Kit for detecting expression typing and expression quantity of HLA-I / II type genes at single cell level

Abstract

The utility model discloses a kit for detecting HLA-I / II type gene expression typing and expression quantity at single cell level, which comprises a kit body and a flip type kit cover, a paper support is arranged in the kit body and divides the interior into an upper layer and a lower layer, an enrichment reagent area and a library building reagent area are arranged on the paper support, the enrichment reagent area contains more than six test tube holes, and the library building reagent area contains more than six test tube holes. An HLA (human leukocyte antigen) Target Mix1, an HLA Target Mix2, a 2X Multiplex PCR (polymerase chain reaction) Master Mix, deionized water and a purification magnetic bead reagent tube are arranged in the kit; the library building reagent area comprises more than 17 test tube holes, a fragmentation and terminal repair system, a connection system and PCR Mix, universal primers, sample index primers, deionized water and purified magnetic bead reagent tubes required by library amplification are arranged in the test tube holes, and all the reagent tubes are provided with tube covers. The method is simple to operate and stable in process, and has the advantages of economic benefit and rapid detection.

Description

Technical Field

[0001] This invention relates to a kit for detecting HLA class I / II gene expression typing and expression levels at the single-cell level, belonging to the field of kits. Background Technology

[0002] High-throughput sequencing technology represents a revolutionary change from traditional sequencing, capable of sequencing hundreds of thousands to millions of DNA molecules at once. Therefore, some literature refers to it as next-generation sequencing, highlighting its epoch-making nature. Furthermore, high-throughput sequencing makes it possible to perform detailed and comprehensive analyses of the transcriptome and genome of a species, hence it is also known as deep sequencing. The advent of high-throughput sequencing technology can be considered a landmark event in the field of genomics research. This technology has drastically reduced the cost of single-base sequencing of nucleic acids compared to first-generation sequencing technologies. For example, the Human Genome Project, conducted at the end of the last century, cost $3 billion to decode the human genome, while second-generation sequencing has brought human genome sequencing into the era of tens of thousands of dollars. Such low single-base sequencing costs allow us to implement genome projects for more species, thereby deciphering the genetic code of more organisms. In species whose genome sequences have been determined, large-scale whole-genome resequencing of other varieties of the same species has become possible, providing an effective tool for studying complex polymorphic regions in the human genome (such as VDJ and HLA).

[0003] The Major Histocompatibility Complex (MHC) is a collective term for a group of genes encoding major histocompatibility antigens in animals. In 1958, Dausset et al. discovered that different specific leukocyte antibodies existed in the serum of patients who had received multiple blood transfusions, multiparous women, and volunteers immunized with allogeneic leukocytes. These antibodies were used to identify many different specific leukocyte antigens, termed Human Leukocyte Antigens (HLA). Through pedigree and population genetic analysis, it was found that the human MHC is located on chromosome 6 and is called the HLA complex. MHC can be divided into two categories: classical MHC and non-classical MHC. Classical MHC includes MHC I, MHC II, and MHC III genes, encoding MHC I, MHC II, and MHC III molecules, respectively.

[0004] Current research on MHC primarily focuses on class I and class II bulk MHC. However, a review of relevant kits reveals only those capable of detecting specific portions of MHC, such as exons 2-4 of class I A gene, exons 2-4 of class I B gene, and exon 2 of the DRB1 gene, as described in patent CN101892317B; and the full lengths of class I A, B, and C genes, as described in patent CN105039332B. Extensive literature review indicates significant polymorphism in HLA class I genes, showing polymorphism in exons 1-7; class II genes are concentrated in exons 2-4 of six genes: DRB1, DRA1, DPA1, DPB1, DQA1, and DQB1. Currently, no kits offering such broad coverage of MHC transcripts have been found.

