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  • Enhancing biomarker discovery with SMART-Seq Pro kit and ICELL8 cx system
  • ICELL8 cx system target enrichment for fusions
  • ICELL8 cx system reagent formulation and dispense guidelines
  • Improved detection of gene fusions, SNPs, and alternative splicing
  • Full-length transcriptome analysis
  • High-throughput single-cell ATAC-seq
  • Protocol: High-throughput single-cell ATAC-Seq
  • Single-cell identification with CellSelect Software
  • Single-cell analysis elucidates cardiomyocyte differentiation from iPSCs
  • Combined TCR profiling and 5’ DE in single cells
  • Automated, high-throughput TCR profiling
SMART-Seq Pro application kit product info SMART-Seq Pro kit product information
Home › Learning centers › Automation systems › ICELL8 introduction › Technical notes › Enhancing biomarker discovery with SMART-Seq Pro kit and ICELL8 cx system

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SMART-Seq Pro application kit product info SMART-Seq Pro kit product information
Tech Note

Enhancing biomarker discovery with the SMART-Seq Pro kit and ICELL8 cx Single-Cell System

Improve your ability to detect important biomarkers with the SMART-Seq Pro application kit.

  • Offers superior detection of gene fusions, splice variants, and SNVs that can be missed by end-counting methods
  • Surpasses performance of popular plate-based methods, such as Smart-seq2, by detecting more genes and transcripts
  • Pairs with Cogent NGS Analysis Pipeline and Cogent NGS Discovery Software for transcript mapping and biomarker discovery
Introduction Results Conclusions Methods References

Introduction  

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, single-cell RNA-seq (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 tools (Figure 1). With the full-length coverage of our SMART-Seq Pro kit, you can identify clinically relevant, novel biomarkers at single-cell resolution with confidence.

Figure 1. The SMART-Seq Pro application kit workflow. Input samples into the ICELL8 cx Single-Cell System to generate Illumina®-ready sequencing libraries. After sequencing on an Illumina platform, use the resulting FASTQ files as input for our free analysis software, Cogent NGS Analysis Pipeline, for mapping and reporting, followed by Cogent NGS Discovery Software for interactive data visualization.

Results  

SMART-Seq Pro uncovers the most biological information from single cells

To determine if the automated, streamlined SMART-Seq Pro workflow could detect genes from single cells at the same rate as or better than other popular plate-based methods such as Takara Bio’s SMART-Seq v4 kit or the Smart-seq2 homebrew method, we used all three methods to prepare single-cell sequencing libraries. Then we analyzed the number of identified genes using Cogent NGS tools. With the SMART-Seq Pro workflow we identified a median of 3,195 genes/cell, while we identified a median of 1,747 genes/cell with the Smart-seq2 workflow, and a median of 2,574 genes/cell with the SMART-Seq v4 workflow, demonstrating that SMART-Seq Pro detects more genes per cell than plate-based methods (Figure 2).

Figure 2. The SMART-Seq Pro application kit offers robust detection of genes and transcripts. The SMART-Seq Pro application kit for the ICELL8 cx system (SS-Pro) identifies more genes than the popular plate-based Smart-seq2 (SS2) and SMART-Seq v4 (SSv4) methods in primary cells with low RNA content such as PBMCs.

SMART-Seq Pro enables transcript-level investigation, revealing information that can be missed with a gene-level-only investigation

To test the ability of the SMART-Seq Pro’s end-to-end workflow to identify information that is missed by gene-level-only analysis, we first identified transcript-level expression changes in a population of human blood mononuclear cells (PBMCs) using the complete SMART-Seq Pro workflow. Briefly, PMBCs were processed using the SMART-Seq Pro application kit on the ICELL8 cx Single-Cell System, which resulted in the creation of full-length scRNA-seq libraries. After sequencing, data were demultiplexed and mapped using the transcript analysis option in the Cogent NGS Analysis Pipeline (Cogent AP), our free-to-use bioinformatics software. The output file from Cogent AP was then used as input for Cogent NGS Discovery Software (Cogent DS). Cogent DS was then used to perform clustering analysis and generate a UMAP plot based on transcript counts (Figure 3, Panel A).

