Application of synthetic sequencing technology in space transcriptome coding microsphere chip

Abstract

The invention relates to the field of biological sequencing, in particular to application of a synthetic sequencing technology to a space transcriptome coding microsphere chip. The sequence of the microsphere probe is determined by adopting a synthetic sequencing technology, and the main implementation thought is as follows: microspheres with the probe are densely laid on a carrier, a primer is added to be hybridized with the probe, and the probe sequence on the microspheres is obtained by utilizing a synthetic sequencing reaction. Specifically, after a microsphere probe on a carrier is incubated through a sequencing primer, a reversible terminator with four-color fluorescence is added, a nucleotide is synthesized in a reaction buffer solution under the action of amplification enzyme, then fluorescence is excited through laser to obtain an optical signal, information of a basic group is obtained, and the detection result is obtained. And then eluting the fluorescence signal and the blocking group on the reversible terminator, and entering a new round of reaction to obtain the information of the next basic group. The experimental scheme is simple and convenient, the time is effectively saved, the cost is reduced, and the probe sequence on the microsphere can be efficiently and accurately determined.

Description

Technical Field

[0001] The present invention relates to the field of biological sequencing, and in particular to the application of synthetic sequencing technology on a spatial transcriptome encoding microsphere chip. Background Art

[0002] Idle-coding microsphere chip probe sequencing technology is a method that combines microsphere technology and nucleic acid probe hybridization technology for high-throughput detection and analysis of DNA or RNA sequences. This technology has a wide range of applications in molecular biology, clinical diagnosis, environmental monitoring and other fields. The following are some existing microsphere probe sequencing technologies: 1. Luminex xMAP technology: Principle: Luminex xMAP (Multiplexed Analyte Profiling) technology uses microspheres with different fluorescence codes, each of which carries a specific DNA or RNA probe. After the target nucleic acid in the sample hybridizes with the probe, the signal is read by the fluorescence detector to achieve simultaneous detection of multiple targets. Application: Widely used in genotyping, pathogen detection, SNP (single nucleotide polymorphism) analysis and other fields. 2. Flow Cytometry: Principle: Use flow cytometry to detect the fluorescent signal on the microsphere. Specific nucleic acid probes are fixed on the surface of the microsphere. After hybridization with the target nucleic acid in the sample, the fluorescence is excited by the laser of the flow cytometer, and the signal is read and analyzed. Application: Commonly used for the detection of cell surface markers, quantitative analysis of intracellular nucleic acids, etc. 3. QuantiArray microsphere array technology: Principle: QuantiArray technology fixes different types of microspheres on an array, and each microsphere carries a specific nucleic acid probe. After the target nucleic acid in the sample hybridizes with the probe, the signal is read by a fluorescent scanner to achieve high-throughput sequence determination. Application: Mainly used for gene expression profiling, mutation detection, etc. 4. NanoString nCounter technology: Principle: NanoString nCounter technology uses barcoded probes, which form complexes after hybridization with the target nucleic acid. The barcode signal is read by an optical imaging system to achieve quantitative analysis of the target nucleic acid. Application: Widely used in gene expression analysis, miRNA detection, pathogen identification, etc. 5. Microarray technology: Principle: Although it is not a traditional microsphere technology, microarray technology can also be regarded as a high-density microsphere probe system. A large number of nucleic acid probes are fixed on a solid support, hybridized with the target nucleic acid in the sample, and the signal is read by a fluorescent scanner to achieve high-throughput sequence determination. Application: Widely used in gene expression profiling, genomics research, etc. The high-throughput detection equipment and reagents involved in the above technologies are relatively expensive. The operation steps are relatively complicated and require professional technicians to perform operations and data analysis. Summary of the invention