[0005] Existing research suggests that differences in bulk MHC genotypes may also be observed at the single-cell level, and single-cell gene expression analysis has significant advantages in studying MHC and its relationship with immune diseases and tumors. However, there are currently no commercially available HLA typing kits specifically developed for single cells. Most HLA typing studies on single cells in the literature use scRNA-Seq data, which requires a large amount of sequencing data to obtain accurate single-cell HLA typing information, increasing unnecessary costs. Utility Model Content

[0006] The purpose of this invention is to provide a kit for detecting HLA class I / II gene expression typing and expression levels at the single-cell level.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by this utility model is as follows:

[0008] A kit for detecting HLA class I / II gene expression genotyping and expression levels at the single-cell level includes a box body and a flip-top lid rotatably connected to the box body. The box body has a paper tray that divides the box body into upper and lower layers. The paper tray has an enrichment reagent area and a library preparation reagent area. The enrichment reagent area has more than 6 test tube wells, and the library preparation reagent area has more than 17 test tube wells. The enrichment reagent area contains wells containing HLA Target Mix 1 tubes for HLA-I / II gene capture, HLA Target Mix 2 tubes for HLA-I / II gene capture, 2X Multiplex PCR Master Mix tubes, deionized ultrapure water, and purified magnetic beads. The library preparation reagent area contains wells containing fragmentation and end-repair enzyme tubes and buffer tubes, 1X TE Buffer tubes, ligation adapter tubes, ligase tubes, ligation buffer tubes, and 2X PCR Mix tubes, Universal Primer tubes, Sample Index Primer tubes, deionized ultrapure water, and purified magnetic beads for library amplification. All tubes are capped.

[0009] All the test tube wells are not arranged in an overlapping manner; each test tube well corresponds to one test tube. HLA Target Mix 1 tube refers to a tube containing HLA Target Mix 1 for HLA-I / II gene capture; HLA Target Mix 2 tube refers to a tube containing HLA Target Mix 2 for HLA-I / II gene capture; 2X Multiplex PCR Master Mix tube refers to a tube containing 2X Multiplex PCR Master Mix; Fragmentation and End Repair Enzyme tube refers to a tube containing fragmentation and end repair enzyme; Fragmentation and End Repair Buffer tube refers to a tube containing fragmentation and end repair buffer; 1X TE Buffer tube refers to a tube containing 1X TE Buffer; Ligator tube refers to a tube containing ligator; Ligase tube refers to a tube containing ligase; Ligation Buffer tube refers to a tube containing ligation buffer; 2X PCR Mix tube refers to a tube containing 2X PCR Mix; Universal Primer tube refers to a tube containing Universal Primer; SampleIndex Primer tube refers to a tube containing Sample Index... Primer test tubes; deionized ultrapure water test tubes refer to test tubes containing deionized ultrapure water; purified magnetic bead test tubes refer to test tubes containing purified magnetic beads.

[0010] To save materials, reduce volume, and meet daily needs, all test tubes are 1.5mL tubes.

[0011] To save materials, reduce size, and facilitate use, the reagent kit has a cubic structure with a length of 20±1cm, a width of 15±1cm, and a height of 12±1cm.

[0012] To simplify the structure, save costs, and ensure the stability of the device, the paper tray includes a support surface and support feet arranged around the bottom of the support surface. The support feet are located on the bottom surface of the assembly. The enrichment reagent area and the library preparation reagent area are located on the support surface.

[0013] In this application, terms such as "top," "bottom," and "upper" all refer to the relative positions of the device during normal use.

[0014] As another improvement of this application, the paper tray includes an upper support surface, a lower support surface, and a support ring. The support ring connects the upper and lower support surfaces into a single structure. The lower support surface is a sponge layer and contacts the bottom surface of the inner box. The enrichment reagent area and the library preparation reagent area are located on the upper support surface. This provides better protection for each test tube.

[0015] For ease of use and better protection of the reagents, the paper tray has 23-26 test tube wells. The inner surface of each well has a first elastic layer, and all wells have a diameter of 9mm. Any extra wells are for future use.

