CN116640855A - Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof - Google Patents
Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof Download PDFInfo
- Publication number
- CN116640855A CN116640855A CN202310394055.4A CN202310394055A CN116640855A CN 116640855 A CN116640855 A CN 116640855A CN 202310394055 A CN202310394055 A CN 202310394055A CN 116640855 A CN116640855 A CN 116640855A
- Authority
- CN
- China
- Prior art keywords
- tcr
- porcine
- sequencing
- sequence
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 claims abstract description 18
- 241000282898 Sus scrofa Species 0.000 claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims description 45
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 239000006285 cell suspension Substances 0.000 claims description 13
- 239000010437 gem Substances 0.000 claims description 10
- 210000004698 lymphocyte Anatomy 0.000 claims description 9
- 239000011324 bead Substances 0.000 claims description 8
- 230000010100 anticoagulation Effects 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 239000000839 emulsion Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000010839 reverse transcription Methods 0.000 claims description 5
- 238000003908 quality control method Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 241000282887 Suidae Species 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 229910052804 chromium Inorganic materials 0.000 claims description 3
- 239000011651 chromium Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 238000002372 labelling Methods 0.000 claims description 2
- 230000002998 immunogenetic effect Effects 0.000 abstract description 3
- 230000003449 preventive effect Effects 0.000 abstract 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 14
- 101150008942 J gene Proteins 0.000 description 11
- 101150117115 V gene Proteins 0.000 description 10
- 230000003321 amplification Effects 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 108091008874 T cell receptors Proteins 0.000 description 4
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000008707 rearrangement Effects 0.000 description 4
- 101150111062 C gene Proteins 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 101150097493 D gene Proteins 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002992 thymic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
- Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
Abstract
The invention relates to a group of primers for sequencing single cells V (D) J of a swine source and application thereof, belongs to the technical field of preventive veterinarian, and at present, in an IMGT (International immunogenetics database), swine source TCR sequence information is still very limited, so that research on swine source TCR diversity is severely restricted. The invention provides a group of porcine single cell V (D) J sequencing TCR sequence primers and a method for annotating sequencing data. Can be used for obtaining porcine TCR sequence information at the level of single cells.
Description
Technical Field
The invention belongs to the technical field of prevention veterinarian, and particularly relates to a group of primers for sequencing swine single cell V (D) J and application thereof.
Background
T cells are important components of peripheral blood lymphocytes and play an important role in the adaptive immune response of the body. T Cell Receptors (TCRs) are specific markers on the surface of all T cells, TCRs are heterodimers consisting of two different peptide chains, which can be divided into αβ T cells and γδ T cells depending on the peptide chain; the alpha chain and the gamma chain consist of a V gene, a J gene and a C gene, and the beta chain and the delta chain are added with one D gene.
TCR gene rearrangement refers to the process in which, during the process of thymic differentiation and maturation of T cells, the germ line-state genes are linked by separate, transcriptionally inactive gene fragments into a complete, transcriptionally active gene under the action of specific recombinases; during rearrangement, the D gene and the J gene are firstly connected in the beta and delta chains, so that a gene with transcriptional activity is generated, and finally, the gene is connected with the C gene, so that a complete beta or delta functional gene is formed; in alpha and gamma, the V gene is directly connected with the J gene and then connected with the C gene to form a complete alpha or gamma functional gene. The gene rearrangement of the TCR only occurs in the early stage of T cell differentiation, and because of the strict time limitation of the specific recombinase, the T cell can only perform one effective gene rearrangement in the differentiation and maturation process, so that one T cell clone can only express one specific TCR. T cells specifically recognize and bind to antigens presented by the major histocompatibility complex (Major Histocompatibility Complex, MHC), 90% -95% of T cells express alpha and beta chains in peripheral blood and a few express gamma and delta chains. The sum of all functionally diverse B-and T-lymphocytes in the circulatory system of an individual at any particular point in time is called the immune repertoire; the diversity of the immune repertoire is positively correlated with the health condition of the organism, and the diversity of the immune repertoire under the influence of different factors such as age, environment, diseases and the like is researched, so that the immune repertoire is beneficial to further understanding of an organism immune regulation mechanism.
