• Unique Molecular Index (UMI) 적용으로, v2 업그레이드 - PCR 혹은 sequence error로 인한 reads 제거로 신뢰도 있는 결과 분석
• 다양한 범위의 샘플 적용 가능 - peripheral blood leukocytes (10 ng - 1 ug), T cell 유래 total RNA (1 - 100ng), whole T cell (1,000 - 10,000 purified cells)
• 간단한 PCR 과정 - TCR-alpha, TCR-beta chain을 동시에 또는 개별적으로 NGS Library 제작 가능
• Unique Dual Index (UDI) 사용으로 분석 양 증가 - multiplexing 샘플 수 증가 및 high-throughput sequencer 적용
• Illumina^® platform 분석용 libraries 제작 - 모든 Illumina^® 기기에 적용 가능 및 MiSeq™ system에서 full-length V(D)J와 CDR3 분석 가능
• Complete workflow - 고품질의 TCR profiling analysis를 위한 Cogent NGS Immune Profiler Software 제공 (연구용, 상업적 용도에서 모두 무상 사용 가능)

제품 설명

SMARTer Human TCR a/b Profiling Kit v2 (TCRv2) 탁월한 감도와 재현성을 가진 TCR repertoire의 분석을 위한 Illumina platform NGS Library 제작 키트이다. 본 제품은 SMART (Switching Mechanism at 5’ End of RNA Template)의 full-length cDNA 합성 기술과 TCR 전사체의 V(D)J 가변영역 (variable region) 내 TRA, TRB 유전자 분석을 위하여 5’ RACE 유사 기술을 접목하였다.
본 제품을 이용하면 RNA 샘플 (RIN값 8 이상)로부터 고품질의 library를 제작할 수 있으며, peripheral blood leukocytes (10 ng - 1 ug), T cell 유래 total RNA (1 - 100 ng), whole T cell (1,000 - 10,000 purified cells)에 적용할 수 있다. 또한, TCR-α, TCR-β chain을 동시에 또는 각 chain에 대한 라이브러리를 제작하여 분석할 수 있다. 제품 내에는 Illumina platform에서 분석 가능한 dual index (UDI)를 포함할 뿐 아니라, unique molecular identifiers (UMIs)를 포함하고 있어, PCR 과정 혹은 sequence error로부터 유래한 reads를 제거하여 분석함으로써 보다 정확하고 신뢰도 있는 결과를 얻을 수 있다.


그림 1. 다양한 RNA 샘플 양으로부터 감도 높게 재현성 있는 clonotype 검출
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%

표 1. TCRv2로 분석했을 때, 감도와 재현성 평가
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^® 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.


그림 2. TCRv2의 우월한 감도와 재현성
We split 5M PBMC cells from two different healthy donors for RNA and gDNA extraction. 1.6 μg 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.

자세한 구성은 CoA를 참고하세요.