some embodiments, the chiplet has a size of 8 mm x 8 mm or 10 mm x 10 mm.

[0029] In some embodiments, the incubation time is 15 to 120 min, for example 15 min.

[0030] In some embodiments, the final concentration of the single-stranded oligonucleotide is 5-40 nM, for example, 5 nM.

[0031] In some embodiments, the single-stranded oligonucleotide is one or more of Poly-nC, Poly-nG, Poly-nT, Poly-nA, and n is a natural number greater than 3, for example, 5.

[0032] In some embodiments, S2 further includes adding controls, wherein the controls include negative controls and positive controls.

[0033] In some embodiments, the negative control is a buffer, such as a HEPES buffer or a MES buffer or a citrate buffer.

[0034] In some embodiments, the positive control is a labeled DNA to be tested that is not incubated on the sequencing chip or chiplet.

[0035] In some embodiments, in S3, the adsorption ratio is calculated using a formula of 1-(XY) / (ZY).

[0036] Beneficial effects brought by the technical solution of the present invention:

[0037] (1) Provide a sequencing chip quality control solution that is platform-independent, simple to operate, low-cost, and fast;

[0038] (2) Provide a method for rapidly optimizing nucleic acid library loading efficiency and loading bias;

[0039] (3) Provide a reaction reagent that can effectively improve the loading efficiency and loading bias of the nucleic acid library.

[0040] Figure 1 is a schematic diagram of sequencing base separation.

[0041] FIG. 2 is a schematic diagram showing the loading of DNB onto a chip.

[0042] FIG. 3 is a schematic diagram of the test of oligo adsorption on a sequencing chip.

[0043] FIG. 4 shows the chips cut from batch 1 (left) and the chips cut from batch 2 (right).

[0044] FIG5 is a schematic diagram showing the effect of chip batch and adsorption time on DNA adsorption.

[0045] FIG. 6 is a schematic diagram showing the test of the adsorption of Oligo on the sequencing chip in solutions with different pH values.

[0046] FIG. 7 is a schematic diagram showing the effect of metal ions on DNA adsorption by a sequencing chip.

[0047] FIG8 is a schematic diagram showing the effect of metal ions on DNA adsorption on a sequencing chip.

[0048] The present invention is described in more detail below in conjunction with the embodiments and the accompanying drawings. In the present invention, all equipment and raw materials can be purchased from the market or are commonly used in the industry. Unless otherwise specified, the methods used in the embodiments are common techniques in the art.

[0049] Table 1. Reagents and consumables

[0050] Name Company Article No. Origin 3' fluorescent labeled Oligo (AAAAA-Cy3) Shanghai Biotech No China Citrate buffer (pH3-6) Self-prepared No China 1M HEPES buffer Shanghai Biotech E607018-0100 China 0.1M MES buffer Canos sj1074 China Ultrapure water MGI No China Sequencing chip - cut into 8mm×8mm MGI No China 96-well ELISA plate Greiner Bio-One655801 Austria 1.5ml centrifuge tube Shanghai Biotech F601620 China Tweezers No No China surface

[0051] Table 2. Instruments

[0052] Name Company Item No. Origin Manual single channel pipette - one set Eppendorf without German microplate reader BMG LABTECHFLUOstar Omega Germany surface

[0053] Here is the process:

[0054] 1. Obtain a fluorescently labeled DNA oligonucleotide, wherein the fluorescent label uses Cy3 fluorescent dye, the DNA oligonucleotide is single-stranded, and can be a poly-oligonucleotide (Poly AAAAA is used in this scheme) or a random combination of four bases ACGT, and the length is preferably ≤30nt;

[0055] 2. Use laser cutting or other methods (such as glass knife cutting) to cut the normally prepared sequencing chip (or its scraps - with photolithography pattern and chemical vapor deposition treatment) into small square sequencing chips (detection chips) of about 8-10 mm square, and store them face up in a nitrogen cabinet;

