CN108913376B

CN108913376B - Reagent for cleaning sequencing chip and preparation method and application thereof

Info

Publication number: CN108913376B
Application number: CN201810542121.7A
Authority: CN
Inventors: 刘晨; 黄金; 田志坚; 许振朋; 丁芬; 吴昊
Original assignee: Wuhan Bgi Medical Laboratory Co ltd
Current assignee: Wuhan Bgi Medical Laboratory Co ltd
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2020-11-24
Anticipated expiration: 2038-05-30
Also published as: CN108913376A

Abstract

The application discloses a reagent for cleaning a sequencing chip, and a preparation method and application thereof. The reagent for cleaning the sequencing chip comprises citric alcohol, a saponin extracting solution, potassium hydroxide, EDTA (ethylene diamine tetraacetic acid), epoxidized soybean oil, polyether modified silicone oil, ethanol and pure water. The sequencing chip cleaning reagent is designed aiming at pollution sources such as oil stains of a sequencing chip, and multiple plant extracts are adopted as active detergents, and the components in the reagent are matched with each other to effectively clean the sequencing chip and remove impurities such as oil stains on the surface of the sequencing chip, so that spots are not left after cleaning, the reagent is free from fogging, and the cleaning effect is good. In addition, the reagent does not contain chloride and low aromatic hydrocarbon, the main active component is plant extract, and the reagent is environment-friendly, safe and stable and does not contain any halogen component, so that the ozone layer is not damaged; provides a special cleaning reagent for the sequencing chip.

Description

Reagent for cleaning sequencing chip and preparation method and application thereof

Technical Field

The application relates to the field of sequencing chip cleaning, in particular to a reagent for sequencing chip cleaning and a preparation method and application thereof.

Background

The sequencing chip is cleaned for ensuring the cleanness and no pollution of the sequencing chip and further ensuring the sequencing quality. At present, no special sequencing chip cleaning reagent exists, and corresponding research and report are lacked. In practice, the chip cleaning is usually carried out by using ethanol and isopropanol according to the recommendations of chip manufacturers. The ethanol and the isopropanol have the functions of dissolving and removing organic matters, accelerating volatilization and achieving the effect of cleaning the sequencing chip. However, isopropanol has high toxicity, has obvious anesthetic action, has stimulation effect on mucous membranes of eyes and respiratory tracts, and can damage retina and optic nerve; has been listed in the list of class 3 carcinogens by the world health organization international agency for the study of cancer. In addition, ethanol and isopropanol have poor cleaning effects, are easy to decompose and volatilize, and particularly, can decompose under storage conditions at high temperatures to cause the cleaning agent to lose efficacy.

Therefore, it is necessary to develop a new reagent dedicated to washing sequencing chips to ensure the washing effect and sequencing quality.

Disclosure of Invention

The purpose of the application is to provide a novel reagent for cleaning a sequencing chip, and a preparation method and application of the reagent.

In order to achieve the purpose, the following technical scheme is adopted in the application:

one aspect of the application discloses a reagent for cleaning a sequencing chip, which comprises citric alcohol, saponin extracting solution, potassium hydroxide, EDTA, epoxidized soybean oil, polyether modified silicone oil, ethanol and pure water.

The sequencing chip cleaning reagent is characterized in that the citric alcohol is an alcohol substance, and the nonpolar molecule can dissolve oil stains on the surface of the sequencing chip and is non-toxic and fresh in smell; the saponin extraction liquid is prepared by extracting plant-derived surfactant from saponin, and can reduce the adsorption degree between dirt and objects and remove stains; the sodium hydroxide can hydrolyze the grease into soluble glycerin and higher fatty acid sodium; EDTA can form stable water-soluble complex with alkali metal, rare earth element, transition metal and the like, and is used for removing metal ions; epoxidized soybean oil and polyether modified silicone oil are used as stabilizers, so that the stability of the solution can be improved, and the thermal stability and the light stability of the sequencing chip cleaning reagent are improved; ethanol and pure water are used as basic solvents, and the components are dissolved and mixed together.