Traditional TCR sequencing is to obtain a batch of sequence information through RNA-seq, but the obtained information is based on a cell population, the sequence information of TCR chain pairing cannot be obtained, cell heterogeneity information cannot be presented, and some of the less clonotype information can be ignored. The 10x genomic company V (D) J sequencing technology based on single cell level can distribute single cells into water-in-oil microsystems by microfluidic technology, and distinguish different single cells by adding unique DNA marker sequences (barcode and UMI) into each microreaction system, and the cDNA library constructed after reverse transcription of the microreaction system can be used for transcriptome sequencing, BCR, TCR sequencing. The external primer and the internal primer are designed in the constant region of the TCR sequence, and the internal primer is used for amplification on the basis of the external primer by a 5 'terminal rapid amplification technology (5' RACE), so that the error amplification of the first reaction can be greatly eliminated, the correctness and the specificity of PCR amplification are ensured, and thousands of single-cell TCR or BCR information can be obtained once after high-throughput second-generation sequencing. Single cell sequencing technology can correlate the transcription profile of single cells with TCR information, thus more accurately studying the role of T cells in immune responses.
Currently, in IMGT (international immunogenetics database), TCR and BCR sequence information of human and mouse species are quite perfect, but on porcine TCRs, only TCR β chain information is recorded, and research on other porcine TCR chain sequence information is still limited, which prevents further research on porcine TCR immune repertoires. At the single cell level, annotating the porcine TCR sequence, discovering the TCR sequence with a rare proportion, revealing pairing information of the porcine TCR chain, helping to further study the characteristics of immune repertoires under the health and disease states of the organism, further knowing the immune system and discovering the change rule of T cells under different organism conditions.
Disclosure of Invention
In view of the above problems, it is an object of the present invention to provide a set of single cell V (D) J sequencing TCR sequence primers of porcine origin, and a method of annotating sequencing data. Can be used for obtaining porcine TCR sequence information at the level of single cells.
In order to achieve the above purpose, the invention adopts the following specific scheme:
in a first aspect, a set of porcine TCR sequence primers for single cell V (D) J sequencing comprising nucleotide sequences as set forth in SEQ ID NO: 1-10, TRA outer-R, TRA inner-R, TRB outer-R, TRB inner-R, TRD outer-R, TRD inner-R, TRG outer-R, TRG inner-R, outer-F and Inter-F.
In a second aspect, the use of a porcine TCR sequence primer as described above in any one of:
(a) Single cell V (D) J sequencing;
(b) Porcine TCR sequences were prepared or identified.
In a third aspect, a method of annotating a TCR sequence of porcine origin based on single cell V (D) J sequencing, comprising the steps of: step one, preparing a cell suspension: anticoagulation was collected through the anterior vena cava of pigs using EDTA anticoagulation tube, lymphocytes were isolated with lymphocyte separation solution, and cells were resuspended using PBS;
step two, counting the cell suspension by using a cell counter, and adjusting the concentration of the cell suspension to be 1-1.5x10 3 cells/μL；
Step three, constructing a single-cell micro-reaction system:
using 10x Genomics Chromium TM The system loads Gel Beads with sequence labels, cell suspension, reagent premix liquid and oil into respective sample injection channels, and finally forms a single-cell micro-reaction system wrapped by GEMs (Gel Bead in emulsion) through a double-cross system formed by a microfluidic channel network; collecting GEMs, carrying out reverse transcription in a PCR instrument to realize labeling, constructing a high-flux second-generation sequencing library, and carrying out high-flux second-generation sequencing by using the sequencing library;
fourth, sequence quality control: the original data uses celrange (7.1) software vdj-denovo parameters to generate a sequence file;
step five, sequence comparison and annotation: and submitting the sequence file to an IMGT/HighV-QUEST database, taking human TCR data as comparison, using R (4.2.1) software to read the comparison result, and eliminating sequences with the gene similarity less than 75%.
In a fourth aspect, the use of the above method in any one of the following:
(a) Bulk annotation for single cell V (D) J sequencing of porcine TCR sequences;
(b) Exploring the diversity of porcine TCR sequences in healthy or diseased states;
(c) Obtaining porcine TCR chain sequence information of single cell level;
(d) And constructing a swine TCR sequence reference database.
The beneficial effects are that:
1. the invention provides a group of 5' RACE primers for amplifying swine TCR sequences, which can greatly ensure the accuracy and the specificity of PCR amplification through two rounds of amplification.
2. In the scheme adopted by the invention, the porcine TCR sequence information at the single cell level and the pairing information of the TCR chain can be obtained by carrying out high-throughput sequencing second generation sequencing on a single cell microreaction system wrapped by GEMs (Gel Bead in emulsion).
3. The single cell-based TCR chain sequences annotated by the methods provided herein can be used to construct a reference porcine-derived TCR sequence library for use in subsequent immune repertoire studies.
Drawings
FIG. 1 is a flow chart of the annotation of porcine TCR sequencing.