[0056] 3. Prepare a working solution of single-stranded DNA oligonucleotides with fluorescent labels, with a final concentration of 5-10 nM. The diluent can be selected according to the needs, and buffer solutions of different pH (3-7.4), different salt concentrations and types (NaCl, MgCl2, CaCl2, etc.) can be used to study solutions to improve DNB adsorption efficiency, adsorption preference, etc.;

[0057] 4. Use tweezers to clamp the small chip onto a flat surface, with the front side facing up;

[0058] 5. Use a 200μl pipette to absorb about 180μl of fluorescent dye solution and carefully drip it onto the surface of the small chip to avoid the liquid dripping to other places;

[0059] 6. After reacting (incubating) at room temperature for 15 min-2 h, use a 200 μl pipette to pipette about 150 μl of the supernatant into an ELISA plate (e.g. Greiner ELISA plate, catalog number: 655801; or a transparent centrifuge tube, e.g. 0.5 ml specification), and add a negative control (buffer only, no fluorescent dye) and a positive control (not incubated on the chip);

[0060] 7. If necessary, prepare a gradient dilution of the standard solution without incubation and add it to the ELISA plate;

[0061] 8. Turn on the microplate reader, select the corresponding detection wavelength (the excitation and emission wavelengths of Cy3 are 530±20nm and 580±30nm) and the microplate, select the corresponding well position, set the gain (i.e., Gain) to an appropriate value (such as 2000), and perform the test, repeating three times;

[0062] 9. Take the average value of the data from the three tests, with the negative control as the background; take the positive control as 100%, and calculate the adsorption ratio of each condition signal respectively, the calculation formula is: adsorption ratio = 1-(test-negative control) / (positive control-negative control). You can also set a negative control on the microplate reader, then there is no need to subtract the value of the negative control. If there is a marker curve, you can calculate its specific adsorption molar molecular number;

[0063] 10. For sequencing chip QC, the buffer conditions can be fixed, and multiple batches of chips (different wafers, different surface treatments, etc.) can be tested at the same time, and a horizontal comparison can be made. The sequencing chip batches with higher adsorption ratios should have stronger ability to load DNBs, and the sequencing chip batches with lower adsorption ratios should have weaker ability to load DNBs.

[0064] 11. The actual machine performance data should be comprehensively considered (too strong will lead to over-adsorption, too weak will lead to failure to load 100% of DNB), and finally a suitable QC plan and DNB loading plan should be formulated.

[0065] Example 1 Oligo adsorption test on sequencing chip

[0066] 1) Prepare the reaction solution:

[0067] 1. Dilute the 3' fluorescently labeled oligo (using AAAAA-Cy3 (abbreviated as Poly 5A-Cy3) as an example, but not limited to this sequence) with ultrapure water to 100 μM and store at -20°C for later use;

[0068] 2. Dilute a small amount of 100 μM Poly 5A-Cy3 to 1 μM with ultrapure water and place at 4°C for later use.

[0069] 3. Dilute 1M HEPES buffer, pH=7.4, to 20mM with ultrapure water for later use;

[0070] 4. Before use, dilute 1 μM Poly 5A-Cy3 with 20 mM HEPES to a final concentration of 5 nM (e.g., add 5 μl Poly 5A-Cy3 to 995 μl 20 mM HEPES buffer) of Poly 5A-Cy3 dye solution;

[0071] 5. Prepare another clean Greiner Bio-one UV ELISA plate for use.

[0072] 2) Adsorption experiment:

[0073] 1. Use tweezers to clamp the 8mm×8mm small chips onto a clean desktop and arrange them in order;

[0074] 2. Carefully pipette 180 μl of 5 nM Poly 5A-Cy3 dye solution and drop it onto the surface of the small chip, making sure that the droplets do not drip (see Figure 33);

[0075] 3. After incubation at room temperature for 15 minutes, use a 200μl pipette to absorb 150μl of dye solution from the small chip and transfer it to the designated wells of the ELISA plate;

[0076] 4. Add 150 μl of 20 mM HEPES buffer and 5 nM Poly 5A-Cy3 dye solution (without any treatment) to the designated wells of the ELISA plate as controls.