This application is used for abluent reagent of sequencing chip, chemical security is stable, does not contain chloride and low aromatic hydrocarbon, and its main active ingredient is plant extracts such as citric alcohol, saponin extract, epoxy soybean oil, and environmental protection, safety, stability, does not contain any halogen composition, consequently can not destroy the ozone layer, and practice proves, adopts this application to be used for abluent reagent of sequencing chip to wash sequencing chip, can effectively get rid of impurity such as surface oil dirt, does not leave the spot after the washing, does not fog, and the cleaning performance is good. Under the same condition, compared with common ethanol and isopropanol cleaning reagents, the sequencing chip cleaning reagent has better cleaning effect on the sequencing chip, so that the sequencing quality can be ensured and improved; in an implementation manner of the present application, in the same sequencing library and sequencing method, the sequencing chip is washed by using the reagent of the present application, and the obtained sequencing Q30 value and FP% are significantly higher than those of the sequencing chip washed by using ethanol and isopropanol. Wherein, the fogging mainly means that droplets are formed on the surface of the chip after cleaning, and the droplets can influence laser focusing photographing in the sequencing process. Therefore, as a reagent for washing a sequencing chip, fogging after washing cannot occur.

Preferably, the reagent for washing the sequencing chip comprises, by mass, 10% -25% of citric alcohol, 5% -14% of Chinese honeylocust fruit extract, 8% -20% of potassium hydroxide, 0.3% -2% of EDTA (ethylene diamine tetraacetic acid), 1% -7% of epoxidized soybean oil, 0.1% -3% of polyether modified silicone oil, 10% -20% of ethanol and 23% -60% of pure water.

Preferably, in one implementation manner of the present application, the reagent for washing the sequencing chip preferably consists of, by mass, 20% of citric alcohol, 8% of saponin extract, 10% of potassium hydroxide, 2% of EDTA, 5% of epoxidized soybean oil, 3% of polyether modified silicone oil, 20% of ethanol, and 32% of pure water.

The key point of the method is that the special reagent for cleaning the sequencing chip is composed of citric alcohol, saponin extract, potassium hydroxide, EDTA, epoxidized soybean oil, polyether modified silicone oil, ethanol and pure water, and the dosage of each component can be adjusted according to the condition of the specifically cleaned sequencing chip; the application provides a formula which is generally applicable to cleaning of various sequencing chips, namely a reagent which consists of 10-25% of citric alcohol, 5-14% of Chinese honeylocust fruit extract, 8-20% of potassium hydroxide, 0.3-2% of EDTA, 1-7% of epoxidized soybean oil, 0.1-3% of polyether modified silicone oil, 10-20% of ethanol and 23-60% of pure water in percentage by mass. Also, a best reference formulation is provided, namely a reagent consisting of, by mass, 20% of citric alcohol, 8% of an extract of Chinese honeylocust fruit, 10% of potassium hydroxide, 2% of EDTA, 5% of epoxidized soybean oil, 3% of polyether modified silicone oil, 20% of ethanol, and 32% of pure water.

It is understood that, firstly, the reagent of the present invention can satisfy the conventional washing of sequencing chips, but other active ingredients or auxiliary ingredients can be added for other purposes or to make the reagent of the present invention have other functions without affecting the washing function of sequencing chips of the reagent of the present invention, and are not specifically limited herein. Secondly, the above specific dosage range or specific dosage ratio is only a ratio with better cleaning effect provided in an implementation manner of the present application, and for other purposes, such as cost saving or poor cleaning effect requirement, each component may be adjusted outside the range of the present application, and is not specifically limited herein.

The other side of the application discloses a reagent preparation method for washing a sequencing chip, which comprises the steps of adding the citric alcohol, the saponin extract, the potassium hydroxide, the EDTA and the ethanol into pure water, carrying out airtight stirring reaction, then adding the epoxidized soybean oil and the polyether modified silicone oil, and uniformly stirring to obtain the reagent for washing the sequencing chip.

The preparation method comprises the steps of adding the citric alcohol, the saponin extracting solution, the potassium hydroxide, the EDTA and the ethanol into pure water, and carrying out closed stirring reaction; wherein, the stirring aims to fully dissolve all components, and the closed environment can prevent the volatilization of the reagent in the stirring process, thereby ensuring the production quality; and finally, adding epoxidized soybean oil and polyether modified silicone oil to improve the stability of the solution, so that the prepared sequencing chip cleaning reagent has better thermal stability and light stability.

Preferably, in the preparation method, the reaction time is 0.5h-1h under closed stirring.

Preferably, in the preparation method, the stirring time for uniformly stirring is 10min to 20 min.

The application also discloses an application of the reagent for sequencing chip cleaning in preparation of an environment-friendly cleaning agent for cleaning glass or hardware.

The reagent for washing the sequencing chip can effectively wash and remove oil stains, so that the reagent can be completely used for washing other materials, such as glass, hardware and the like, besides being used for washing the sequencing chip, and also has the characteristics of environmental protection, safety, stability and the like.

The application discloses a glass or hardware abluent environmental protection cleaner again, contains this application in this environmental protection cleaner and is used for the abluent reagent of sequencing chip.