Figure 3. Transcript-level analysis of PBMCs with the SMART-Seq Pro workflow. Panel A. UMAP plots showing Human PBMC cell clusters based on transcript expression. Transcript-based Cluster 1 (red circle) and Cluster 5 (blue circle) were chosen for further analysis. Panel B. Example 'Gene Discovery' menu and downloadable transcript list for Cluster 1 from Cogent DS.

Among the eight different transcript-based clusters identified, Cluster 1 (red circle) and Cluster 5 (blue circle) were chosen for further analysis. From the 'Gene Discovery' menu in Cogent DS (Figure 3, Panel B), two lists of differently expressed transcripts were downloaded, one for Cluster 1 and one for Cluster 5. For Cluster 1, 4,935 differently expressed transcripts were identified, while 2,598 transcripts were identified for Cluster 5. Then, transcripts that were detected in both clusters but displayed opposite expression levels were extracted, which resulted in a list of 959 differentially expressed transcripts.

Then, we identified expression changes in the same PBMC population using a gene-level-only analysis akin to the type of analysis that would be performed using data generated from 3′DE or 5′DE single-cell RNA sequencing. Cogent DS was used to generate a UMAP plot showing the results of gene-based clustering (Figure 4, Panel A). From the 'Gene Discovery' menu in Cogent DS (Figure 4, Panel B), a list of differently expressed genes from all gene-based clusters was downloaded. A unique gene list was created, which resulted in 5,987 genes. This list of genes represents expression changes that could be identified by 3′DE or 5′DE methods.

Figure 4. Gene-level analysis of PBMCs with the SMART-Seq Pro workflow. Panel A. UMAP plots showing Human PBMC cell clusters based on gene expression. Panel B. Example downloadable gene list for Cluster 1 from Cogent DS.

To demonstrate the information gained by transcript-level analysis over a gene-level-only analysis, transcripts differentially expressed between transcript-based Clusters 1 and 5 whose parental gene was not differentially expressed were extracted, which resulted in a list of 52 transcripts. These 52 transcripts are expression changes that were only identified through a full-length, transcript-level analysis like the Smart-Seq Pro.

SMART-Seq Pro empowers novel biomarker discovery with transcript-level insight

To demonstrate that the SMART-Seq Pro empowers novel, clinically relevant biomarker discovery, the 52 transcripts that were identified as being differentially expressed at the transcript level but not the gene level between transcript-based Clusters 1 and 5 were further investigated. Of these 52 transcripts, several PTPRC isoforms were identified, including PTPRC-201, PTPRC-207, and PTPRC-209. PTPRC encodes a transmembrane protein tyrosine phosphatase known as CD45 that is required for T-cell antigen receptor signal transduction (Li et al. 2020). Previous studies have shown that different PTPRC isoforms generated through alternative splicing depend on the state of T-cell activation (Li et al. 2020).

By using the 'Gene Discovery' module and choosing PTPRC in Cogent DS, the expression of the PTPRC gene across gene-based clusters was examined. High expression of PTPRC was observed across all gene-based clusters, which was consistent with our initial analysis (Figure 5, Panel A). However, when the expression of PTPRC-201, PTPRC-207, or PTPRC-209 across transcript-based clusters was examined, PTPRC-201 demonstrated higher expression in Cluster 1 (Figure 5, Panel B; red) compared to PTPRC-207 and PTPRC-209, while PTPRC-207 and PTPRC-209 demonstrated higher expression in Cluster 5 (Figure 5, Panel B; blue) compared to PTPRC-201. These data illustrate why an ultra-sensitive, full-length approach such as SMART-Seq Pro is needed for the detection of novel biomarkers.

Figure 5. SMART-Seq Pro transcript-based analysis reveals differential expression of PTPRC isoforms. Panel A. UMAP and violin plot showing expression of the PTPRC gene across gene-based clusters. Panel B. Expression of different PTPRC isoforms generated through alternative splicing depends on the state of activation and differentiation of hematopoietic cells. UMAP and violin plots show the different expression levels of distinct PTPRC isoforms, PTPRC-201, PTPRC-207, and PTPRC-209, by transcript-based clustering analysis. Transcript-based Clusters 1 and 5 are highlighted by red circles/squares and blue circles/squares, respectively.