[0077] 3) Signal detection and analysis

[0078] 1. Open the BMG microplate reader and select the FL endpoint mode. Select the Cy3 dye and Gain value as 2000 in the program and select the corresponding Layout;

[0079] 2. Click the detection button and repeat three times;

[0080] 3. Copy the values ​​of the three tests into an Excel spreadsheet;

[0081] 4. The following is the calculation method of adsorption ratio:

[0082] a) Let the absorbance under a certain condition be X, the absorbance of 20 mM HEPES buffer be Y, and the absorbance of 5 nM Poly 5A-Cy3 be Z;

[0083] b) adsorption ratio = 1-(XY) / (ZY);

[0084] 5. It can be calculated that in 20mM HEPES buffer, after incubation at room temperature for 15min, the adsorption ratio of the currently treated small chip is 53.67%, proving that this scheme can be used to study the adsorption of DNA on the small chip.

[0085] Table 3.

[0086] Table 3. Oligo adsorption test on small chip (Gain 2000)

[0087]

[0088]

[0089] Example 2 Sequencing chip quality control and machine verification

[0090] 1) Prepare the reaction solution:

[0091] 1. Prepare 20 mM MES buffer using 1 M MES buffer, pH = 4;

[0092] 2. Prepare 20nM Poly 5A-Cy3 dye solution using 20mM MES buffer, 4000μl per portion;

[0093] 3. Prepare 400 μl of 20 mM MES buffer as a negative control.

[0094] 2) Preparation of quality control chips

[0095] Using the laser hidden cutting scheme, two different batches of sequencing chips (defined as batch 1 and batch 2, respectively) were cut into a size of 10 mm × 10 mm ( FIG. 4 ).

[0096] 3) Adsorption experiment:

[0097] 1. Use tweezers to clamp the small chips onto a clean table and arrange them in order;

[0098] 2. Carefully pipette 180 μl of 5 nM Poly 5A-Cy3 dye solution and drop it onto the surface of the small chip, making sure that the droplets do not drip (see Figure 33);

[0099] 3. After incubation at room temperature for 15 min-2 h, use a 200 μl pipette to pipette 150 μl of dye solution from each small chip and transfer it to the designated wells of the ELISA plate;

[0100] 4. Add 150 μl of 20 mM MES buffer and 20 nM Poly 5A-Cy3 dye solution to the designated wells of the ELISA plate. These reagents were not treated in any way as a control.

[0101] 4) Signal detection and analysis

[0102] 1. Turn on the BMG microplate reader and select the FL endpoint mode. Select the Cy3 dye and Gain value as 2000 in the program;

[0103] 2. Click the detection button and repeat three times;

[0104] 3. Copy the values ​​of the three tests into an Excel spreadsheet;

[0105] 4. The following is the calculation method of adsorption ratio:

[0106] a) Let the absorbance value of the supernatant solution under different pH conditions be X, and set the buffer solution (without dye solution) as the blank control, so no calculation is required, and the absorbance value of no adsorption is Z;

[0107] b) adsorption ratio = 1-X / Z;

[0108] 5. From the results, it can be seen that compared with batch 1, the adsorption amount of batch 2 is greater than that of batch 1 in all time periods of adsorption ≥15 min, as shown in Figure 55.

[0109] 5) Computer verification

[0110] 1. The two batches of chips were used to prepare MGISEQ-2000 sequencing chips. For distinction, the chip prepared from batch 1 in the adsorption experiment was named batch 1-1, and the chip prepared from batch 2 in the adsorption experiment was named batch 2-1;

[0111] 2. Use the MGISEQ-2000 sequencer standard sequencing process (refer to the "MGISEQ-2000RS High-throughput Sequencing Reagent Set Instructions") to prepare, load and sequence DNBs using PE100;

[0112] 3. After comparing the indicators such as Q30, DNB loading, and effective DNB number, it was found that the indicators of batch 2-1 were significantly better than those of batch 1-1, as shown in Table 4;

[0113] 4. Comprehensive adsorption test and on-machine test show that the present invention can be used for quality control of sequencing chips, and has the advantages of low cost and short time.

