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Home > ÀüÁ¦Ç°º¸±â > NGS °ü·Ã > Immune-Seq > UMI Àû¿ë Human TCR profiling(v2)
TCR repertoire¸¦ Ź¿ùÇÑ °¨µµ·Î ºÐ¼®

UMI Àû¿ë Human TCR profiling(v2)

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
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Clontech
634478
SMARTer¢ç Human TCR a/b Profiling Kit v2
°ü·ÃÇмú ±¸¸ÅÇϱâ
12 ȸ
(Package)
3,769,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x109519
Clontech
634479
SMARTer¢ç Human TCR a/b Profiling Kit v2
°ü·ÃÇмú ±¸¸ÅÇϱâ
48 ȸ
(Package)
8,229,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â

* Human TCR a/b Profiling Kit v2´Â SMART-Seq® Human TCR (with UMIs)·Î ¸®´º¾óµÇ¾ú½À´Ï´Ù.


  • Unique Molecular Index (UMI) Àû¿ëÀ¸·Î, v2 ¾÷±×·¹À̵å - PCR ȤÀº sequence error·Î ÀÎÇÑ reads Á¦°Å·Î ½Å·Úµµ ÀÖ´Â °á°ú ºÐ¼®
  • ´Ù¾çÇÑ ¹üÀ§ÀÇ »ùÇà Àû¿ë °¡´É - peripheral blood leukocytes (10 ng - 1 ug), T cell À¯·¡ total RNA (1 - 100ng), whole T cell (1,000 - 10,000 purified cells)
  • °£´ÜÇÑ PCR °úÁ¤ - TCR-alpha, TCR-beta chainÀ» µ¿½Ã¿¡ ¶Ç´Â °³º°ÀûÀ¸·Î NGS Library Á¦ÀÛ °¡´É
  • Unique Dual Index (UDI) »ç¿ëÀ¸·Î ºÐ¼® ¾ç Áõ°¡ - multiplexing »ùÇà ¼ö Áõ°¡ ¹× high-throughput sequencer Àû¿ë
  • Illumina¢ç platform ºÐ¼®¿ë libraries Á¦ÀÛ - ¸ðµç Illumina¢ç ±â±â¿¡ Àû¿ë °¡´É ¹× MiSeq¢â system¿¡¼­ full-length V(D)J¿Í CDR3 ºÐ¼® °¡´É
  • Complete workflow - °íÇ°ÁúÀÇ TCR profiling analysis¸¦ À§ÇÑ Cogent NGS Immune Profiler Software Á¦°ø (¿¬±¸¿ë, »ó¾÷Àû ¿ëµµ¿¡¼­ ¸ðµÎ ¹«»ó »ç¿ë °¡´É)
Á¦Ç° ¼³¸í
SMARTer Human TCR a/b Profiling Kit v2 (TCRv2) Ź¿ùÇÑ °¨µµ¿Í ÀçÇö¼ºÀ» °¡Áø TCR repertoireÀÇ ºÐ¼®À» À§ÇÑ Illumina platform NGS Library Á¦ÀÛ Å°Æ®ÀÌ´Ù. º» Á¦Ç°Àº SMART (Switching Mechanism at 5¡¯ End of RNA Template)ÀÇ full-length cDNA ÇÕ¼º ±â¼ú°ú TCR Àü»çüÀÇ V(D)J °¡º¯¿µ¿ª (variable region) ³» TRA, TRB À¯ÀüÀÚ ºÐ¼®À» À§ÇÏ¿© 5¡¯ RACE À¯»ç ±â¼úÀ» Á¢¸ñÇÏ¿´´Ù.
º» Á¦Ç°À» ÀÌ¿ëÇϸé RNA »ùÇà (RIN°ª 8 ÀÌ»ó)·ÎºÎÅÍ °íÇ°ÁúÀÇ library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖÀ¸¸ç, peripheral blood leukocytes (10 ng - 1 ug), T cell À¯·¡ total RNA (1 - 100 ng), whole T cell (1,000 - 10,000 purified cells)¿¡ Àû¿ëÇÒ ¼ö ÀÖ´Ù. ¶ÇÇÑ, TCR-¥á, TCR-¥â chainÀ» µ¿½Ã¿¡ ¶Ç´Â °¢ chain¿¡ ´ëÇÑ ¶óÀ̺귯¸®¸¦ Á¦ÀÛÇÏ¿© ºÐ¼®ÇÒ ¼ö ÀÖ´Ù. Á¦Ç° ³»¿¡´Â Illumina platform¿¡¼­ ºÐ¼® °¡´ÉÇÑ dual index (UDI)¸¦ Æ÷ÇÔÇÒ »Ó ¾Æ´Ï¶ó, unique molecular identifiers (UMIs)¸¦ Æ÷ÇÔÇÏ°í ÀÖ¾î, PCR °úÁ¤ ȤÀº sequence error·ÎºÎÅÍ À¯·¡ÇÑ reads¸¦ Á¦°ÅÇÏ¿© ºÐ¼®ÇÔÀ¸·Î½á º¸´Ù Á¤È®ÇÏ°í ½Å·Úµµ ÀÖ´Â °á°ú¸¦ ¾òÀ» ¼ö ÀÖ´Ù.