Detailed Description
Currently, in IMGT (international immunogenetics database), porcine TCR sequence information is still very limited, severely restricting the study of porcine TCR diversity. The invention aims to provide a group of porcine single cell V (D) J sequencing TCR sequence primers and a method for annotating sequencing data. Can be used for obtaining porcine TCR sequence information at the level of single cells.
1 the primers for sequencing the porcine TCR single cell V (D) J provided by the invention are as follows.
2 as a preferred embodiment of the invention, primer sets are provided for use in the preparation or identification of porcine TCR sequences.
3 As a preferred embodiment of the invention, the downstream primer provided may amplify the swine TCR sequence by 5' RACE techniques.
4 As a preferred embodiment of the present invention, the accuracy of the sequence is improved by two rounds of amplification, the first round of amplification using the downstream Outer primer (Outer-R), the second round of amplification using the downstream inner primer (inner-R), and the upstream primer using the upstream universal primer (Outer-F, inter-F) supplied by 10xgenomic corporation.
5 As a preferred embodiment of the invention, the sequencing sample is porcine Peripheral Blood Mononuclear Cells (PBMC) or sorted T lymphocytes.
6 the method for annotating porcine TCR sequences based on single cell V (D) J sequencing provided by the invention comprises the following steps:
(1) Preparation of cell suspension: anticoagulation was collected through the anterior vena cava of pigs using EDTA anticoagulation tube, lymphocytes were isolated with lymphocyte separation solution, and cells were resuspended using PBS;
(2) Counting the cell suspension by using a cell counter, and adjusting the concentration of the cell suspension to be 1-1.5x10 3 cells/μL；
(3) The cell suspension with the adjusted concentration is entrusted to a single-cell micro-reaction system wrapped by GEMs (Gel Bead in emulsion) with a sequence tag, GEMs are collected and subjected to reverse transcription in a PCR instrument to realize tagging, then a high-flux second-generation sequencing library is constructed, and the sequencing library is used for high-flux second-generation sequencing;
(4) Sequence quality control: the original data uses celrange (7.1) software vdj-denovo parameters to generate a sequence file;
(5) Sequence annotation: and submitting the sequence file to an IMGT/HighV-QUEST database, taking human TCR data as comparison, using R (4.2.1) software to read the comparison result, and eliminating sequences with the gene similarity less than 75%.
7 according to the method of the invention, the diversity of TCR sequences of porcine origin in healthy or diseased states can be explored.
8 according to the method of the invention, porcine TCR chain sequence information at the single cell level can be obtained.
9 the sequences obtained according to the method of the invention can be used to construct a swine TCR sequence reference database for subsequent immune repertoire studies.
The technical scheme of the invention will be clearly and completely described in the following in connection with the embodiments of the invention.
Example 1
1. Isolation of peripheral blood mononuclear cells:
transferring the collected 10mL of anticoagulated blood into a 50mL centrifuge tube, adding 10mL of PBS solution for dilution, and lightly mixing; taking 15mL centrifuge tube, adding 5mL lymphCell separation liquid. Then, 5mL of diluted blood is gently added to the upper layer of lymphocyte separation liquid in the centrifuge tube; 2000rpm,20min; after centrifugation, the plasma layer, lymphocyte separation layer and erythrocyte and granulocyte layers are respectively arranged from top to bottom. Sucking the lymphocyte layer into another 15mL centrifuge tube, adding PBS to 10-15mL, centrifuging at 1500rpm for 10min, removing supernatant, adding culture medium, and repeating the above steps to clean cells; counting was performed using a cytometer, and the concentration was adjusted to 1-1.5x10 using PBS as a diluent 3 cells/μL。
2. Single cell micro-reaction system construction:
using 10x Genomics Chromium TM And loading Gel Beads with sequence labels, cell suspension, reagent premix liquid and oil into respective sample injection channels, and finally forming a GEMs (Gel Bead in emulsion) wrapped single-cell micro-reaction system through a double-cross system formed by a microfluidic channel network. GEMs were collected for reverse transcription in a PCR instrument and all single cells were labeled by molecular tags.
3. Single cell TCR immune repertoire sequencing:
the reverse transcribed GEMs are entrusted with Shanghai crystal energy biotechnology limited company to construct a sequencing high-throughput second-generation sequencing library, and high-throughput second-generation sequencing is carried out.
4. Raw data assembly: sequence files were generated using celrange (7.1) software vdj-denovo parameters.
5. Data quality control and annotation: the sequence file is read by using R (4.2.1) software, sequences containing TCR inner primers are screened by using R packet reverse, the screened sequences are submitted to an IMGT/HighV-QUEST database, the humanized TCR data are used as comparison, genes with the gene similarity less than 75% are removed, and the gene fragments are named according to the comparison result.