[0016] For ease of differentiation and operation, preferably, all reagent tubes in the enrichment reagent area, except for ion-rich ultrapure water and purified magnetic beads, have yellow caps; all reagent tubes in the library construction reagent area, except for ion-rich ultrapure water and purified magnetic beads, have green caps; the top of the deionized ultrapure water test tube has a colorless cap; and the top of the purified magnetic bead test tube has a red cap.

[0017] Further optimization involves the following wells in the enrichment reagent area: one HLATarget Mix 1 tube for HLA-I / II gene capture, one HLA Target Mix 2 tube for HLA-I / II gene capture, one 2X Multiplex PCR Master Mix tube, two deionized ultrapure water tubes, and one purification magnetic bead tube. All tubes are equipped with caps.

[0018] Further optimization involves the following wells in the library preparation reagent area: one tube for fragmentation and end-repair enzyme, one tube for fragmentation and end-repair buffer, one tube for 1X TE buffer, one tube for ligation adapter, one tube for ligase, one tube for ligation buffer, one tube for 2X PCR Mix, one tube for Universal Primer, six tubes for Sample Index Primer, two tubes for deionized ultrapure water, and one tube for purified magnetic beads. All tubes are equipped with caps.

[0019] Any technologies not mentioned in this utility model are based on existing technologies.

[0020] This kit for single-cell level detection of HLA class I / II gene expression typing and expression levels makes sequencing libraries more targeted, fully revealing the HLA polymorphism and combination status of each cell. The operation method is simple and the process is stable, which can bring considerable economic benefits and achieve rapid detection. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the kit for detecting HLA class I / II gene expression typing and expression levels at the single-cell level according to this invention;

[0022] Figure 2 This is a schematic diagram of the paper tray structure of the kit for single-cell level detection of HLA class I / II gene expression typing and expression level according to this utility model;

[0023] In the diagram: 1. Box body; 2. Box lid; 3. Paper tray; 3-1. Fragmentation and end-repair enzyme test tube; 3-2. Fragmentation and end-repair buffer test tube; 3-3. 1X TE Buffer test tube; 3-4. Ligation adapter test tube; 3-5. Ligase test tube; 3-6. Ligation buffer test tube; 3-7. Library amplification 2X PCR Mix test tube; 3-8. Universal Primer test tube; 3-9. Sample Index Primer test tube; 3-10. Deionized ultrapure water test tube; 3-11. Purification magnetic bead test tube; 3-12. HLA Target Mix 1 test tube for HLA-I / II gene capture; 3-13. HLA Target Mix 2 test tube for HLA-I / II gene capture; 3-14. 2X Multiplex PCR Master Mix test tube; 3-15. Deionized ultrapure water test tube; 3-16. Purification magnetic bead test tube.

[0024] Figure 3 A schematic diagram illustrating the Labchip detection of fragment sizes in next-generation sequencing libraries;

[0025] Figure 4 Comparison of HLA-I / II genotyping results with mainstream single-cell level gene expression detection reagents on the market;

[0026] Figure 5 Comparison of HLA-I / II genotype expression levels with mainstream single-cell level gene expression detection reagents on the market;

[0027] Figure 6 Single-cell level detection of HLA class I / II gene expression genotyping and expression level distribution map;

[0028] Figure 7 A violin plot showing the expression distribution of a certain HLA subtype in different cell types. Detailed Implementation

[0029] To better understand this utility model, the following embodiments further illustrate the content of this utility model, but the content of this utility model is not limited to the following embodiments.

