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Home > ÀüÁ¦Ç°º¸±â > NGS °ü·Ã > Immune-Seq > [Version 2] Human TCR a/b Profiling Kit v2
TCR repertoire¸¦ Ź¿ùÇÑ °¨µµ·Î ºÐ¼®

[Version 2] Human TCR a/b Profiling Kit 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
2,899,200¿ø 
3,624,000¿ø
°¡°ÝÇÒÀÎ
05.02 ~ 06.30
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
6,329,600¿ø 
7,912,000¿ø
°¡°ÝÇÒÀÎ
05.02 ~ 06.30
Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â

* º» Á¦Ç°Àº ±âÁ¸ SMARTer¢ç Human TCR a/b Profiling Kit (Code 635014)ÀÇ ¾÷±×·¹ÀÌµå ¹öÀü Á¦Ç°ÀÔ´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 - °íǰÁúÀÇ 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 °íǰÁúÀÇ 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÷ÇÔÇϰí 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.

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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