T-cell receptor profiling

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Why single-cell TCR analysis is needed

Benefits of studying T cells at the single-cell level. Panel A. Schematic of a T-cell receptor comprised of an alpha chain (TCR-α) and a beta chain (TCR-β). Panel B. Schematic representing the difficulties of obtaining pairing information for alpha and beta chains from bulk sequencing data. Rare clonotypes can allow for assessing the pairing of some cells (clonotypes in orange). However, for the more highly expressed clonotypes (TCRA V2J1, TCRA V1J2, TCRB V3D1J1, TCRB V2D1J2) there are four possible pairing combinations.

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Bioanalyzer traces from example TCR libraries

Electropherogram profiles of TCR sequencing libraries. Electropherograms from three different cell pools (see technical note for more details): Pool 9 (Panel A), 10 (Panel B), and 11 (Panel C). The variation in the profiles reflects the abundance of amplified TCRa (peak at ~900 bp) and TCRb (peak at ~700 bp). Panel D. Positive Control RNA TCR library (generated with 8 x 5 pg Control Jurkat Total RNA). Panel E. Negative Control TCR library shows no library is produced.

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Cell-type calling and alpha-beta pairing analysis of a mixed population of cells

Analysis of a mixed cell population from a 96-well plate. Panel A. Cell-type calling based on the identified clonotypes for each well. The seven omitted cells did not have clonotype calls for either TCRa or TCRb with read numbers that were above the threshold. Panel B. Analysis of pairing information. Paired TCR-αβ chains were obtained for 34 cells in the plate. Panel C. The alpha, beta, or alpha-beta pairing information represented as a percent of cells analyzed. Omitted cells were not included in this analysis.

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The SMARTer scTCR workflow uses optimized indexing to allow for pooling 96 cells into 12 libraries.

SMARTer Human scTCR a/b Profiling Kit workflow and pooling strategy. Panel A. First-strand cDNA synthesis is dT-primed (RT Primer) and performed by an MMLV-derived reverse transcriptase (RT), which adds nontemplated nucleotides to the 5' end of each mRNA template. The SMART-Seq Indexed Oligos anneal to these nontemplated nucleotides and serve as a template for the incorporation of an additional sequence of nucleotides into the first-strand cDNA by the RT (this is the template-switching step). Each of the eight different SMART-Seq Indexed Oligos provided in the kit each contains a unique six-base in-line index that serves as a cell barcode to allow downstream cell identification after pooling. The additional sequence added to the cDNA by the RT—referred to as the "SMART sequence"—serves as a primer-annealing site for subsequent rounds of PCR, ensuring that only sequences from full-length cDNAs undergo amplification. After pooling (described in Panel B) and a cleanup step, two rounds of gene-specific PCR are performed in succession to amplify cDNA sequences corresponding to variable regions of TCRa and/or TCRb transcripts. The first gene-specific PCR uses the amplified double-stranded cDNA as a template and includes a forward primer with complementarity to the SMART sequence—which also incorporates the Illumina Read 2 sequence (TCR Primer 1)—and reverse primers that are complementary to the constant (i.e., nonvariable) region of TCRa and TCRb (TCR a/b Human Primer 1). The second round of PCR takes the product from the first PCR as a template and uses a forward primer that binds to the Read 2 sequence added by the previous PCR step. The reverse primers bind in the constant region, internal to the PCR1 primers (TCR a/b Human Primer 2 Reverse HT Index) allowing amplification of the entire variable region and a portion of the constant region of TCRa and TCRb cDNA. The forward and reverse primers include adapter and index sequences that are compatible with the Illumina sequencing platform and allow for multiplexing of up to 96 samples in a single flow-cell lane. Panel B. Samples are pooled by column, such that each pool contains eight cells each with a differently indexed SMART-Seq Indexed Oligo. Different combinations of the Forward and Reverse HT indexes are used during PCR 2 to allow multiplexing of the samples into a single flow-cell lane (see the User Manual for more details).

