Splice variants or alternative isoforms play an important role in the understanding of human health and disease. It is estimated that nearly all protein-coding genes in the human genome are alternatively spliced, providing an essential source of protein diversity (Pan et al. 2008; Wang et al. 2008). As many cancer-associated genes are regulated by alternative splicing, tumor-specific splice variants have clear diagnostic value as biomarkers and may serve as potential drug targets in novel therapeutics (Zhang et al. 2021).
One of the toughest challenges in biology and medicine today is to map genotypes to phenotypes, which can be tackled by performing transcriptomics analysis via single-cell mRNA sequencing (Hwang et al. 2018). Full-transcriptome mRNA sequencing enables the discovery of rare biological events such as gene fusions, SNPs, and alternative splicing, which is an essential first step to new advancements in medicine.
Though there are plate-based methods for full-length scRNA-seq, they often lack integrated analysis tools. Our complete, automated solution for enhanced biomarker detection includes optimized SMART-Seq chemistry on the ICELL8 cx Single-Cell System and Cogent NGS bioinformatics tools. This streamlined workflow maintains the robust detection of genes and transcripts seen with plate-based kits like Takara Bio’s SMART-Seq v4 and surpasses common homebrew methods like Smart-seq2 (Figure 1). With the full-length coverage of our SMART-Seq Pro kit, you can identify clinically relevant novel biomarkers with confidence.
To demonstrate automation of in-depth scRNA-seq using the SMART-Seq Pro kit for the ICELL8 cx Single-Cell System, Takara Bio scientists recreated part of a transcript isoform study from Peking University (Li et al. 2020). This research focused on identifying and characterizing gene isoforms related to non-small cell lung cancer. The study required a full-length transcriptomics approach to explore these isoforms of interest and used plate-based RNA-seq to complement the limitations of 10x Chromium data.
Their study focused on the Protein Tyrosine Phosphatase Receptor Type C (PTPRC) gene, which encodes the transmembrane tyrosine phosphatase, CD45, and has several isoforms with variations across many exons. PTPRC isoforms are critical for T-cell receptor (TCR) signal transduction and are important for regulating tumor-infiltrating T cells. The authors further performed clonal analysis to show that isoform switching occurs during T-cell activation and differentiation.
They observed that phenotypic naïve CD8+ T cells—cells that are mature but not yet activated—were represented dominantly in the PTPRC-209 isotype. Exhausted CD8+ T cells, cells with progressive loss of function due to prolonged antigen stimulation, were most represented in the PTPRC-201 isotype. Unique exon spanning profiles for PTPRC-201 and PTPRC-209 that were uncovered through the study are shown in Figure 2.
Characterization of PTPRC using the SMART-Seq Pro kit and ICELL8 cx system
We adapted the reference study’s approach for the ICELL8 cx Single-Cell System, Takara Bio’s automated platform for creating full-length scRNA-seq libraries. Our comparative study focused on the PTPRC gene to see if isoform patterns similar to the reference study would be found in PBMCs.
The experiment was carried out using the SMART-Seq Pro protocol for the ICELL8 cx system. Data were analyzed using Cogent NGS Analysis Pipeline (CogentAP) and Cogent NGS Discovery Software (CogentDS) tools, freely available for ICELL8 cx users.