The reagent for cleaning the sequencing chip can be used for cleaning glass or hardware, so that the reagent can be used for preparing a corresponding environment-friendly cleaning agent; it is understood that other active or auxiliary components may be added to the reagent of the present application for glass or hardware cleaning, which is not specifically limited herein, so as to facilitate the use or add or enhance the function. For example, the agent of the present application may be prepared into a foaming agent for convenience of use, and auxiliary components of the foaming agent may be added accordingly, which is not specifically limited herein.

Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:

the reagent of the application is designed aiming at pollution sources such as oil stains of a sequencing chip, and adopts various plant extracts as active detergents, and all the components in the reagent can be matched with each other to effectively clean the sequencing chip, so that impurities such as the oil stains on the surface of the sequencing chip are removed, spots are not left after cleaning, fogging is avoided, and the cleaning effect is good. In addition, the reagent does not contain chloride and low aromatic hydrocarbon, the main active component is plant extract, and the reagent is environment-friendly, safe and stable and does not contain any halogen component, so that the ozone layer is not damaged; provides a special cleaning reagent for the sequencing chip.

Detailed Description

At present, ethanol and isopropanol recommended by sequencing chip manufacturers are mainly adopted for cleaning sequencing chips, and no special sequencing chip cleaning reagent is available; however, isopropyl alcohol has potential safety hazard in use, is a carcinogenic substance named in international cancer research organization of world health organization, has poor cleaning effect on ethanol and isopropyl alcohol, and can be decomposed to cause the cleaning agent to lose efficacy under the storage condition with high temperature.

Based on the above recognition, the inventors of the present application have specifically analyzed the contaminating components of the sequencing chip to determine the main contamination or residue of the sequencing chip including: dust, lubricating oil steam, dust, Tris and NaCl salt ions, bacterial colonies, viruses and other biological substances in a chip production workshop. In view of the above, the present application develops a reagent capable of effectively dissolving and removing the above residual components, and the reagent is used for cleaning a sequencing chip to improve the cleaning effect; namely, the reagent for washing the sequencing chip comprises the citric alcohol, the saponin extraction solution, the potassium hydroxide, the EDTA, the epoxidized soybean oil, the polyether modified silicone oil, the ethanol and the pure water. The application provides a special cleaning reagent for a sequencing chip, and has the advantages of good cleaning effect, good functional stability under high-temperature storage conditions, environmental protection, safety and the like.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Example one

The reagent for cleaning the sequencing chip comprises, by mass, 10% -25% of citric alcohol, 5% -14% of saponin extract, 8% -20% of potassium hydroxide, 0.3% -2% of EDTA (ethylene diamine tetraacetic acid), 1% -7% of epoxidized soybean oil, 0.1% -3% of polyether modified silicone oil, 10% -20% of ethanol and 23% -60% of pure water; in this example, sequencing chip cleaning reagents of multiple formulations were prepared according to the amounts of the components in table 1, and the surface volatilization time, light transmittance, and intensity a performance of the sequencing chip cleaning reagents were respectively tested, as a comparison, and the surface volatilization time, light transmittance, and intensity a performance of pure water and Illumina recommended cleaning reagents were simultaneously tested, wherein Illumina recommended cleaning reagents are ethanol and isopropanol mixed at equal volumes. In addition, some formulas which also adopt the citric alcohol, the saponin extraction solution, the potassium hydroxide, the EDTA, the epoxidized soybean oil, the polyether modified silicone oil, the ethanol and the pure water as the sequencing chip cleaning reagents are arranged, but the dosage of each component is not completely within the dosage range as a control, and each test and a comparative test are shown in the table 1. The citric alcohol, the saponin extract, the potassium hydroxide, the EDTA and the epoxidized soybean oil were obtained from Yanghui cosmetics, Inc., Shanghai Huilou trade, and the polyether-modified silicone oil was obtained from Aiyota silicone oil, Inc., Ji' an, in this example.

The reagent for washing the sequencing chip in the embodiment is prepared by the following steps:

firstly, cleaning stirring equipment, and preparing pure water, citric alcohol, saponin extract, potassium hydroxide, EDTA, epoxidized soybean oil, polyether modified silicone oil and ethanol according to the proportion in table 1.

And secondly, injecting pure water into a clean stirring device, and simultaneously adding the citric alcohol, the saponin extracting solution, the potassium hydroxide, the EDTA and the ethanol, and then starting closed stirring.

And thirdly, after stirring for 1 hour in a closed manner, adding epoxidized soybean oil and polyether modified silicone oil into stirring equipment, and then stirring for 10 minutes to obtain the reagent for washing the sequencing chip.