[0003] In view of this, the present invention provides the application of synthetic sequencing technology on spatial transcriptome encoding microsphere chips. The present invention uses synthetic sequencing technology to determine the sequence of microsphere probes, and its main implementation idea is: the probe microspheres are densely spread on the carrier, primers are added to hybridize with the carrier probes, and the probe sequences are obtained by synthetic sequencing reaction. Specifically, a reversible terminator with four-color fluorescence is added to the probe microspheres on the carrier, and a nucleotide is synthesized in the reaction buffer under the action of an amplification enzyme, and then a light signal is obtained by laser excitation fluorescence to obtain the information of this base, and then the fluorescent signal and the blocking group on the reversible terminator are eluted, and a new round of reaction is entered to obtain the next base information. The present invention has a simple experimental scheme, which effectively saves time and reduces costs, and can efficiently and accurately complete the determination of the probe sequence.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a sequencing method, comprising the following steps:

[0006] S1: connecting the microspheres coupled with the probes to the carrier to obtain a microsphere chip;

[0007] S2: hybridizing the hybridization primer with the probe to obtain a probe-primer complex;

[0008] S3: four nucleotides carrying different optical detection labels are combined with the probe-primer complex through a polymerization reaction, and single base extension is performed to obtain an extension product;

[0009] S4: exciting the optical detection label to generate an optical signal, imaging the extension product, and obtaining an image;

[0010] S5: Clear the optical detection mark;

[0011] S6: Repeat S3 to S5 until sequencing is completed;

[0012] S7: melt the chain and obtain sequencing results;

[0013] The probe comprises: a sequence to be detected and a fixed sequence; the fixed sequence has a sequence as shown in SEQ ID NO: 1;

[0014] The hybridization primer and the fixed sequence have complementary sequences.

[0015] In some embodiments of the present invention, the sequence of SEQ ID NO: 1 is: TAGCTGCTTCAGCGT.

[0016] In some embodiments of the present invention, in the above sequencing method, the number of the microspheres is greater than or equal to 1.

[0017] In some embodiments of the present invention, in the above sequencing method, when the microspheres are different, the sequences to be detected are different.

[0018] In some embodiments of the present invention, in the above sequencing method, the optical detection label is selected from DAPI, FITC, Alexa fluor 488, AF532, AF700, Cy3, Cy5, TRITC, and Cy7.

[0019] In some embodiments of the present invention, the connection in the above sequencing method S1 is performed by centrifugation at a speed of 1000 g for 10 min.

[0020] In some embodiments of the present invention, the hybridization time in the above sequencing method S2 is 5 min to 10 min, and the temperature is 40 to 44°C.

[0021] In some embodiments of the present invention, the hybridization time in the above sequencing method S2 is 5 minutes and the temperature is 42°C.

[0022] In some embodiments of the present invention, the amplification in the above sequencing method S3 uses an amplification buffer, an amplification enzyme and dNTPs.

[0023] In some embodiments of the present invention, the amplification buffer of the above sequencing method comprises: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2~10 mM MgSO4, and 0.05~0.2% (w / w) Triton X-100.

[0024] In some embodiments of the present invention, the dNTPs in the above sequencing method carry a fluorescent group and a blocking group.

[0025] In some embodiments of the present invention, the fluorescent group and / or the blocking group in the above sequencing method is modified with an azide group or a terminal disulfide bond.

[0026] In some embodiments of the present invention, the fluorescent group and / or the blocking group in the above sequencing method is modified with an azide group.

[0027] In some embodiments of the present invention, the cleaning time in the above sequencing method S4 is 1 min to 5 min, and the temperature is 42 to 65°C.

[0028] In some embodiments of the present invention, the cleaning time in the above sequencing method S4 is 1 min and the temperature is 42-65°C.

[0029] In some embodiments of the present invention, the cleaning in the above sequencing method S4 uses 25mM~50mM TCEP.

[0030] In some embodiments of the present invention, the cleaning in the above sequencing method S4 uses 50 mM TCEP.

[0031] The present invention has a simple experimental scheme, effectively saves time, reduces costs, and can efficiently and accurately complete the determination of the probe sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0033] Figure 1 1. The sequence flow chart of the present invention is shown;

[0034] Figure 2 Schematic diagram of the carrier is shown;

[0035] Figure 3 Schematic diagram of the gasket frame is shown;

[0036] Figure 4 Figure 4 shows the test of amplification reaction conditions, and the effect of different reaction buffers on the imaging effect was tested. A black and white camera was used to record the fluorescence signal. The images shown in ABC are the signals released by the bases labeled with Cy5 fluorescent group. Among them: A shows that the reaction conditions are 20 mM Tris-HCl; 10 mM (NH4)2SO4; 10 mM KCl; 2 mM MgSO4; 0.1% Triton® X-100, and the imaging effect is poor; B shows that the reaction conditions are 20 mM Tris-HCl; 10 mM (NH4)2SO4; 10 mM KCl; 2 mMMgSO4; 0.05% Triton® X-100, and the imaging effect is good; C shows that the reaction conditions are 20 mM Tris-HCl; 10 mM (NH4)2SO4; 10 mM KCl; 2 mM MgSO4; 0.2% Triton® X-100, and the imaging effect is general.

[0037] Figure 5 The figure shows the effect of test temperature on the elution of fluorescent groups and blocking groups. A black and white camera was used to take pictures and record the intensity of the Cy5 residual fluorescence signal. Among them: A shows elution at 42°C, with a poor effect; B shows elution at 55°C, with a good effect; C shows elution at 65°C, with a general effect. DETAILED DESCRIPTION

[0038] The invention discloses the application of synthetic sequencing technology on a spatial transcriptome encoding microsphere chip.

[0039] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0040] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0041] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0042] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0043] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0044] The present invention provides a method for microsphere sequencing, which specifically comprises the following steps:

[0045] (1) Preparation of microsphere carriers: Spreading microspheres with specific probes on the carrier;

[0046] (2) Microsphere primer hybridization: Add amplification primers and buffer to hybridize with the chip microsphere probes;

[0047] (3) Amplification: Add an amplification system containing dNTPs with fluorescent groups and reversible blocking groups to amplify a single nucleotide;

[0048] (4) Fluorescence photography: Capture the fluorescence signal generated by laser-excited fluorescent groups through a camera;

[0049] (5) Elution: eluting the fluorescent group and blocking group;

[0050] (6) Repeat the above amplification-fluorescence photography-elution process until the target sequence is determined;

[0051] (7) Melting: Add melting reagent to remove the synthetic chain and finally obtain the sequence information of the microsphere probe.

[0052] In Example 1 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0053] The present invention will be further described below in conjunction with embodiments:

[0054] Example 1

[0055] 1. Preparation of microsphere chip: Place a gasket frame on an acrylic polymer double-sided adhesive carrier, add microspheres into the gasket frame, and make them adhere to the carrier by centrifugation.

[0056] (1) About microspheres:

[0057] This embodiment uses PMMA microspheres with a size of 10 μm;

[0058] The microspheres carry probes with the same structure. The probe sequence includes a segment of random bases and a segment of fixed bases. The length and fixed sequence information can be customized. The sequence used in this embodiment is 5'-NNNNNNNNNNNNNNNTAGCTGCTTCAGCGT-3';

[0059] The random base sequences of the probes carried by the same microsphere are the same, and the random base sequences of the probes carried by different microspheres are different;

[0060] The probe sequence can be generated by solid phase synthesis technology. Assuming the number of random bases is n, the corresponding number of probe types is 4 to the power of n;

[0061] (2) About the carrier:

[0062] The carrier is a sheet material with an adhesive layer, and this embodiment adopts an acrylic polymer double-sided adhesive carrier;

[0063] The carrier size is not limited, this embodiment uses 25cm*75cm. Figure 2 shown.

[0064] (3) About the gasket frame

[0065] The gasket frame material can be made of silicone;

[0066] The specifications of the gasket frame are not limited. In this embodiment, a square frame is used with an outer side length of 6.5 cm and an inner side length of 6 cm. Figure 3 shown.

[0067] (4) About centrifugal

[0068] The centrifugal force was 1000 g, the centrifugal time was 10 min, and the temperature was room temperature.

[0069] 2. Microsphere primer hybridization: Add hybridization buffer, hybridize the hybridization primer with the 3' end of the microsphere probe, the reaction temperature is 42°C, and the reaction time is 5 minutes;

[0070] The hybridization buffer was 4xSSC;

[0071] The hybridization primer sequence was 5'-ACGCTGAAGCAGCTA-3' (as shown in SEQ ID NO: 2), and the concentration was 1 μM.