±×¸² 1. ´Ù¾çÇÑ RNA »ùÇà ¾çÀ¸·ÎºÎÅÍ °¨µµ ³ô°Ô ÀçÇö¼º ÀÖ´Â clonotype °ËÃâ
TRA and TRB libraries were generated from 1, 10, and 100 ng of human CD3+ T-cell total RNA and 1,000 and 10,000 CD3+ T cells. The sequence reads were processed by the Cogent NGS Immune Profiler Software.

% Jurkat RNA spiked in to 100 ng of PBMC RNA

Total read count (TRA/TRB)

Without UMI collapse

With UMI collapse

# of TRB raw reads

# of reads for TRBV12-3-TRBJ1-2

Detected percentage of Jurkat reads

# of detected UMIs

# of UMIs for TRBV12-3-TRBJ1-2

Detected percentage of Jurkat UMIs

10.0%

2,500,000

1,565,005

397,179

25.0%

281,280

62,629

22.0%

1.0%

2,500,000

1,422,102

47,160

3.3%

219,776

6,426

2.9%

0.1%

2,500,000

1,366,127

5,412

0.4%

189,580

631

0.33%

0.01%

2,500,000

1,218,025

521

0.043%

196,615

74

0.038%

0.001%

2,500,000

1,331,465

909

0.068%

197,870

6

0.003%

0.0001%

2,500,000

1,409,199

-

0%

124,149

-

0%

0%

2,500,000

1,222,245

-

0%

197,933

-

0%


Ç¥ 1. TCRv2·Î ºÐ¼®ÇßÀ» ¶§, °¨µµ¿Í ÀçÇö¼º Æò°¡
Spike-in analysis was performed in replicate on PBMC RNA samples spiked at varying concentrations (10%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001%) with RNA obtained from a homogeneous population of leukemic Jurkat T cells (containing TRBV12-3-TRBJ1-2 clonotypes). TRB CDR3 regions were amplified from 100 ng of total RNA using the TCRv2 kit and sequenced. Reads of 2 x 150 bp were obtained on an Illumina NextSeq¢ç system. The sequencing reads were downsampled to 2.5 M reads. Read results for spike-in concentrations identified as the reliable concentration limit for each criterion (without and with UMI collapse) have data highlighted in gray. Without UMI collapse, PCR duplicates of TRBV12-3 were observed in 0.0010% of the raw reads.


±×¸² 2. TCRv2ÀÇ ¿ì¿ùÇÑ °¨µµ¿Í ÀçÇö¼º
We split 5M PBMC cells from two different healthy donors for RNA and gDNA extraction. 1.6 ¥ìg of gDNA was used for library preparation according to the manufacturer's instructions (15% of the total amount of extracted gDNA). 100 ng of RNA was used for library preparation (2% of the total amount of extracted RNA). Clonotype numbers for TCRa/b libraries are shown from each company (NT: not tested). In the comparison, TCRv2 generated an average of 48.7K and 163K clonotypes for TRA and TRB, respectively, representing a 290% increase against Company X's RNA-based approach and a 145% increase against Company Y¡¯s gDNA-based approach. Importantly, the RNA methods used only 2% of the total RNA from the 5M PBMCs.
ÀÚ¼¼ÇÑ ±¸¼ºÀº CoA¸¦ Âü°íÇϼ¼¿ä.
Citation
‘SMARTer® Human TCR a/b Profiling Kit v2’ Á¦Ç°Àº ‘SMART-Seq® Human TCR (with UMIs)’À¸·Î Á¦Ç°¸íÀÌ º¯°æµÇ¾ú½À´Ï´Ù (ÀÚ¼¼ÇÑ ³»¿ëÀº Á¦Ç°ÆäÀÌÁö¸¦ ÂüÁ¶). CitationÀÌ µ¿ÀÏÇÕ´Ï´Ù.