[0114] Table 4. Performance of sequencing chip batches 1-1 and 2-1

[0115] Condition Batch 1-1 Batch 2-1 Cycle Number 2020 Total Reads (M) 513.73 548.73 Q30 (%) 93.06 94.26 ESR (%) 86.66 90.21 Read Number 5137 256 185 487 257 94 Base Number 10 27 45 123 60 10 97 45 158 80 surface

[0116] Example 3 DNA adsorption test of sequencing chip at different pH

[0117] 1) Prepare the reaction solution:

[0118] 1. Prepare citrate buffers of different pH values, ranging from 3 to 6, wherein the concentration of sodium citrate is 20 mM; at the same time, prepare two other buffers: 20 mM MES buffer at pH 4 and 20 mM HEPES buffer at pH 7.4;

[0119] 2. Prepare 5 nM Poly 5A-Cy3 dye solution using citrate buffer of different pH values, 400 μl per aliquot;

[0120] 3. Prepare non-fluorescent and fluorescent (Poly-5A-Cy3) citrate buffers at different pH as negative and positive controls.

[0121] 2) Adsorption experiment:

[0122] 1. Use tweezers to clamp the 8mm×8mm small chips onto a clean desktop and arrange them in order;

[0123] 2. Carefully pipette 180 μl of 5 nM Poly 5A-Cy3 dye solution and drop it onto the surface of the small chip, making sure that the droplets do not drip (see Figure 6);

[0124] 3. After incubation at room temperature for 15 minutes, use a 200μl pipette to absorb 150μl of dye solution from the small chip and transfer it to the designated wells of the ELISA plate;

[0125] 4. Add negative control: 20mM HEPES buffer without fluorescence; add positive control: 20mM HEPES buffer containing 5nM Poly 5A-Cy3, 20mM MES buffer and pH 3-6 citrate buffer respectively in the designated wells of the ELISA plate. None of these reagents were subjected to any adsorption treatment.

[0126] 3) Signal detection and analysis

[0127] 1. Open the BMG microplate reader and select the FL endpoint mode. Select the Cy3 dye and Gain value as 2000 in the program, and select the corresponding Layout according to Table 3;

[0128] 2. Click the detection button and repeat three times;

[0129] 3. Copy the values ​​of the three tests into an Excel spreadsheet;

[0130] 4. The following is the calculation method of adsorption ratio:

[0131] For each pH condition, the absorbance values ​​were processed separately according to the following conditions: let the absorbance of the supernatant solution after Poly 5A-Cy3 adsorption be X, the absorbance of the buffer solution (without dye HEPES) be Y, and the absorbance of the unadsorbed dye be Z; adsorption ratio = 1-(XY) / (ZY);

[0132] 5. It can be calculated that as the pH decreases, the overall ability of the small chip to adsorb DNA gradually increases, increasing from 36.36% to >68%. However, between pH 4.5 and pH 5, as the pH decreases, the DNA adsorption rate remains basically unchanged. This data shows that the pH should be appropriately lowered for the small chip to adsorb DNA. In actual sequencing, when the pH is less than 4, abnormal loading may occur, so the DNB loading conditions between pH 4-4.5 can be focused on. In addition, compared with citric acid, although the pH is the same, MES has a higher proportion of adsorbed DNA Oligo, so it has more advantages.

