- CSHL Genome Engineering
- CSHL Single Cell Analyses
- LakePharma Symposium
- Molecular Med Tri-Con
- Next Generation Dx Summit
- Oxford Global NGS Congress
- PharmSci 360
- Single Cell Genomics
- Sake and Science 2018
Association of Biomolecular Resource Facilities (ABRF) Annual Meeting
Leading the way with automated solutions for next-generation sequencing workflows
The Association of Biomolecular Resource Facilities (ABRF) Annual Meeting brings together key decision makers, technology users, and leaders in scientific core disciplines to address important issues encountered in biomolecular resource facilities. We are proud to be a platinum sponsor of the ABRF 2020 meeting, "Empowering Team Science," from February 29 through March 3 in Palm Springs, California.
We are excited to help you find the best solutions for your research at ABRF 2020. In the meantime, we invite you to review our upcoming and past talks and posters, and you can sign up here to meet with one of our experts at the conference.
ABRF 2020: talks and posters
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
Previous ABRF talks and posters
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.
Advancements in NGS Library Preparation for Challenging Samples
Speaker: Suvarna Gandlur, Associate Director, NGS Marketing, Takara Bio USA, Inc.
Advancements in next-generation sequencing (NGS) have made the technology widely available to scientists, researchers, and clinicians trying to elucidate unanswered questions in life sciences, understand disease mechanisms, and develop more effective methods for diagnosis and treatment. Improvements to methods for preparing nucleic acids for sequencing have been an essential part of these efforts, allowing even smaller, degraded, and complex samples to be investigated by sequencing. Our SMARTer NGS portfolio has contributed to these advancements by enabling the generation of NGS libraries from single cells, low-input, and other challenging sample types and providing streamlined workflows that are efficient, convenient, automation-friendly, and less prone to errors. In this talk, we presented recent developments to our SMARTer ThruPLEX portfolio, which uses a novel chemistry and adapter design to enable an addition-only, true single-tube protocol that is highly sensitive and free of any intermediate transfer, cleanup, or optimization steps. We introduced a new technology that minimizes ligation-induced bias in microRNA library preparation, allowing microRNAs to be captured and analyzed with high accuracy, sensitivity, and reproducibility.
Confident detection of low-frequency mutations in cell-free DNA using SMARTer ThruPLEX technology with unique molecular tags
Liquid biopsies provide a noninvasive method to acquire the genetic information provided in cell-free DNA (cfDNA). Access to this genetic information through next-generation sequencing (NGS) identifies mutations and alterations that may play a role in cancer and other diseases. The key to identifying rare mutations is improved sequencing accuracy and the ability to distinguish between biological and PCR duplicates. SMARTer ThruPLEX Tag-seq was developed with the addition of unique molecular tags (UMTs) to improve sequencing accuracy by accounting for polymerase and sequencing errors and to increase confidence in rare allele identification. Libraries were prepared with SMARTer ThruPLEX Tag-seq using 10–30 ng of Horizon Discovery's Multiplex I cfDNA Reference Standard Set containing six single nucleotide variants (SNV) for four different genes (EGFR, KRAS, NRAS, PIK3CA) present at 0.5–5% allele frequency. The libraries were enriched with either a 110-kb or 240-kb custom panel or the Agilent ClearSeq Comprehensive Cancer Panel. Enriched libraries were sequenced with an average total read coverage of approximately 5,000X and analyzed with and without the UMTs. Deduplication without molecular tags reduced coverage to 295X; whereas, deduplication with UMTs allowed separation of biological duplicates from PCR duplicates and increased coverage to 2,110X, a significant reduction in false positives, 73% elimination of background noise, and a 10-fold increase in unique coverage compared to deduplication without UMTs. Employing UMT consensus reads, the sensitivity to detect 70 SNVs at 1% MAF was increased from 30% to 95% reads, and at 0.2% MAF increased from 7% to 75% and false positive calls reduced by 32X. Therefore, use of UMTs in the preparation of NGS libraries from cfDNA enhances sequencing accuracy: by distinguishing between biological duplicates and PCR duplicates, increasing read coverage and decreasing background noise, reducing false positives, and in more confident mutation calls.
An unbiased and highly reproducible method for constructing microRNA NGS libraries for accurate expression profiling
MicroRNA sequencing (miRNA-seq) is a useful tool for aiding researchers in the examination of miRNA expression patterns, the characterization of novel miRNAs, and for uncovering miRNA-disease associations. Since miRNAs are also unusually well-preserved in a range of specimens (e.g., urine, FFPE tissue, plasma), profiling their expression could become a powerful diagnostic tool. However, current methods for sequencing miRNA require large amounts of total RNA, are not very reproducible, and more importantly, have considerable systematic bias resulting in loss of many prospective biomarkers. This bias severely affects the trustworthiness of results as libraries are not a true representation of the biological state of the sample.