Conclusions  

We successfully used the SMART-Seq Pro kit, together with the ICELL8 cx Single-Cell System and Cogent NGS analysis tools, to generate high-quality data that can be easily used for characterizing clinically relevant isoforms. The findings show how the ICELL8 cx system offers an efficient way to scale up biomarker discovery by leveraging the full-length sequencing coverage and unparalleled sensitivity of SMART-Seq Pro. With the freely available Cogent NGS tools, we streamlined the discovery of novel biomarkers by maximizing detection power, reproducibility, and resolution.

Methods  

Cell staining and preparation

Cryopreserved PBMCs (Cat. # HUMAN-PBMC-M-170046, lot # 00PB000450, BioIVT) were thawed in a 37°C water bath for 60 seconds and then topped off with pre-warmed RPMI-1640 Complete Medium (20% FBS). After centrifugation and subsequent removal of the supernatant, the concentration of recovered PBMCs was determined via a Moxi automated cell counter. PBMCs were then stained with Hoechst 33342 and propidium iodide as described in the SMART-Seq Pro application kit user manual.

Cell dispensation and imaging

The PBMCs and controls were then dispensed into 5,184 nanowells of the ICELL8 350v Chip using the ICELL8 cx system and ICELL8 cx CELLSTUDIO v2.5 Software. The nanowells were then imaged by the ICELL8 cx system with both blue and red wavelength filters. After imaging, the chip was frozen at –80°C while the ICELL8 cx CellSelect v2.5 Software was used to analyze the resulting images with the automated threshold detection. After selecting candidate wells, the software generated a filter file to use for all the remaining dispenses.

Library construction

The chip was thawed and returned to the ICELL8 cx system, where RT reagents were dispensed only into the nanowells defined as candidates by the CellSelect filter file. The chip was run through a program to perform first-strand cDNA synthesis on the ICELL8 cx Thermal Cycler initiated by SMART-Seq Pro CDS (an oligo-dT primer). Following first-strand cDNA synthesis, the SMART-Seq Pro Oligonucleotide was hybridized to the 3′ end of the full-length cDNA and mediated template switching, serving as a priming site for second-strand cDNA synthesis. Once synthesized, the second-strand cDNA was amplified through PCR, resulting in copies of unbiased, full-length cDNA. After amplification, the full-length cDNA was tagmented by Illumina Bead-Linked Transposome (BLT). The tagmented cDNA was then amplified using forward and reverse indexing primers, generating the final library construct.

The resulting libraries were extracted from the chip, purified, amplified, and purified again. After validation steps, the libraries were loaded on the Illumina NextSeq® 500 system. The resultant sequencing reads were downsampled to 100,000 reads per cell to see the number of genes detected.

Gene expression analysis

Cogent NGS Analysis Pipeline (CogentAP) v1.5 was used for gene expression analysis. As part of the pipeline, steps including adapter trimming (using cutadapt tool), genome alignment (using STAR), gene read counting (using featureCounts), and transcript read counting (using RSEM) were performed. The resulting gene/transcript matrices were then input into Cogent NGS Discovery Software (CogentDS) v1.5 to perform clustering analysis and generate UMAP plots and clusters. 

References  

Hwang, B. et al. Single-cell RNA sequencing technologies and bioinformatics pipelines. Exp. Mol. Med. 50, 1–14 (2018).

Li, J. et al. Landscape of transcript isoforms in single T cells infiltrating in non-small-cell lung cancer. Genet. Genomics 47, 373–388 (2020).

Pan, Q. et al. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat. Genet. 40, 1413–1415 (2008).

Wang, E. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).

Zhang, Y. et al. Alternative splicing and cancer: a systematic review. Sig. Transduct. Target Ther. 6, 78 (2021).

Related Products

Cat. # Product Size License Quantity Details
640257 SMART-Seq® Pro Application Kit - 2 Chip 2 Chips USD $4950.00

License Statement

ID Number  
275 SMART-Seq2 Technology. This product is sold under exclusive license from Ludwig Institute of Cancer Research, Ltd. and is covered by US Patent No. 10266894, Japanese Patent No. 6336080, and European Patent No. 3036336, and pending U.S. patent application and/or pending claims of foreign counterparts. For license information, please contact a Takara Bio USA, Inc. licensing representative by e-mail at licensing@takarabio.com.