The sequencing chip cleaning reagent has the following performance tests:

1. surface volatilization time test

And placing 2 mu L of the sequencing chip cleaning reagent on the surface of the sequencing chip, and measuring the time of complete volatilization of the sequencing chip cleaning reagent so as to characterize the surface volatilization time of the sequencing chip cleaning reagent. The test results are shown in table 1.

For comparison, 2. mu.L of pure water and 2. mu.L of Illumina recommended wash reagent were also placed on the surface of the sequencing chip, and the time to complete volatilization was measured, thereby characterizing the respective surface volatilization times. The test results are shown in table 1.

2. Light transmittance test

And (3) carrying out light transmittance test on the sequencing chip by using an OU4220 visible/infrared/ultraviolet light transmittance instrument, specifically, starting up for 10min, and placing the sequencing chip into a measurement port of the OU4220 visible/infrared/ultraviolet light transmittance instrument for testing. Detailed test methods refer to the OU4220 instructions for use. The specific technical parameters are as follows:

the application range is as follows: glass, coating materials, organic materials and coatings; measurement range: 0 to 100 percent; light source: infrared light, visible light, near ultraviolet light; resolution ratio: 0.1 percent; repeatability: 0.2 percent; indicating value error: 2 percent; working voltage: a fifth dry cell (AA) or rechargeable battery; area measurement: 13mm minimum; and (3) measuring the thickness: 20mm maximum; ambient temperature: 0 to 40 ℃.

In this example, the light transmittances of 10 sequencing chips before washing were respectively detected to be 44.5%, 46.1%, 45.1%, 44.1%, 43.6%, 43.9%, 43.8%, 44.1%, 44.2%, 44%, and the average was 44.34%.

Then, the sequencing chips were washed with the respective washing reagents for the sequencing chips, and the washing reagents recommended for pure water and Illumina, and the light transmittance of each washed sequencing chip was measured, and the results are shown in table 1.

The cleaning mode of the sequencing chip is as follows: dipping the cleaning reagent in kimtech dust-free paper, slightly wiping the sequencing chip by the dust-free paper dipped with the cleaning reagent, completing wiping, and measuring the light transmittance after the cleaning reagent is dried.

3. intensity A test

The light intensity of the basic group of the chip constant engineering area A, namely intensity A, is an important parameter for reflecting the light transmission reading of the sequencing chip. In the embodiment, the light intensity of the A base in the same area obtained in the photographing process of the BGISEQ-500 sequencer is used as the intensity A value of the sequencing chip.

In this example, the intensity A values of the sequencing chips washed with the respective sequencing chip washing reagents, and pure water and Illumina recommended washing reagent were measured, and the results are shown in Table 1. The washing mode of the sequencing chip is the same as that of the 2 light transmittance test.

TABLE 1 sequencing chip cleaning reagent and its performance test results

The results in table 1 show that the sequencing chip cleaning reagent containing 10-25% of the mass fraction of the citric alcohol, 5-14% of the mass fraction of the saponin extract, 8-20% of the mass fraction of the potassium hydroxide, 0.3-2% of the mass fraction of the EDTA, 1-7% of the mass fraction of the epoxidized soybean oil, 0.1-3% of the mass fraction of the polyether modified silicone oil, 10-20% of the mass fraction of the ethanol and 23-60% of the mass fraction of the pure water has better comprehensive performance in all aspects; particularly, each property of the formula 14 is optimal; formula 14 has a surface volatilization time of 3s, except that the surface volatilization time of formula 14 is 1s longer than that of the Illumina recommended cleaning agent, both the light transmittance and intensity a values are significantly better than that of the Illumina recommended cleaning agent, and the surface volatilization time of 3s is also acceptable.

The formulas 29 and 30 have longer volatilization time due to higher content of the polyether modified silicone oil; also, formulation 30 had a lower light transmittance. Formula 34 has better light transmittance and intensity a value, but has longer surface volatilization time due to higher content of epoxidized soybean oil. The citric alcohol content of formula 35 was too low and that of formula 36 was too high, so both the light transmittance and the intensity a value were significantly poorer; the saponin extract of formula 37 has a low content, so that the intensity A value is low; the saponin extract of formula 38 has a slightly higher content, so that the light transmittance is slightly lower; it can be seen that the formulations 29, 30, 34-38 were not in the preferred ranges of the present application due to their partial components, so that the sequencing chips were all poorly cleaned. In addition, pure water for comparison has a long surface volatilization time; the Illumina recommended cleaning reagent has good detection effects, but has the problems of high toxicity, easy volatilization, decomposition and the like.

