To educate and inform the scientific community about our cutting-edge NGS solutions, Takara Bio staff frequently presents scientific posters at major conferences around the world. Included below are scientific posters featuring methods and data for a wide array of our NGS applications.
A complete, ultra-low input RNA-seq solution for full-length transcriptome analysis and RNA counting
Objective: RNA sequencing (RNA-seq) is a powerful way to investigate transcriptional highs and lows, allelic origins, and isoform preferences in the transcriptome that can underlie key biological states. One current limitation of single-cell RNA-seq methodologies is either the absence of unique molecular identifiers (UMIs), or the inability to maintain the yield, sensitivity, and reproducibility when UMIs are employed.
Methods: To test the yield, sensitivity, and reproducibility, we benchmarked a new SMART-Seq method, SMART-Seq mRNA LP (with UMIs), against the existing SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (SSv4), and the Smart-seq 2 homebrew method (SS2). We included our novel library prep method in the testing to determine if a complete, end-to-end solution improved the data outcome. In addition, we tested the performance of this new method on common automation platforms.
Results: Gene count and read distribution across major RNA-seq output components were comparable between SMART-Seq mRNA LP (with UMIs) and SSv4. However, the new method showed significantly increased sensitivity compared to the SS2 homebrew method. In addition, we demonstrate that SMART-Seq mRNA LP (with UMIs) can enable RNA counting, and while optimized for low RNA input, is compatible with single-cell RNA-seq analysis. Finally, we show that SMART-Seq mRNA LP (with UMIs) is compatible with common automation platforms.
Conclusions: Our data demonstrate that SMART-Seq mRNA LP (with UMIs) leveraging SMART technology with UMIs for cDNA generation and our unique library preparation protocol, combined with our Cogent NGS analysis software (CogentAP), is a complete, robust, and sensitive solution for full-length transcriptome studies. The inclusion of UMIs allowed for RNA counting without compromising data quality, and lead to superior sensitivity compared to homebrew SS2 chemistries.
Development of a complete, end-to-end, automated single-cell system for biomarker discovery
Many RNA transcripts undergo post-transcriptional modification such as alternative splicing, with tumor-specific isoforms showing clear diagnostic value as biomarkers. Full-length single-cell mRNA sequencing has contributed to the discovery of these rare biological events; however, automated, high-throughput workflows are desired. Conventional plate-based methods cannot satisfy increasing research needs due to limited sample throughput and lengthy hands-on time. We developed the SMART-Seq Pro Application Kit, which carries out full-length transcriptome analysis on the ICELL8 cx Single-Cell System. Combining the application kit with the included Cogent NGS bioinformatics tools creates an end-to-end, automated solution for biomarker discovery. We compared the sensitivity of the SMART-Seq Pro Application Kit against the Smart-seq2 (SS2)1 homebrew method and assessed the ability of the SMART-Seq Pro kit to correctly identify clinically relevant splice variants of the PTPRC gene. We showed that SMART-Seq Pro kit detects more genes than SS2, highlighting the kit’s ability to reveal rare biological events. The SMART-Seq Pro kit also successfully identified PTPRC isoforms of interest, which were missed by 3' end-counting methods. These data illustrate how the SMART-Seq Pro kit for the ICELL8 cx Single-Cell System can characterize clinically relevant isoforms and, together with Cogent NGS analysis tools, offer an efficient way to accelerate translational research by providing insight of important biomarkers in single-cell samples.
Efficient and sensitive high-throughput human B-cell receptor repertoire profiling using SMART technology
Objective: B-cell receptor (BCR) repertoire profiling is increasingly used in health and pathogenic contexts with the goal of biomarker discovery. However, current sequencing technologies are limited in their ability to generate data accurately and reproducibly for all BCR isotypes. To overcome these limitations, we have developed a new kit to accurately profile all heavy (A, D, E, G, M) and light-chain (K, L) isotypes—an end-to-end solution, from library preparation to streamlined data analysis. Here we present data on an updated approach for efficient and high-throughput BCR repertoire profiling of human samples.
Methods: Libraries were prepared from human peripheral blood cells (10 ng–1 μg total RNA) or from B-cells (1 ng–100 ng total RNA) using our new human BCR repertoire profiling kit (~2.5 hours hands-on time). Prepared libraries were then analyzed on the Illumina® Miseq® benchtop sequencer using 300-bp paired-end reads.
Results: For each library, >90% of sequencing reads were on- target while the most highly represented clonotype was found to remain consistent among technical duplicates across a range of input amounts. In comparison to the previous version of our BCR-sequencing kit, the new approach enabled a ~4x increase in total clonotype count observed across various RNA inputs. Furthermore, a sensitivity assay demonstrated that B-cell RNA corresponding to a single clonotype could be detected above background levels when spiked into input total RNA at a relative concentration of 0.001%.
Conclusions: Our new human BCR repertoire profiling kit was found to accurately and reproducibly profile B-cell clones and provide information on the diversity of BCR repertoire in human samples.
Highly reproducible TCR profiling using rNA from rhesus macaque PBMC
Non-human primates (NHP) such as the rhesus macaque (Macaca mulatta) have long been key translational models in biomedical research because of their genetic and physiological similarity to humans. Studies using rhesus macaques have contributed significantly to our understanding of T-cell responses to vaccines, cancer, and infectious diseases. More recently, these NHP have emerged at the forefront of COVID-19 vaccine research.
Increasingly complex information can now be gleaned from immune system processes. High-throughput TCR sequencing (TCR-seq) profiles T-cell responses in exquisite detail. A comprehensive understanding of immune responses in such a closely related organism as the rhesus macaque would be a significant advance in science.
Numerous tools exist for performing TCR-seq in human samples, but equivalent tools for rhesus samples have been lacking. Because rhesus macaques often serve as surrogates in the lead-up to human studies, there is an industry need for a complete TCR-seq solution for these NHP samples.
Due to strong species homology, our SMARTer Human TCR a/b Profiling Kit v2 (TCRv2) can generate high-quality TCR sequencing libraries using human or rhesus macaque RNA.
Previous years' posters sorted by application
Whole transcriptome analysis
High-throughput automation of single-cell mRNA-seq
DNA-seq from cfDNA
ChIP-seq for low-input DNA
Whole exome sequencing
High-throughput automation of DNA-seq
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