We have recently developed the SMARTer microRNA-Seq Kit that uses MAGIC (Mono-Adapter liGation and Intramolecular Circularization) technology to efficiently capture miRNA species with extremely low bias. Libraries prepared using an equimolar mixture of 963 miRNAs, sequenced on Illumina platforms and analyzed for read distribution reveal that >70% of miRNAs captured fall within a +/– 2-fold variation of the expected read number they should receive. This means that the expression level of ~70% of miRNAs in the equimolar mixture was truly and accurately represented. In contrast, frequency distribution analyses for kits from competitors N, I, and B revealed that 49–79% of miRNAs are greatly under-represented (i.e., less than 2X fewer reads than expected), 13–35% of miRNAs are represented within a +/– 2-fold variation of the expected read number, and around 8–14% are over-represented by more than 2X. These findings highlight the importance of understanding the current technical state of miRNA sequencing technologies to better prepare for analyzing and validating miRNA expression data. Additionally, the SMARTer microRNA-Seq Kit is designed and developed to more accurately reflect the true biological state of a sample, which will be an important factor as miRNA research moves toward diagnostic tools specific for personalized medicine.
High-throughput single-cell transcriptomics with SMART‑Seq technology
Since the emergence of next-generation sequencing (NGS), the importance and demand for single-cell analysis have risen rapidly. Single-cell RNA-seq generates data for various analysis methods such as differential gene expression, alternative splicing, gene fusion identification, and so on, which allow for cell profiling in great detail. As a result, single-cell RNA-seq has been gaining prominence not only in basic research fields but also in clinical fields. Extracting meaningful biological information from the small amount of mRNA present in a single cell requires a library preparation method with exceptional sensitivity and reproducibility. By providing the capability to obtain full-length mRNA sequence information (as opposed to merely capturing transcript 3' ends), the SMART‑Seq v4 Ultra Low Input RNA Kit for Sequencing (SMART‑Seq v4) offers the most advanced single-cell RNA-seq method on the market. However, this method is relatively low-throughput, while researchers are interested in analyzing hundreds or thousands of individual cells for any given experiment. To address this need, our single-cell RNA-seq technology was further modified to create a simplified, high-throughput workflow with very little hands-on time. The reverse transcription (RT) and PCR amplification steps were combined into a single RT-PCR step so that users can simply set up the RT-PCR and walk away. The updated workflow, available in the SMART‑Seq HT Kit, is extremely fast, convenient, and generates a higher cDNA yield than its predecessor, all while providing the same unparalleled sensitivity and reproducibility.
A SMARTer solution to stranded single-cell RNA‑seq
Since the emergence of next-generation sequencing (NGS), the importance and demand for single-cell analysis have risen rapidly. Single-cell RNA-seq generates data for various analysis methods such as differential gene expression, alternative splicing, gene fusion identification, and so on, which allow for cell profiling in great detail. As a result, single-cell RNA-seq has been gaining prominence not only in basic research fields but also in clinical fields. Extracting meaningful biological information from the small amount of mRNA present in each cell requires a library preparation method with exceptional sensitivity and reproducibility. The SMART‑Seq v4 Ultra Low Input RNA Kit for Sequencing offers the most advanced single-cell RNA-seq method on the market, in part due to its incomparable capability to retrieve information from full-length mRNA and not just the 3' end. However, this method can only capture polyadenylated mRNA, and thus works best with high-quality RNA and cells. In addition, it does not preserve strand-of-origin information. To address these problems, we have further modified our SMART RNA-seq technology to create the SMART‑Seq Stranded Kit, a single-cell-capable workflow that relies on random priming instead of oligo(dT) priming, hence capturing any RNA regardless of polyadenylation status. The SMART‑Seq Stranded Kit preserves the strand-of-origin information, making it more amenable to distinguishing overlapping genes and for comprehensively annotating and quantifying lncRNAs. The SMART‑Seq Stranded Kit offers a workflow that delivers a more accurate representation of the single-cell transcriptome than is achievable with current methods.
Evaluation of RNA sequencing methods for use with highly degraded formalin-fixed, paraffin-embedded (FFPE) tissue samples
Genomics research requires a significant sample size to provide robust biological signal, leaving researchers clamoring for access to large sample sets. As this research continues to expand into the clinical arena, the demand to sequence RNA from banked tissue samples, such as formalin-fixed paraffin-embedded blocks, is unavoidable. Recovering DNA and RNA from such samples can be challenging depending on age of the sample block and fixture protocols. To fulfill the need for increased recovery of usable reads, several manufacturers (including Takara Bio) have developed solutions to address these challenges, including FFPE-specific extraction kits, as well as library synthesis and quality control reagents. In this study, the authors (Dragon et al.) analyzed the quality and outcome of RNA-seq data generated from three library synthesis kits of FFPE-derived human thyroid tumors with storage times from 3–6 years.
Comparative analysis of single-cell RNA sequencing platforms and methods
The ABRF Genomics Research Group (GRG) generated data from hundreds of individual SUM149PT cells treated with the histone deacetylase inhibitor TSA vs. untreated controls across several scRNA-Seq platforms (Fluidigm C1, Takara Bio ICELL8 system, 10X Genomics Chromium Controller, and Illumina/BioRad ddSEQ). The goals of this project were to demonstrate RNA sequencing (RNA-Seq) methods for profiling the ultra-low amounts of RNA present in individual cells, and RNA amplification using the various currently available platforms. The authors (Chittur et al.) discussed the results of the study as well as technical challenges/lessons learned and presented general guidelines for best practices in sample preparation and analysis.
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