[0133] Table 5. Proportion of Oligo adsorbed on sequencing chip at different pH (gain 2000)

[0134]

[0135] Example 4 Adsorption preference and efficiency of sequencing chip for DNA

[0136] For the present invention, due to the advantages mentioned above, the present invention provides a good experimental platform for optimizing the efficiency and adsorption preference of DNA samples adsorbed by sequencing chips. Therefore, the following experiment was designed:

[0137] 1. Tested the adsorption of four bases ACGT on the surface of the sequencer chip

[0138] A (adenine deoxyribonucleotide), C (cytosine deoxyribonucleotide), G (guanine deoxyribonucleotide), T (thymine deoxyribonucleotide) are the components of DNA, among which A and T are complementary pairs, and C and G are complementary pairs. Considering the randomness of DNA extraction and library construction, assuming that DNB loading is fully random, in the sequence obtained by sequencing, A=T, C=G, but in actual sequencing, due to various reasons, A and T, C and G are often not equal, partly because when DNB is loaded, the chip has a selection preference for different bases (Figure 1). In this regard, Poly-5A-Cy3, Poly-5C-Cy3, Poly-5G-Cy3 and Poly-5T-Cy3 were used, and the method in Example 1 was used to perform adsorption verification on the sequencing chip. The results show that all four bases can be adsorbed and detected, among which Poly-5C-Cy3 not only has a high signal, but also has a faster adsorption rate. It can also be seen that the adsorption rate of the chip to different bases is inconsistent, which may be related to the action site (the action site of pyrimidine is smaller than that of purine), as shown in Table 6.

[0139] Table 6. Adsorption of four bases ACGT on the surface of sequencing chip (Gain 2000)

[0140]

[0141] 2. Test the effect of metal ions on DNA adsorption and verify it on the machine

[0142] Metal ions carry positive charges, while the bases and phosphate groups of DNA generally carry negative charges in solution (such as pH 4-8). Therefore, they can bind to DNA through electrostatic interactions, assist DNA folding, and promote the binding of DNA molecules to the surface of the sequencing chip through salt bridges or electrostatic shielding. + Mg 2+ Metal ions, but the problem of low DNB loading efficiency still often occurs, which affects the data output. As shown in Figure 2, the loading efficiency in the left figure is not high, resulting in many empty spots; in the right figure, the loading efficiency is high and there are basically no empty spots.

[0143] In the present invention, six divalent metal ions and one trivalent metal ion were tested, including five transition metal ions, as shown in Table 7. The metal ions were prepared into a 50 μM solution using 20 mM HEPES at pH 7.4, and 5 nM Poly-5A-Cy3 was prepared using the solution, and adsorption verification was performed on a sequencing chip, as shown in FIG7 .

[0144] The experimental data show that ZnCl2 and CaCl2 have a certain effect on promoting the adsorption of DNA by the sequencing chip, but the effects of other tested ions are not significant and may have a certain impact on the fluorescence signal, as shown in Figure 8. The ion concentration gradient was not verified in this test, and it is expected that the ion concentration gradient will also have a certain effect on adsorption.

[0145] Table 7. Effect of metal ions on DNA adsorption on sequencing chips

[0146] Metal ion concentration Buffer solution Reaction time Remarks MgCl250mM20mM HEPES10minCeCl350mM20mM HEPES10minTransition metal ZnCl250mM20mM HEPES10minTransition metal MnCl250mM20mM HEPES10minTransition metal CaCl250mM20mM HEPES10minCuCl250mM20mM HEPES10minTransition metal NiCl250mM20mM HEPES10minTransition metal surface

[0147] Furthermore, two metal salts, ZnCl2 and CaCl2, were tested on the MGISEQ-2000 sequencer, with existing standard conditions as the control group. The test scheme is as follows:

[0148] (1) After the DNB preparation is completed, ZnCl2 and CaCl2 with a final concentration of 15-40 mM are added to the DNB loading system, and the DNB is loaded on the MGISEQ-2000 sequencing chip;

[0149] (2) SE20 sequencing was performed on the MGISEQ-2000 sequencer, and indicators such as Q30, effective data volume, and DNB number were analyzed.

