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Advances in genomics and transcriptomics continue to catapult basic and clinical research into a new era. A more comprehensive examination of genomic and transcriptional states allows for high-resolution mapping of disease-driving cellular and molecular events and boosts the potential for novel biomarker detection.
Two complementary advances—full-length transcriptome and whole-genome sequencing of single cells at large scale—will have a lasting impact on oncology research as these technologies facilitate unprecedented characterization of heterogeneity in malignancies. Full-length transcript coverage is needed to identify RNA splicing isoforms and different RNA species like long-noncoding RNAs that underlie tumorigenesis, prognosis, and therapeutic response. Single-cell whole-genome amplification plays an important role in the detection of CNVs and SNVs for tumor subclonal analysis.
The choice of library preparation and sequencing approaches greatly influences the quality and depth of information produced. For example, commonly used library preparation chemistries for short-read sequencing, including those for droplet-based end counting, lack the required sensitivity and coverage that full gene-body single-cell RNA-seq affords.
Automated, High-Throughput Library Prep
Takara Bio’s Shasta™ Single Cell System is a simplified, high-throughput NGS library preparation solution with well-validated chemistries and intuitive bioinformatics tools. The system allows researchers to mine more genomic and transcriptomic information, efficiently and cost-effectively, from many more cells than previously possible.
“We’re very excited about this technology,” said early-access user Ting Wang, PhD, Head of the Department of Genetics at Washington University School of Medicine in St. Louis. “We’re fully committed to continue pushing the limits of the technology to do the best science.”
The Shasta Single Cell System has three core elements: a state-of-the-art, automated dispensing and imaging platform that isolates single cells; an array of optimized NGS library prep chemistries; and easy-to-use Cogent™ bioinformatics software for comprehensive data analysis and production of publication-quality figures.
High-Throughput Single-Cell Total RNA Profiling with Full Gene-Body Coverage
Current single-cell RNA-seq library prep methods with full gene-body coverage are not sufficiently scalable to satisfy high-throughput needs (i.e., existing methods are limited to up to 384 single cells per run). Plus, widely used end-counting technologies lack full gene-body coverage, providing only partial information.
Based on random priming, the Shasta Total RNA-Seq Kit is the first high-throughput full gene-body total RNA-seq kit scaled to support library prep for up to 100,000 single cells and up to 12 samples per experiment. Full-length transcript coverage allows detection of critical events such as alternative splicing and gene fusions. Capturing total RNA information, including from protein-coding and noncoding genes, is particularly valuable for the discovery of novel biomarkers.
High-Throughput Single-Cell Whole-Genome Amplification
Single-cell DNA-seq was developed to understand driving mutations such as CNVs in individual cells of tumor tissues to identify actionable biomarkers and support the development of novel interventions. Although current targeted panels can profile single-cell CNVs in a high-throughput manner, they lack whole-genome coverage and cannot be used to detect segmental CNVs. Existing whole-genome amplification methods, which are plate-based, do not satisfy high-throughput needs.
The Shasta Whole-Genome Amplification (WGA) Kit allows preparation of single-cell WGA libraries for up to 1,500 single cells per experiment. This facilitates the reliable detection of CNVs for tumor subclonal analysis at a shallow sequencing depth of only 250,000 read pairs per cell. Moreover, Cogent software can assess single-cell SNVs using pseudobulk data from cells clustered based on their CNV profile. Nanoliter-scale total reaction volume per cell and automated dispensing offer a significant reduction in reagent use and hands-on time compared to plate-based methods.
Together, these two novel high-throughput Shasta library prep and analysis workflows assist biomarker discovery for researchers beginning to take advantage of single-cell profiling.
Explore Shasta system data at takarabio.com/more.