The screened sequence is shown as SEQ ID NO:11 to 35. The results of the comparison are shown in tables 1-4 below.
Table 1: partial TCR alpha chain V gene, J gene similarity to human TCR alpha chain V gene, J gene.
Table 2: partial TCR beta chain V gene, J gene and porcine TCR alpha chain V gene, J gene similarity.
Table 3: TCR delta chain V gene, J gene and human delta chain V gene, J gene similarity.
Table 4: TCR gamma sequence V gene, J gene and human delta chain V gene, J gene similarity.
The primer provided by the invention can be used for single cell V (D) J sequencing or 5' end RACE amplification to obtain a swine TCR sequence.
The sequence annotation method provided by the invention can be used for batch annotation of single-cell V (D) J sequencing swine TCR sequences, and the obtained sequences can further obtain TCR chain pairing information through molecular tags.
It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.
Claims (4)
1. A set of porcine TCR sequence primers for single cell V (D) J sequencing, characterized by: comprises nucleotide sequences shown in SEQ ID NO: 1-10, TRA outer-R, TRA inner-R, TRB outer-R, TRB inner-R, TRD outer-R, TRD inner-R, TRG outer-R, TRG inner-R, outer-F and Inter-F.
2. Use of a porcine TCR sequence primer according to claim 1 in any of the following:
(a) Single cell V (D) J sequencing;
(b) Porcine TCR sequences were prepared or identified.
3. A method for annotating a porcine TCR sequence based on single cell V (D) J sequencing, characterized by: the method comprises the following steps:
step one, preparing a cell suspension: anticoagulation was collected through the anterior vena cava of pigs using EDTA anticoagulation tube, lymphocytes were isolated with lymphocyte separation solution, and cells were resuspended using PBS;
step two, counting the cell suspension by using a cell counter, and adjusting the concentration of the cell suspension to be 1-1.5x10 3 cells/μL；
Step three, constructing a single-cell micro-reaction system:
using 10x Genomics Chromium TM The system loads Gel Beads with sequence labels, cell suspension, reagent premix liquid and oil into respective sample injection channels, and finally forms a single-cell micro-reaction system wrapped by GEMs (Gel Bead in emulsion) through a double-cross system formed by a microfluidic channel network; collecting GEMs, carrying out reverse transcription in a PCR instrument to realize labeling, constructing a high-flux second-generation sequencing library, and carrying out high-flux second-generation sequencing by using the sequencing library;
fourth, sequence quality control: the original data uses celrange (7.1) software vdj-denovo parameters to generate a sequence file;
step five, sequence comparison and annotation: and submitting the sequence file to an IMGT/HighV-QUEST database, taking human TCR data as comparison, using R (4.2.1) software to read the comparison result, and eliminating sequences with the gene similarity less than 75%.
4. A use of the method according to claim 3 in any of the following:
(a) Bulk annotation for single cell V (D) J sequencing of porcine TCR sequences;
(b) Exploring the diversity of porcine TCR sequences in healthy or diseased states;
(c) Obtaining porcine TCR chain sequence information of single cell level;
(d) And constructing a swine TCR sequence reference database.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310394055.4A CN116640855A (en) | 2023-04-13 | 2023-04-13 | Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310394055.4A CN116640855A (en) | 2023-04-13 | 2023-04-13 | Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116640855A true CN116640855A (en) | 2023-08-25 |
Family
ID=87621898
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310394055.4A Pending CN116640855A (en) | 2023-04-13 | 2023-04-13 | Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116640855A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150141261A1 (en) * | 2012-06-15 | 2015-05-21 | Board Of Regents, The University Of Texas Systems | High throughput sequencing of multiple transcripts of a single cell |
| CN104673892A (en) * | 2014-12-30 | 2015-06-03 | 南方科技大学 | Primer group for developing rhesus T cell immune repertoire, high-flux sequencing method and application of method |
| US20180258492A1 (en) * | 2017-03-06 | 2018-09-13 | Karkinos Precision Oncology LLC | Diagnostic methods for identifying t-cell lymphoma and leukemia by high-throughput tcr-beta sequencing |
| US20200392479A1 (en) * | 2017-10-23 | 2020-12-17 | The Broad Institute, Inc. | Single cell cellular component enrichment from barcoded sequencing libraries |