Myelodysplastic Syndrome Progression to Acute Myeloid Leukemia at the Stem Cell Level
Nature medicine    |    2018 Oct 31    |    PubMed ID: 30510255    |    Read Article

An immunodominant NP 105-113 -B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease
Nature immunology    |    2021 Oct 27    |    PubMed ID: 34853448    |    Read Article

Polyclonal expansion of TCR Vbeta 21.3 + CD4 + and CD8 + T cells is a hallmark of Multisystem Inflammatory Syndrome in Children
Science immunology    |    2022 Feb 04    |    PubMed ID: 34035116    |    Read Article

Clinical outcome following checkpoint therapy in renal cell carcinoma is associated with a burst of activated CD8 T cells in blood
Journal for Immunotherapy of Cancer    |    2022 Dec 14    |    PubMed ID: 35863822    |    Read Article

Evaluating the efficacy of a priming dose of cyclophosphamide prior to pembrolizumab to treat metastatic triple negative breast cancer
Journal for Immunotherapy of Cancer    |    2022 Dec 14    |    PubMed ID: 35121644    |    Read Article

Epstein–Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report
Frontiers in Immunology    |    2021 Nov 16    |    PubMed ID: 34868006    |    Read Article

Functional changes in cytotoxic CD8+ T-cell cross-reactivity against the SARS-CoV-2 Omicron variant after mRNA vaccination
Frontiers in Immunology    |    2023 Jan 04    |    Read Article

Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data
Frontiers in Immunology    |    2022 Nov 18    |    Read Article

Features of repertoire diversity and gene expression in human cytotoxic T cells following allogeneic hematopoietic cell transplantation
Communications Biology    |    2021 Oct 11    |    PubMed ID: 34635773    |    Read Article

Assay and Isolation of Single Proliferating CD4+ Lymphocytes Using an Automated Microraft Array Platform
IEEE Transactions on Biomedical Engineeri…    |    2020 Aug 01    |    Read Article

Abstracts from the 53rd European Society of Human Genetics (ESHG) Conference: Interactive e-Posters
European Journal of Human Genetics    |    2020 Dec 01    |    PubMed ID: 33262485    |    Read Article

Staphylococcus aureus Isolated from Skin from Atopic-Dermatitis Patients Produces Staphylococcal Enterotoxin Y, Which Predominantly Induces T-Cell Receptor Vα-Specific Expansion of T Cells
Infection and Immunity    |    2020 Feb 01    |    PubMed ID: 31740530    |    Read Article

Genomic profiling of intestinal T-cell receptor repertoires in inflammatory bowel disease.
Genes and immunity    |    2021 Jan 06    |    PubMed ID: 32029881    |    Read Article

Clinical and immunological response to checkpoint therapy in renal cell carcinoma is associated with TCF1+ CD8 T-cells in the tumor
ppr0375699    |    2021 Jul 28    |    Read Article

Intratumoral mregDC and CXCL13 T helper niches enable local differentiation of CD8 T cells following PD-1 blockade
bioRxiv    |    2022 Jun 26    |    Read Article

Superantigenic TCR Vbeta 21.3 signature in Multisystem Inflammatory Syndrome in Children
medRxiv    |    2021 Feb 12    |    Read Article

Human thymopoiesis selects unconventional CD8 + α/β T cells that respond to multiple viruses
bioRxiv    |    2020 Jul 29    |    Read Article

>> ³í¹® ´õ º¸±â (¿¬°áµÇ´Â ÆäÀÌÁö ÇÏ´ÜÀ» Âü°íÇØÁÖ¼¼¿ä)

Keyword : TCR,CDR3,T cell receptor,repertoire,variable region,Immune cell,Immune-seq,Immunology,UMI,Unique molecular index,Cogent.,SMART,smart-seq,NGS,Next generation sequencing,cancer,Cancer research,¾Ï¿¬±¸,vaccine,¹é½Å,¹é½Å¿¬±¸

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