[0150] The results are shown in Table 8. From the results, it can be seen that both ZnCl2 and CaCl2 can increase the number of DNBs, indicating that the efficiency of DNB loading on the chip is improved. The concentrations of ZnCl2 and CaCl2 can be 15-50 mM.

[0151] In addition, after adding ZnCl2 and CaCl2, the bias of DNB loading on the sequencing chip was improved, which was reflected in the reduction of AT values, which was more consistent with the distribution of bases in the library and reference genome. Therefore, the sequencing quality and customer satisfaction can be further improved.

[0152] Table 8. Effect of metal ions on improving DNB loading efficiency

[0153]

[0154] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

A method for detecting the ability of a sequencing chip to adsorb nucleic acids, characterized in that: The method comprises at least one of the following conditions: (i) the pH value of the nucleic acid loading reagent is between 3 and 5; (ii) the nucleic acid loading reagent contains metal ions, and the metal ions are, for example, from one or more of the following solutions: MgCl2, CeCl3, ZnCl2, MnCl2, CaCl2, CuCl2 and NiCl2; (iii) the chip is incubated with the nucleic acid loading reagent for more than 10 minutes, preferably 15 to 120 minutes. The method according to claim 1, characterized in that In (i), the pH value is 3 or the pH value is 5. The method according to claim 1, characterized in that In (ii), the metal ions are from ZnCl2 or CaCl2 solution. The method according to claim 3, characterized in that The final concentrations of the ZnCl2 and CaCl2 solutions are 15mM-50mM, for example, the ZnCl2 solution is 15mM, 20mM and 40mM, preferably 15mM; the CaCl2 solution is 20mM and 30mM, preferably 30mM. The method according to claim 3, characterized in that The nucleic acid loading reagent is diluted with a buffer, such as one or more of a HEPES buffer, a citrate buffer and a MES buffer. The method according to claim 5, characterized in that The pH of the buffer is 3-7.

4. The method according to claim 5, characterized in that The buffer is a citrate buffer, and the concentration of the citrate is, for example, 20 mM. A nucleic acid loading reagent, characterized in that: The nucleic acid loading reagent comprises a buffer as defined in any one of claims 1 to 7 and metal ions. Use of the nucleic acid loading reagent as claimed in claim 8 in sequencing or preparing sequencing reagents. A method for rapidly detecting the ability of a chip to adsorb nucleic acid, characterized in that: The method comprises the following steps: S1: aspirating the nucleic acid loading reagent described in claim 7, adding the labeled DNA to be tested, dripping it onto the surface of the sequencing chip or a small chip cut therefrom, and incubating; S2: Pipetting the supernatant of the nucleic acid loading reagent into a detection container for detection; S3: Calculate the adsorption ratio. The method according to claim 10, characterized in that In S1, the DNA to be tested is a single-stranded oligonucleotide; and / or the label is a fluorescent label, preferably Cy3; and / or the detection container is an ELISA plate or a 0.5 ml transparent centrifuge tube; the detection is performed in an ELISA reader or a Qubit fluorescence quantifier; and / or the size of the small chip is 8 mm×8 mm or 10 mm×10 mm; and / or the incubation time is 15 to 120 min, for example, 15 min. The method according to claim 11, characterized in that The final concentration of the oligonucleotide single strand is 5-40 nM, for example, 5 nM; and / or the oligonucleotide single strand is one or more of Poly-nC, Poly-nG, Poly-nT, Poly-nA, where n is a natural number greater than 3, for example, 5. The method according to any one of claims 10 to 12, characterized in that S2 also includes adding controls, which include negative controls and positive controls. The method according to claim 13, characterized in that The negative control is a buffer, such as HEPES buffer or MES buffer or citrate buffer; and / or, the positive control is a labeled DNA to be tested that is not incubated on a sequencing chip or a small chip. The method according to any one of claims 10 to 14, characterized in that In S3, the calculation formula of the adsorption ratio is 1-(XY) / (ZY).

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