| CN112852936A (en) * | 2020-06-24 | 2021-05-28 | 广州华银健康医疗集团股份有限公司 | Method for analyzing sample lymphocyte or plasma cell by using immune repertoire sequencing method, application and kit thereof |
| CN113026111A (en) * | 2019-12-25 | 2021-06-25 | 苏州绘真生物科技有限公司 | Kit for constructing human single cell TCR sequencing library and application thereof |
| CN114107458A (en) * | 2013-03-15 | 2022-03-01 | 血统生物科学公司 | Method for sequencing an immune repertoire |
| CN114410743A (en) * | 2022-01-24 | 2022-04-29 | 武汉菲沙基因信息有限公司 | Whole-length library construction and sequencing method for single-cell TCR immune repertoire |
-
2023
- 2023-04-13 CN CN202310394055.4A patent/CN116640855A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150141261A1 (en) * | 2012-06-15 | 2015-05-21 | Board Of Regents, The University Of Texas Systems | High throughput sequencing of multiple transcripts of a single cell |
| CN114107458A (en) * | 2013-03-15 | 2022-03-01 | 血统生物科学公司 | Method for sequencing an immune repertoire |
| CN104673892A (en) * | 2014-12-30 | 2015-06-03 | 南方科技大学 | Primer group for developing rhesus T cell immune repertoire, high-flux sequencing method and application of method |
| US20180258492A1 (en) * | 2017-03-06 | 2018-09-13 | Karkinos Precision Oncology LLC | Diagnostic methods for identifying t-cell lymphoma and leukemia by high-throughput tcr-beta sequencing |
| US20200392479A1 (en) * | 2017-10-23 | 2020-12-17 | The Broad Institute, Inc. | Single cell cellular component enrichment from barcoded sequencing libraries |
| CN113026111A (en) * | 2019-12-25 | 2021-06-25 | 苏州绘真生物科技有限公司 | Kit for constructing human single cell TCR sequencing library and application thereof |
| CN112852936A (en) * | 2020-06-24 | 2021-05-28 | 广州华银健康医疗集团股份有限公司 | Method for analyzing sample lymphocyte or plasma cell by using immune repertoire sequencing method, application and kit thereof |
| CN114410743A (en) * | 2022-01-24 | 2022-04-29 | 武汉菲沙基因信息有限公司 | Whole-length library construction and sequencing method for single-cell TCR immune repertoire |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Swanson et al. | Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq | |
| Fan et al. | Combinatorial labeling of single cells for gene expression cytometry | |
| James et al. | Distinct microbial and immune niches of the human colon | |
| US11591652B2 (en) | System and methods for massively parallel analysis of nucleic acids in single cells | |
| US20220176335A1 (en) | Methods and compositions for tagging and analyzing samples | |
| Bai et al. | Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL | |
| US20150154352A1 (en) | System and Methods for Genetic Analysis of Mixed Cell Populations | |
| EP3262189A1 (en) | Methods and compositions for barcoding nucleic acids for sequencing | |
| Dash et al. | Single-cell analysis of T-cell receptor αβ repertoire | |
| Dietlein et al. | Usp22 is an intracellular regulator of systemic emergency hematopoiesis | |
| Wu et al. | Comparative transcriptome analysis of the transcriptional heterogeneity in different IgM+ cell subsets from peripheral blood of Nile tilapia (Oreochromis niloticus) | |
| US20220005547A1 (en) | Multiplexed droplet-based sequencing using natural genetic barcodes | |
| Hurtado et al. | Efficient isolation of rare B cells using next-generation antigen barcoding | |
| Imamichi et al. | Interleukin-27 induces interferon-inducible genes: analysis of gene expression profiles using Affymetrix microarray and DAVID | |
| CN116640855A (en) | Porcine TCR sequence primer for single cell V (D) J sequencing and application thereof | |
| Pollastro et al. | Characterization and monitoring of antigen-responsive T cell clones using T cell receptor gene expression analysis | |
| Walsh et al. | Single-Cell–based high-throughput ig and TCR repertoire sequencing analysis in rhesus macaques | |
| Gierahn et al. | Seq-well: A portable, low-cost platform for high-throughput single-cell rna-seq of low-input samples | |
| WO2021188384A1 (en) | Methods of amplifying paired transcript sequences from single cells | |
| Tian et al. | Genomic Architecture of Cells in Tissues (GeACT): Study of Human Mid-gestation Fetus | |
| Jia et al. | Isolating peripheral blood mononuclear cells from HIV-infected patients for single-cell RNA sequencing and integration analysis | |
| US11613783B2 (en) | Systems and methods for detecting multi-molecule biomarkers | |
| US20220028492A1 (en) | Systems and methods for calling cell-associated barcodes | |
| US20220145285A1 (en) | Compartment-Free Single Cell Genetic Analysis | |
| Ainciburu-Fernández | Application of single cell transcriptomics to characterize the progression of hematopoietic cells to myeloid malignancies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |