Association of Biomolecular Resource Facilities (ABRF) Annual Meeting
Takara Bio was proud to be a platinum sponsor of the 2022 ABRF Annual Meeting
The ABRF Annual Meeting brought together key decision makers, technology users, and leaders in scientific core disciplines to address important issues encountered in biomolecular resource facilities. We shared our complete, novel solutions that drive biomarker discovery for research cores and their customers.
2022 talks and posters
Platinum Sponsor seminar: a complete, ultra-low input RNA-seq solution for full-length transcriptome analysis and RNA counting
Abstract
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. To address these problems, we developed a new SMART-Seq method that includes the use of unique molecular identifiers (UMIs) for RNA counting to allow for allelic and transcript isoform resolution analysis. We benchmarked the new SMART-Seq method against the existing SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (SSv4), and two relevant homebrew methods, Smart-seq2 (SS2) and Smart-seq3 (SS3). The inclusion of UMIs did not compromise data quality and led to superior sensitivity compared to homebrew SS2 and SS3 chemistries. In addition, we show that our new SMART-Seq method can enable RNA counting, while optimized for low RNA input, is compatible with single-cell RNA-seq analysis. Our data demonstrate that the new SMART-seq method leveraging SMART technology with UMIs for cDNA generation and our unique library preparation protocol, combined with our Cogent NGS analysis software, is a complete, robust, and sensitive solution for full-length transcriptome studies.
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 (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 (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 (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 (with UMIs) is compatible with common automation platforms.
Conclusions: Our data demonstrate that SMART-Seq mRNA (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.
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.
Previous talks and posters
2020
Platinum Sponsor seminar: library preparation workflows for the new age of high-throughput sequencing
We presented the latest solutions from Takara Bio for DNA-seq and RNA-seq with low-input and challenging samples. This includes the SMART-Seq Single Cell Kit, a system that enables library preparation from single cells with unprecedented sensitivity. We also presented the new ThruPLEX HV family of products for high-performance DNA library preparation from challenging samples like FFPE, cell-free DNA, and microbial samples.
Innovation Theater seminar: single-cell application development with the ICELL8 cx system
We presented our solutions for plate-based and automated single-cell sequencing: our SMART-Seq Single Cell Kit, designed to generate high-quality, full-length cDNA directly from single cells with very low input, and the ICELL8 cx Single-Cell System, with its open-platform software allowing researchers to design custom automated, targeted assays on up to eight samples per run, for up to 1,500 sample wells.
ThruPLEX HV: a simplified system for preparation of molecular-tagged NGS libraries from FFPE and cell-free DNA
We presented a complete, fast, modular NGS library prep system that enables accurate, reproducible sequencing readout from highly challenging sample types. The system's features include: 1) a higher input volume to maximize library complexity, 2) tunable enzymatic fragmentation that does not add any time to the workflow, 3) optional molecular tags for detection of low-frequency mutations, and 4) optimized conditions throughout to ensure a broad range of accurate GC representation. Performance data is demonstrated for cell-line DNA, FFPE DNA, cell-free DNA, and microbial DNA.
Pushing the limits of single-cell RNA-seq with SMART-Seq single cell technology
Since the emergence of next-generation sequencing (NGS), the importance of and demand for single-cell analysis have risen rapidly. Extracting meaningful biological information from the small amount of mRNA present in each cell requires an RNA-seq preparation method with exceptional sensitivity and reproducibility. 3' droplet-based sequencing has been the primary method used to date. However, using droplet sequencing and full-length mRNA information in parallel has become an emerging requirement to help generate and better understand rich single-cell datasets. To address this need, we developed the SMART-Seq Single Cell Kit (SSsc) using new chemistry with unparalleled sensitivity and a highly scalable, easily automatable workflow.
The SMART-Seq Single Cell Kit outperforms all current commercial and noncommercial full-length methods, particularly with as little as 2 pg of total RNA. When validating with a B lymphocyte cell line or peripheral blood mononuclear cells (PBMC) from a healthy donor, we were able to detect 50–60% more genes with the new chemistry compared to current methods. The improvement in sensitivity was associated with a clear reduction of the dropout rate as well as an increase in reproducibility. This unparalleled sensitivity continues to be seen with automation and miniaturized workflows.
These features make SSsc chemistry extremely useful for difficult cells—e.g., clinical research samples that often have very low RNA content—making it ideal for highly detailed characterization of precious samples.
Efficient high-throughput sequencing for quantitative immune profiling using unique molecular identifiers
Next-generation sequencing (NGS) for immune repertoire profiling has become a powerful tool for understanding the role of the adaptive immune system in health and disease. Additionally, unique molecular identifiers (UMI) have become a vital aspect of this approach and are used for preserving quantitative information (i.e., accurate clonotype counts) of the repertoire by removing PCR/sequencing errors and duplicates. In the experiment presented in this poster, we integrated UMI with our SMART technology to detect genuine low-frequency events in full-length variable regions of B-cell receptor (BCR) genes and T-cell receptor (TCR) genes. For each library, >90% of reads were on-target, and the most highly represented clonotypes remained consistent among the technical duplicates in the range of 10 ng–1 µg of input RNA or 50–10,000 cells. A sensitivity assay demonstrated RNA transcripts corresponding to multiple UMIs could be detected when spiked into input RNA at a relative concentration of 0.001%. We also developed software to analyze multiple sequence data with UMI to generate detailed stats for reads, clonotype, UMI, and mapping rate as output. The updated human TCR profiling kit, SMARTer Human TCR a/b Profiling Kit v2 (TCRv2), is designed to be compatible with any Illumina platform using 2 x 150 bp reads. Moreover, unique dual indexes (UDI) were incorporated to avoid crossover contamination caused by index hopping. Thus, our immune profiling technology can be used to observe clonal selection and hypermutation events in rare clonotypes found in blood and tumor tissues. These methods could also serve as a basis for the discovery of antibody-based therapeutics.
Robust and sensitive detection of gene fusions using high-throughput SMART-Seq chemistry on the ICELL8 cx system
Isolating single cells at high-throughput levels and obtaining their full-length transcript information has become critical to the scientific community to generate rich single-cell datasets. We automated SMART-Seq chemistry on the ICELL8 cx Single-Cell System to address this need and show this workflow provides useful information that end-capture technologies cannot. We present performance data showing that capturing junction and spanning reads with this automated, full-length mRNA-seq method enables confident and robust identification of gene fusions in a breast cancer tumor cell line.
Utilizing the Rheonix NGS OnePrep Solution to automate the Takara Bio ThruPLEX Tag-Seq HV library preparation kit
2019
Advances in performance and simplifications of workflows for single-cell RNA-seq and DNA-seq
In the past decade, we have observed significant advances in our knowledge of the activity of individual cells in heterogeneous tissue or culture. As more and more attention is paid to these differences, methods for extracting the most useful data are in high demand. Here we present application data demonstrating state-of-the-art workflow simplifications and high-resolution accuracy in data from RNA-seq using the SMART-Seq Stranded RNA-seq kit, and from DNA-seq using the PicoPLEX Gold Single Cell DNA-seq Kit.
SMART-Seq Stranded Kit performance with ovarian cancer
Single-cell RNA sequencing (scRNA-seq) approaches are increasingly being used to characterize the abundance and functional state of tumor-associated cell types, and have provided unprecedented detail into cellular heterogeneity. Extracting meaningful biological information from the small amount of RNA in single cells requires a library preparation method with exceptional sensitivity and reproducibility. The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (SMART-Seq v4) is an extremely sensitive scRNA-seq library preparation method in part due to its capability to retrieve information from full-length mRNA and not just the 3' end. However, this method can only capture polyadenylated mRNA. To address this, we have modified our SMART RNA-seq technology to create the SMART-Seq Stranded Kit, a single-cell RNA-seq library preparation method that relies on random priming instead of oligo dT priming. The SMART-Seq Stranded Kit captures any RNA regardless of polyadenylation status and preserves strand-of-origin information, making it more amenable for distinguishing overlapping genes and comprehensive annotation and quantification of long noncoding RNA (lncRNAs). To show the applicability of the SMART-Seq Stranded Kit in characterizing tumor heterogeneity, we analyzed single cells dissociated from a solid tumor in stage IV ovarian cancer (serous carcinoma). We sorted CD45+ leukocytes and EpCAM+ tumor cells in 96-wells plates. After library preparation, sequencing, and analysis, we detected an average of 4,717 genes in the CD45+ cells and 8,039 genes in the EpCAM+ tumor cells. This analysis enabled identification of well-accepted markers of tumor-infiltrating lymphocytes (TILs) associated with ovarian carcinoma.
Next-generation whole genome amplification methods for CNV and SNV detection from single cells
Preparation of amplified genomic material from small amounts of DNA or single cells is an essential research tool in assisting genetic analyses of clinical samples aimed at identifying the best treatment regimen and molecular diagnoses of diseases such as cancer. Technologies that allow for accurate, reproducible detection of single nucleotide variation (SNV) and copy number variation (CNV) of genomic material from limited samples need to do so with high fidelity and high genome coverage. Additionally, they should be flexible enough to be used in a variety of analysis platforms. To address these research needs, we have developed the PicoPLEX WGA V2 System, a platform-agnostic whole genome amplification system, and the PicoPLEX Gold Single Cell DNA-Seq Kit, a complete cells-to-library solution for Illumina sequencers. These systems use optimized enzymes, primers, and protocols for optimal sequencing coverage, uniformity, and accuracy to detect SNVs, all the while increasing the resolution for CNV detection relative to previous versions. Both systems maintain the technology's simple workflow and unmatched cell-to-cell reproducibility that is a hallmark of the PicoPLEX technology. In this study, we have demonstrated CNV detection to 5-mb resolution at a depth of 1 million read pairs in a single run, with validated copy-number gains and losses. SNV detection and reproducibility are shown to be superior to competitive technologies.
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