免疫组库(Immune Repertoire,IR)是指在任何指定时间,某个个体的循环系统中所有功能多样性B细胞和T细胞的总和。免疫组库测序(Immune Repertoire sequencing(IR-SEQ))是以 T/B 淋巴细胞为研究目标,以多重PCR或5’RACE技术目的扩增决定B细胞受体(BCR)或T细胞受体(TCR)多样性的互补决定区(CDR区),再结合高通量测序技术,全面评估免疫系统的多样性,深入挖掘免疫组库与疾病的关系。
我们的优势
整体解决方案:提供从CDR的扩增、建库、测序、后续信息分析一系列全方位的服务;
稳定的测序平台:可根据数据量和速度的要求选择Hiseq2500或Miseq平台;
全面深度的研究方法:专门针对CDR3的V区及J区两端设计引物,目的扩增CDR3区域
高保真的扩增技术:采用多重PCR或5’RACE技术,实现不同克隆等效扩增,Unique克隆数高达10^5。
B细胞生物反应原理

Figure 1 Antibody structure and sequence diversification mechanisms. (a) Schematic of IgG structure. In the top chains, domains encoded from germline V, D, J and C segments are indicated. Nontemplated N-nucleotides are shown in red. These top chains delineate the 5′ to 3′ genetic composition of the antibody. In the bottom chains, framework (FR) and complementarity-determining regions (CDRs) are indicated. These bottom chains delineate the N-terminal to C-terminal protein sequence. Dashed lines denote disulfide bonds. (b) Key steps in antibody diversification. The primary antibody heavy chain repertoire is created predominantly by the somatic recombination of variable (V), diversity (D) and joining (J) gene segments,
and by the random nontemplated addition of N-nucleotides. The antigen-binding site of a heavy chain is formed by the juxtaposition of the hypervariable complementarity-determining regions (CDR-H1, H2 and H3) and the framework 3 region (FR3). After productive IgH rearrangement, recombination of the light chain (IgL) ensues, and the heterodimeric pairing of H and L chains forms the complete antibody of the IgM isotype that is expressed on the surface of a newly formed immature B cell. Eμ: IgM intronic enhancer; Sμ: tandem repeats critical for class-switch recombination. Numbers in parentheses refer to estimates of human germline VH DH and JH segments.
T细胞生物反应原理

Figure 2 Antigen recognition by the T-cell receptor and probing antigen specificity with peptide–MHC multimers. (a) Antigen-specific T-cell responses are initiated through the interaction of a TCR, expressed on T cells, and the corresponding peptide-MHC protein complex expressed by antigen-presenting cells. TCR engagement initiates a complex cell-signaling cascade that results in T-cell activation. (b) Binding affinity of TCR to its specific peptide–MHC ligand is very low (~1–100 μM) and has very fast dissociation kinetics (t1/2 usually much less than a minute). Thus, monomeric staining reagents are insufficiently stable for the detection of antigen-specific T cells. In contrast, by taking advantage of cooperative binding, multimeric complexes of peptide-MHC allow for remarkably sensitive and accurate detection of antigen-specific T cells
技术服务流程
Strategies for high-throughput single-cell analysis of TCR sequences and identification of TCR ligands.

(a) As demonstrated for B-cell immunoglobulin genes, sequencing of V regions of endogenously paired TCRα and TCRβ genes can be performed in high-throughput by mRNA capture and emulsion linkage RT-PCR, or by direct cellular-emulsion-linkage PCR. Cell-specific barcoded tags can also be introduced at the single-cell stage as a general means of increasing the throughput of this type of approach.
(b) Libraries of yeast clones displaying 105–108 unique peptides tethered to MHC molecules enable high-throughput screening for peptides capable of binding MHC (because these peptides result in proper folding and surface expression of peptide–MHC complexes) and for peptides capable of binding to a TCR of interest (here the TCR is used as a tetrameric staining reagent). After multiple rounds of selection, hits are sorted, cloned and sequenced; the peptide sequences can then be analyzed.
技术参数
一、样本要求
总量:DNA≥ 2.5 µg ;RNA≥ 5µg
浓度:DNA≥ 37.5 ng/µL;RNA≥ 200 ng/µL
DNA样本要求无降解或轻微降解;RNA样本要求无降解,RIN≥ 7.0,28S/18S≥ 1.0。
二、测序策略
Hiseq2500一般推荐选择PE101,也可根据项目实际需要选择MiSeq平台和测序策略。
三、推荐数据量
推荐数据量:至少2G clean data。
四、项目执行周期
按标准流程(不包括高级信息分析),100个样本的运转周期约为50个工作日。
五、项目合格指标
产生不低于合同规定的clean data。
提交项目结题报告文档,说明项目完成情况
更多详情:http://www.biogenius.cn/htm/products/genomics/immnoseq/

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