[0030] Example 1

[0031] like Figure 1-2 As shown, a kit for single-cell level detection of HLA class I / II gene expression typing and expression level is disclosed. The kit has a cubic structure, with a length of 20±1cm, a width of 15±1cm, and a height of 12±1cm. The kit includes: a box body and a flip-top lid rotatably connected to the box body. Inside the box body is a paper tray that divides the box body into upper and lower layers. The paper tray includes a support surface and support feet arranged around the bottom of the support surface, which are located on the bottom surface of the box body. The support surface has an enrichment reagent area and a library construction reagent area. The enrichment reagent area has more than 6 test tube wells, the inner surface of which has a second elastic layer, and the pore diameter of each test tube well is 9mm. The library construction reagent area has more than 17 test tube wells, the inner surface of which has a second elastic layer, and the pore diameter of each test tube well is 9mm.

[0032] like Figure 1-2As shown, a kit for single-cell level detection of HLA-I / II gene expression genotyping and expression levels includes: In the enrichment reagent section, each well contains one HLA Target Mix 1 tube for HLA-I / II gene capture, one HLA Target Mix 2 tube for HLA-I / II gene capture, one 2X Multiplex PCR Master Mix tube, two deionized ultrapure water tubes, and one purified magnetic bead tube; In the library preparation reagent section, each well contains one fragmentation and end-repair enzyme tube, one fragmentation and end-repair buffer tube, one 1X TE buffer tube, one ligation adapter tube, one ligase tube, one ligation buffer tube, and for library amplification, one 2X PCR Mix tube, one Universal Primer tube, six Sample Index Primer tubes, two deionized ultrapure water tubes, and one purified magnetic bead tube.

[0033] All test tubes are equipped with caps. The remaining wells are reserved for future kit upgrades and to accommodate additional reagent wells. All test tubes are 1.5 mL. Different colors are used to distinguish different reagents by their caps for ease of use. All tubes in the enrichment reagent area (except for ion-enriched ultrapure water and purified magnetic beads) have yellow caps, while all tubes in the library construction reagent area (except for ion-enriched ultrapure water and purified magnetic beads) have green caps. Deionized ultrapure water tubes have a colorless cap, and purified magnetic bead tubes have a red cap.

[0034] Example 2

[0035] The paper tray is basically the same as in Example 1, except that: the paper tray includes an upper support surface, a lower support surface and a support ring, the support ring connects the upper support surface and the lower support surface into a whole structure, the lower support surface is a sponge layer, the lower support surface is in contact with the bottom surface of the assembly, and the enrichment reagent area and the library construction reagent area are located on the upper support surface.

[0036] Primer information for this utility model:

[0037] Table 1. Primer information required for the first targeted enrichment

[0038]

[0039] Table 2. Primer information required for the second targeted enrichment

[0040]

[0041] Application Example 1. Obtaining and amplifying cDNA with cell-derived markers and molecular tags.

[0042] This step requires a cDNA sample carrying single-cell information. To fully demonstrate the beneficial effects of this invention and the conditions of our laboratory, this invention selects the cDNA product obtained from the 10X Chromium Single Cell V(D)J Reagent Kits (V1.0) of the 10X Genetics platform as the initial input sample. The specific process follows the instructions to obtain amplified cDNA with cell-derived markers and molecular tags. The sample source can be fresh human blood, fresh or frozen tissue, etc. The amplified cDNA needs to be purified using Agencourt AMPure XP Beads at a 1X concentration according to the instructions to remove impurities. The purified cDNA concentration needs to be detected using a Qubit assay.

[0043] In this application example, the detected cDNA concentration should be 1 ng / µl or higher. The purified cDNA obtained can be stored at 4°C for 72 hours or at -20°C for long-term storage, avoiding repeated freeze-thaw cycles.

[0044] Application Example 2. Targeted capture and amplification of HLA-I / II

[0045] 1. First targeted enrichment of HLA-I / II

[0046] The first targeted enrichment was performed using the Hieff® Multiplex PCR Kit. Following the instructions, the PCR components required for the first enrichment, the amplified cDNA obtained by the 10X platform method after purification with magnetic beads, the 5' universal upstream primer (corresponding to the universal sequence carried at the 5' end of the template cDNA), and the downstream primer combination in HLA-I / II Target Mix 1 were added to the reaction tube respectively. The first targeted enrichment was performed on the PCR instrument under the conditions shown in Table 3 below.