[0072] 3. Amplification: Add amplification buffer, amplification enzyme, dNTP with fluorescent group and reversible blocking group, synthesize a nucleotide on the probe mother chain, the reaction temperature is 55℃, and the reaction time is 1min

[0073] The amplification enzyme used was Therminator DNA polymerase (NEB);

[0074] Amplification buffer is:

[0075] 20mM Tris-HCl, 10mM (NH4)2SO4, 10mM KCl, 2.5mM MgSO4, 0.1% Triton® X-100;

[0076] Both the fluorescent group and the blocking group of dNTP are modified with azide groups

[0077] The fluorescent groups of the nucleotides are selected from AF532, AF700, Cy3, and Cy5, and each nucleotide is labeled with a different fluorescent group;

[0078] The working concentration of dNTPs was 200 μM;

[0079] The amplification reaction conditions are shown in Table 1. The effects of temperature, buffer, and reaction time on fluorescence imaging are mainly tested. Figure 4 shown.

[0080] Table 1

[0081]

[0082] 4. Fluorescence photography: The dNTP with fluorescent groups on the probe is excited by laser, and the released fluorescent signal is captured by the camera and converted into base information through an algorithm.

[0083] 5. Elution: Elute the fluorescent group and blocking group at a reaction temperature of 55°C for 1 min and an elution reagent of 50 mM TCEP.

[0084] The conditions of the elution test temperature are shown in Table 2, and the results are as follows Figure 5 shown.

[0085] Table 2

[0086]

[0087] 6. Repeat the above amplification-fluorescence photography-elution process until the target sequence is determined.

[0088] 7. Melting: Add melting reagent to remove the synthetic chain. The reaction temperature is 30°C and the reaction time is 5 min. The melting reagent is 0.2 mol NaOH.

[0089] 8. Examples of some results obtained from sequence measurement, where ycoord and xcoord represent the relative coordinate position information of the microspheres:

[0090] Table 3

[0091]

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A sequencing method, characterized in that The steps include: S1: connecting the microspheres coupled with the probes to the carrier to obtain a microsphere chip; S2: hybridizing the hybridization primer with the probe to obtain a probe-primer complex; S3: four nucleotides carrying different optical detection labels are combined with the probe-primer complex through a polymerization reaction, amplified, and an extension product is obtained; S4: exciting the optical detection label to generate an optical signal, imaging the extension product, and obtaining an image; S5: Clear the optical detection mark; S6: Repeat S3 to S5 until sequencing is completed; S7: melt the chain and obtain the sequencing results; The probe comprises: a sequence to be detected and a fixed sequence; the fixed sequence has a sequence as shown in SEQ ID NO: 1; The hybridization primer and the fixed sequence have complementary sequences.

2. The sequencing method according to claim 1, characterized in that The number of the microspheres is greater than or equal to one.

3. The sequencing method according to claim 1 or 2, characterized in that: The optical detection label is selected from DAPI, FITC, Alexa fluor 488, AF532, AF700, Cy3, Cy5, TRITC, and Cy7.

4. The sequencing method according to any one of claims 1 to 3, characterized in that The connection in S1 is carried out by centrifugation at a speed of 1000 g for 10 min.

5. The sequencing method according to any one of claims 1 to 4, characterized in that: The hybridization time described in S2 is 5 min to 10 min, and the temperature is 40 to 44°C.

6. The sequencing method according to any one of claims 1 to 5, characterized in that: The amplification described in S3 uses an amplification buffer, an amplification enzyme, and dNTPs.

7. The sequencing method according to claim 6, characterized in that The amplification buffer comprises: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2~10 mM MgSO4, and 0.05~0.2% (w / w) Triton X-100.

8. The sequencing method according to claim 6 or 7, characterized in that: The dNTP carries a fluorescent group and a blocking group.

9. The sequencing method according to any one of claims 1 to 8, characterized in that: The cleaning time in S4 is 1 min to 5 min, and the temperature is 42 to 65°C.

10. The sequencing method according to claim 9, characterized in that The clearance described in S4 used 25 mM~50 mM TCEP.

Images