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From a pool of activated T cells transduced with two different, FDA-approved CAR T-cell products, samples were processed with the leading two TCR single cell technologies. Results were compared for performance and data quality.
Human T cells were isolated from healthy PBMC donors, in vitro activated (anti-CD3, anti-CD28, IL2) and transduced with a lentivirus to introduce a CAR T-cell receptor. The constructs CD19 4-bb zeta, and CD19 CD28 zeta, target CD19 as therapy for B-cell lymphoma.
After ex vivo expansion, the cell cultures were prepared for the Parse Evercode TCR workflow or 10x Genomics Chromium Next GEM Single Cell 5' Kit v2 with a Chromium Single Cell Human TCR Amplification Kit.
The libraries were then sequenced on an Illumina NextSeq 550 instrument, with FASTQ files processed using each technology’s specific pipeline. A single Parse sub-library was compared with a lane from the Chromium product. Sequencing results were compared after down-sampling to approximately 8k mean reads per cell for the whole transcriptome and 4k mean reads for the TCR data.
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The Parse assay includes separate libraries and pipelines for the TCR and whole transcriptome data. Compared to the Chromium results, a highly similar fraction of cells with paired TCR chains and the number of genes per cell, were identified. All the major T cell subtypes are observed in both approaches.
A significantly higher fraction of ribosomal proteins were observed in the Chromium dataset.
Using the Evercode TCR assay, 5-fold more unique cells and clonotypes were obtained from a single sub-library than a single lane of the Chromium V(D)J assay. We note that a large TCR sample size is needed to observe rare clonal types and to track clones over time. A large sample size is important for observing sufficient numbers of T cells, given the highly variable proportion of T cells in different tissues, and effects of different treatment regimes. In addition, larger TCR samples provide a more accurate measure of T cell repertoire diversity (i.e., clonality) and V, D, and J gene usage.
With the Evercode TCR assay, samples are collected over time, fixing and running them all at once. This reduces batch effects and enables us to scale a research program.
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