[0047] Table 3. Conditions for the first targeted enrichment reaction

[0048]

[0049] 2. Purification of products after the first targeted enrichment of HLA-I / II

[0050] The reaction product was purified using 0.8X Agencourt AMPure XP Beads according to the instructions to remove excess oligos and PCR components, thus obtaining the first enriched product.

[0051] 3. Second targeted enrichment of HLA-I / II

[0052] Add the purified first enrichment products to the reaction tubes and perform a second targeted enrichment on the PCR instrument according to the conditions in Table 4 below, using the Hieff® Multiplex PCR Kit. For the second targeted enrichment, the 5' universal upstream primer is still used, and the downstream primer combination in HLA-I / II Target Mix 2 is used.

[0053] Table 4. Conditions for the second targeted enrichment reaction

[0054]

[0055] 4. Purification of the product from both ends after the second targeted enrichment of HLA-I / II

[0056] The reaction product was purified using 0.5X and 0.8X Agencourt AMPure XP Beads according to the instructions, and fragments of the desired length were screened out.

[0057] Enriched product analysis: The peak chromatogram of the enriched product was analyzed using Labchip, and the Qubit concentration was determined. The enriched product can be stored at 4°C for 72 hours or at -20°C for one week.

[0058] Application Example 3. Next-Generation Sequencing Library Construction and Quality Control for Illumina Platform

[0059] 1. Sequencing library preparation and purification

[0060] The enriched DNA was digested and fragmented using a fragmentation enzyme under appropriate conditions, the ends were repaired, and an A was added to the 3' end. Then, the ligase was used to ligate the fragments, and the ligation product was purified using 0.8X Agencourt AMPure XP Beads. Sequencing adapters and sample indexes were then added to both ends of the fragments by PCR, and the reaction product was purified once more using 0.8X Agencourt AMPure XP Beads to obtain the sequencing library.

[0061] 2. The obtained next-generation sequencing library needs to be quantified using qPCR. The library concentration is detected using Qubit, and the product fragment size is detected using Labchip. Figure 3 )

[0062] Application Example 4. Preprocessing of raw offline FastQ data.

[0063] Based on previous bioinformatics analysis experience, the raw FastQ data were initially quality controlled using software such as FastQC, FastP, Trimmatic, and Bowtie2 to remove low-quality data and adapters, and aligned to the reference genome. The quality of the aligned data was then further controlled.

[0064] 1. HLA typing analysis

[0065] Single-cell matrices were constructed using software such as CellRanger and HLA-VBseq, and HLA typing and expression levels were statistically analyzed.

[0066] 2. Comparative analysis of HLA results obtained by different methods

[0067] HLA typing and expression levels obtained from different methods were compared end-to-end. The results are shown in [link to analysis]. Figure 4-7 .

[0068] This invention relates to a kit for detecting HLA-I / II gene expression genotyping and expression levels at the single-cell level. The use of this kit allows for more targeted sequencing libraries, fully showcasing the HLA polymorphism of each cell and effectively demonstrating the HLA combination state during single-cell expression, rather than the HLA state contained within bulk cells at the DNA level. HLA genotyping during transcription is more accurate in reflecting cellular immune status. The kit comprehensively includes genotyping of all nine HLA-I and HLA-II genes, providing a large and comprehensive sample size for a more accurate assessment of single-cell immune status. The kit is simple to operate, has a stable process, and offers both considerable economic benefits and rapid detection.