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634431: SMARTer Human scTCR a/b Profiling Kit

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640182: ICELL8 Human TCR a/b Profiling Reagent Kit

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Sequencing reads on target and correlation of clonotype count data for varying sample input amounts

Sequencing reads on target and correlation of clonotype count data for varying sample input amounts. Panel A. Percentages of sequencing reads that map to CDR3 regions in either TCR-α (blue) or TCR-β (purple) or that represent off-target reads (gray). The experimental protocol was performed on three different amounts of peripheral blood RNA: 10 ng, 100 ng, and 1,000 ng. Panel B. Correlation of clonotype count data for 100 ng input RNA vs. 1,000 ng input RNA. Pearson (R) and Spearman (ρ) correlation coefficients are included.

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Assessing the sensitivity and reproducibility of the SMARTer approach

Assessing the sensitivity and reproducibility of the SMARTer approach. The experimental protocol was performed in replicate on PBMC RNA samples spiked at varying concentrations (10%, 1%, 0.1%, 0.01%, and 0.001%) with RNA obtained from a homogenous population of Leukemic Jurkat T cells (TRAV8-4-TRAJ3, TRBV12-3-TRBJ1-2 clonotype). Panel A. Correlation between concentration of spiked-in Jurkat RNA and number of TRBV12-3-TRBJ1-2-specific sequence reads. Numbers along the X-axis indicate serial-diluted concentrations of spiked-in Jurkat RNA (by mass): 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = 0.01%; 5 = 0.0.01. Count data for TRBV12-3-TRBJ1-2-specific sequence reads were normalized by subtracting the number of corresponding reads obtained for negative control samples consisting of unspiked PBMC RNA. Normalized count data were then Log₁₀ transformed. Circles and triangles correspond to experimental replicates for each sample concentration. Results of linear regression analysis are indicated in the upper right region of the graph. Panel B. Count data, signal-to-noise ratios, and statistical analysis for TRBV12-3-TRBJ1-2-specific sequence reads obtained from spiked RNA samples. Signal-to-noise ratios were generated using the mean counts of TRBV12-3-TRBJ1-2-specific sequence reads for each pair of experimental replicates. Rows highlighted in yellow include concentrations of spiked-in Jurkat RNA for which statistically significant elevations in TRBV12-3-TRBJ1-2-specific sequence reads were detected relative to background counts observed for unspiked negative control RNA samples.

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Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach

Library preparation workflow and PCR strategy for TCR profiling using the SMARTer approach. Panel A. Reverse transcription and PCR amplification of TCR subunit mRNA sequences. First-strand cDNA synthesis is primed by the TCR dT Primer and performed by an MMLV-derived reverse transcriptase (RT). Upon reaching the 5′ end of each mRNA molecule, the RT adds non-templated nucleotides to the first-strand cDNA. The SMART-Seq v4 Oligonucleotide contains a sequence that is complementary to the non-templated nucleotides added by the RT, and hybridizes to the first-strand cDNA. In the template-switching step, the RT uses the remainder of the SMART-Seq v4 Oligonucleotide as a template for the incorporation of an additional sequence on the end of the first-strand cDNA. Full-length variable regions of TCR cDNA are selectively amplified by PCR using primers that are complementary to the oligonucleotide-templated sequence (SMART Primer 1) and the constant region(s) of TCR-α and/or TCR-β subunits (TCR a/b Human Primer 1). A subsequent round of PCR is performed to further amplify variable regions of TCR-α and/or TCR-β subunits and incorporate adapter sequences, using TCR Primer 2 and TCR a/b Human Primer 2. Included in the primers are adapter and index sequences (read 2 + i7 + P7 and read 1 + i5 + P5, respectively) that are compatible with the Illumina sequencing platform. Following purification, size selection, and quality analysis, the TCR cDNA library is ready for sequencing. Panel B. Semi-nested PCR approach for amplification of TCR-α and/or TCR-β subunits. The primer pairs used for the first round of amplification capture the entire variable region(s) and most of the constant region(s) of TCR-α and/or TCR-β cDNA. The second round of amplification retains the entire variable region(s) of TCR-α and/or TCR-β cDNA, and a smaller portion of the constant region(s). The anticipated size of final TCR library cDNA (inserts + adapters) is ~700–800bp.