SMART-Seq Pro Application kit contains the necessary chips, reagents, and consumables required to run two complete experiments of single-cell, full-length transcriptome analysis on the ICELL8 cx Single-Cell System (Cat. No. 640188).

The protocols for performing SMART-Seq chemistry on ICELL8 systems leverage SMART technology for full-length cDNA synthesis and Nextera® technology for Illumina library preparation with dual 72 x 72 indexes (Nextera Tagmentation bead-linked transposome not included). The protocols generate indexed libraries that are ready for sequencing on Illumina platforms. Provided index plates allow for two chip libraries to be processed simultaneously in a single sequencer run.

ICELL8/MSND 384-Well Source Plate and Seals (Cat. Nos. 640192, 640018, or 640037) need to be purchased separately. A total of four plates and seals are required to process each individual chip.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Comparison of genes detected from PBMCs using SMART-Seq Pro or Smart-seq2 (SS2) workflows.

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Comparison of genes detected from PBMCs using SMART-Seq Pro or Smart-seq2 (SS2) workflows.

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Comparison of isoform data generated using the SMART-Seq Pro kit or 10x Chromium.

Comparison of isoform data generated using the SMART-Seq Pro kit or 10x Chromium.

Comparison of isoform data generated using the SMART-Seq Pro kit or 10x Chromium. The SMART-Seq Pro method using the ICELL8 cx system can identify junction reads across exons 3, 4, 5, 6, and 7 and shows distinct profiles for PTPRC-201 and PTPRC-209 isoforms. Data generated from 10x Chromium end-counting methods could not identify junctions in these same exon regions, with peaks representing noise from misaligned reads.

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640257: SMART-Seq Pro Application Kit - 2 Chip

640257: SMART-Seq Pro Application Kit - 2 Chip
640258 SMART-Seq® Pro Indexing Primer Set - A 1 Chip USD $950.00

The SMART-Seq Pro Indexing Primer Set - A enables users to analyze full-length transcripts using ICELL8 single-cell systems and can generate indexed libraries that are ready for sequencing on Illumina platforms.

The plate contains 72 forward and 72 reverse indexes and a plate seal to process samples on one ICELL8 250v Chip or one ICELL8 350v Chip. This plate has been made available separately for customized protocol development. Primers are not resuspended to maximize flexibility with volumes and concentrations for assay developers.

This set contains different index primers than the SMART-Seq Pro Indexing Primer Set - B plate (Cat. # 640260). The SMART-Seq Pro protocol allows for the option to multiplex two libraries in a single sequencing run, one generated using Set A indexes and one generated using Set B indexes.

NOTE: This plate is already included with SMART-Seq Pro Application Kit - 2 Chip (Cat. No. 640257) and required for that workflow.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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640258: SMART-Seq Pro Indexing Primer Set - A

640258:  SMART-Seq Pro Indexing Primer Set - A
640188 ICELL8® cx Single-Cell System Each Inquire for Quotation

License Statement

ID Number  
329 This product is protected by one or more of these U.S. Patents 9,132,427, 9,909,171, 9,447,925, 9,951,381 and/or corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
397 This product is protected by U.S. Pat. No. 11,460,405 and corresponding foreign patents pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
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The ICELL8 Single-Cell System is an advanced, integrated automation platform that combines the power of imaging and dispensing with the isolation of single cells in 5,184-nanowell chips. This open-platform, high-throughput system is prevalidated for use with several NGS applications, and also provides the flexibility to enable users to develop applications of their choice.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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640188: ICELL8 cx Single-Cell System

640188: ICELL8 cx Single-Cell System
650002.V2.5 ICELL8® cx CellSelect® v2.5 Software Each USD $700.00

License Statement

ID Number  
397 This product is protected by U.S. Pat. No. 11,460,405 and corresponding foreign patents pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

ICELL8 cx CellSelect v2.5 Software analyzes the images generated by the ICELL8 cx Single-Cell System, providing users with insight into the distribution of sample cells in the nanowells of the ICELL8 cx chip. Included natively with the ICELL8 cx instrument, ICELL8 cx CellSelect v2.5 Software may also be purchased separately for installation on a standalone Windows 10 PC, allowing users to perform and review candidate selection independent of the instrument.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

Clontech, TaKaRa, cellartis

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