Claims

1. A kit for detecting HLA-I / II class gene expression typing and expression level at single cell level, characterized in that: The kit comprises a box body and a flip cover connected with the box body, a paper support is arranged in the box body, and the box body is divided into two layers by the paper support; the paper support is provided with a reagent enrichment area and a library building reagent area; the reagent enrichment area is provided with more than six test tube holes, and the library building reagent area is provided with more than seventeen test tube holes; the test tube holes of the reagent enrichment area are provided with an HLA Target Mix1 test tube for HLA-I / II class gene capture, an HLA Target Mix2 test tube for HLA-I / II class gene capture, a 2X Multiplex PCR Master Mix test tube, a deionized ultrapure water test tube and a purification magnetic bead test tube; the test tube holes of the library building reagent area are provided with a fragmentation and end repair enzyme test tube, a fragmentation and end repair buffer test tube, a 1X TE buffer test tube, a linker test tube, a ligase test tube, a ligation buffer test tube, a 2X PCR Mix test tube required for library amplification, a Universal Primer test tube, a Sample Index Primer test tube, a deionized ultrapure water test tube and a purification magnetic bead test tube, and all the test tubes are provided with test tube covers.

2. The kit for detecting HLA-I / II class gene expression typing and expression amount at single cell level according to claim 1, characterized in that: All the test tubes are 1.5ml test tubes.

3. The kit for detecting HLA-I / II class gene expression typing and expression amount at single cell level according to claim 1 or 2, characterized in that: The kit has a cubic structure, and the length, width and height thereof are 20±1cm, 15±1cm and 12±1cm respectively.

4. The kit for detecting HLA-I / II class gene expression typing and expression amount at single cell level according to claim 1 or 2, characterized in that: The paper support comprises a support surface and support feet arranged along the bottom of the support surface, and the support feet are arranged on the bottom surface in the box body; the reagent enrichment area and the library building reagent area are arranged on the support surface.

5. The kit for detecting HLA-I / II class gene expression typing and expression amount at single cell level according to claim 1 or 2, characterized in that: The paper support comprises an upper support surface, a lower support surface and a support ring, the support ring connects the upper support surface and the lower support surface into an integral structure, the lower support surface is a sponge layer, the lower support surface is in contact with the bottom surface in the box body, and the reagent enrichment area and the library building reagent area are arranged on the upper support surface.

6. The kit for detecting HLA-I / II class gene expression typing and expression amount at single cell level according to claim 1 or 2, characterized in that: The paper support is provided with 23-26 test tube holes, and the diameters of all the test tube holes are 9mm.

7. The kit for detecting HLA-I / II class gene expression typing and expression amount at single cell level according to claim 1 or 2, characterized in that: All the reagent tubes of the reagent enrichment area are provided with yellow caps except the deionized ultrapure water and the purification magnetic beads, all the reagent tubes of the library building reagent area are provided with green caps except the deionized ultrapure water and the purification magnetic beads, the test tube cover of the deionized ultrapure water test tube is provided with a colorless cap at the top, and the test tube cover of the purification magnetic bead test tube is provided with a red cap at the top.

8. The kit for detecting HLA-I / II class gene expression typing and expression amount at single cell level according to claim 1 or 2, characterized in that: The test tube holes of the reagent enrichment area are provided with one HLA Target Mix1 test tube for HLA-I / II class gene capture, one HLA Target Mix2 test tube for HLA-I / II class gene capture, one 2X Multiplex PCR Master Mix test tube, two deionized ultrapure water test tubes and one purification magnetic bead test tube, and all the test tubes are provided with test tube covers.

9. The kit for detecting HLA-I / II class gene expression typing and expression amount at single cell level according to claim 1 or 2, characterized in that: The tube holes in the reagent area of the library building device are provided with one tube of fragmentation and end repair enzyme, one tube of fragmentation and end repair buffer, one tube of 1X TE buffer, one tube of ligation adapter, one tube of ligation enzyme, one tube of ligation buffer, one tube of 2X PCR Mix, one tube of Universal Primer, six tubes of Sample Index Primer, two tubes of deionized ultrapure water, and one tube of magnetic bead purification.

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