Example two

In this example, the sequencing chip cleaning reagent (hereinafter referred to as formula 14) of the formula of test 14 in the first example was used for the test, and meanwhile, the same Illumina recommended cleaning reagent in the first example was used for comparison to compare the influence of the sequencing chips cleaned by different reagents on the subsequent sequencing quality. Specifically, this example was tested using three libraries, each of the phix control libraries, identified by the illumina kit cat No.: FC-110 and 3001. Each library was divided equally into two parts, one part was sequenced using the sequencing chip washed with the sequencing chip washing reagent of formula 14, and the other part was sequenced using the sequencing chip washed with the Illumina recommended washing reagent. The method for washing the sequencing chip is the same as that of the first embodiment. Except that the reagents used for washing the sequencing chip are different, the sequencing chip, the sequencing reagents, the sequencing platform and the sequencing method are the same.

After the sequencing was completed, the Q30 value and FP% value of each sequencing off-line data were counted. Wherein Q30 is a mass value of sequencing; q30 mathematically means that the probability of misidentification is 0.1%, i.e. the error rate is 0.1%, or the accuracy is 99.9%; in the sequencing evaluation, in order to evaluate the accuracy of the off-line reads, Q30 and the proportion of all base quality values larger than Q30 are evaluated, and the higher the value is, the better the sequencing quality is. The FP% value, passfilter, is the percentage of clusters that meet the criteria, correlated with the final data volume throughput of the sequencing; the higher this number reflects better sequencing quality and the greater the amount of data obtained.

The statistical results are shown in table 2.

TABLE 2Q 30 values and FP% values for each library sequencing

Test group	Q30	FP％
			Library one: illumina recommended washing reagent	74.47	83.9％
Library one: formulation 12	83.1	90.3％
			Library two: illumina recommended washing reagent	70.6	84.5％
Library two: formulation 12	83.5	90.8％
			Library three: illumina recommended washing reagent	86.6	86.9％
Library three: formulation 12	90.8	96.2％

In table 2, "library one: illumina recommended cleaning reagent "refers to a method in which a sequencing chip cleaned by Illumina recommended cleaning reagent is used for sequencing, and the sequence of a library i: formula 12 "means that the first library was sequenced using the sequencing chip washed with the sequencing chip washing reagent of formula 12, and so on for the second library and the third library.

The results in table 2 show that in the three libraries, the sequencing chip washed with the sequencing chip washing reagent of formula 12 was used for sequencing, and both the Q30 value and the FP% value were evident due to the washing of the sequencing chip with the Illumina recommended washing reagent; the sequencing chip cleaning reagent with the formula 12 can be used for cleaning a sequencing chip better and cleaner and more thoroughly, so that better guarantee is provided for subsequent sequencing.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

Claims

1. A reagent for washing a sequencing chip, comprising: comprises citric alcohol, fructus Gleditsiae Abnormalis extractive solution, potassium hydroxide, EDTA, epoxidized soybean oil, polyether modified silicone oil, ethanol and pure water;

the mass fraction of the citric alcohol is 10% -25%, the mass fraction of the saponin extraction solution is 5% -14%, the mass fraction of the potassium hydroxide is 8% -20%, the mass fraction of the EDTA is 0.3% -2%, the mass fraction of the epoxidized soybean oil is 1% -7%, the mass fraction of the polyether modified silicone oil is 0.1% -3%, the mass fraction of the ethanol is 10% -20%, and the mass fraction of the pure water is 23% -60%.

2. The reagent according to claim 1, characterized in that: the mass fraction of the citric alcohol is 20%, the mass fraction of the saponin extract is 8%, the mass fraction of the potassium hydroxide is 10%, the mass fraction of the EDTA is 2%, the mass fraction of the epoxidized soybean oil is 5%, the mass fraction of the polyether modified silicone oil is 3%, the mass fraction of the ethanol is 20%, and the mass fraction of the pure water is 32%.

3. The method for preparing a reagent according to claim 1 or 2, characterized in that: adding the citric alcohol, the saponin extracting solution, the potassium hydroxide, the EDTA and the ethanol into pure water, stirring for reaction in a closed manner, then adding the epoxidized soybean oil and the polyether modified silicone oil, and stirring uniformly to obtain the reagent for cleaning the sequencing chip.

4. The production method according to claim 3, characterized in that: the reaction time is 0.5h-1h under closed stirring.

5. The production method according to claim 3 or 4, characterized in that: the stirring time for stirring uniformly is 10min-20 min.