Shop All Sequencing Kits & Reagents

Ion 510™ & Ion 520™ & Ion 530™ Kit for Agrigenomics – Chef (1 sequencing run per initialization) (Ion Torrent™)

The Ion 510 & Ion 520 & Ion 530 Kit-Chef (1 sequencing run per initialization) enables robust and automated template preparation and sequencing of up to 400 base-read libraries using the Ion Chef System and the Ion S5 or Ion S5 XL sequencing system. The Ion 510 & Ion 520 & Ion 530 Kit contains pre-packaged single-use template and sequencing reagent cartridges with integrated sample tracking, delivering an automated workflow for sequencing of 8 loaded chips with complete run traceability. This kit accommodates 1 sequencing run per initialization. For 2 sequencing runs per initialization (200 base-read applications only), see Cat. No. A44875.

The Ion 510 & Ion 520 & Ion 530 Kit-Chef workflow:
• Provides template preparation and chip loading reagents for up to 400 base-read libraries
• Simplifies your research with ready-to-use, disposable reagent cartridges for sequencing
• Enables fast and simple sequencing instrument set-up, with less than 15 minutes hands-on time
• Leverages the latest advancements in data quality and accuracy
• Maximizes flexibility with compatibility for the Ion 510, Ion 520, and Ion 530 chips

The Ion S5 and Ion S5 XL sequencers, along with the Ion Chef System, offer reproducible sequencing workflows, with minimal hands-on time. Choose to pair the Ion 510 & Ion 520 & Ion 530 Kit with the Ion 510 Chip Kit (up to 3 million reads per chip), the Ion 520 Chip Kit (up to 5 million reads per chip), or Ion 530 Chip Kit (up to 20 million reads), allowing for flexibility of total throughput. For even greater flexibility, combine different chip types in a single Ion Chef run when using Torrent Suite Software 5.6 and above. For de novo research applications, add the Ion S5 Calibration Standard to prepared libraries for improved base-calling accuracy.

Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent™)

The Ion AmpliSeq™Cancer Hotspot Panel v2 is a single pool of primers used to perform multiplex PCR for preparation of amplicon libraries from genomic "hot spot" regions that are frequently mutated in human cancer genes (download the list of targeted genes and mutations for the Ion AmpliSeq™Cancer Hotspot Panel v2). Building on the mutations included in our original Ion AmpliSeq™Cancer Panel, the latest Ion AmpliSeq™ready-to-use panel provides:
• Maintained compatibility with FFPE samples while expanding mutational content for broader coverage of additional genes and "hot spot" mutations
• Extremely uniform coverage for more efficient sequencing and cost savings
• Enhanced primer design with optimization of new primer sets and even lower strand bias for added confidence in accurate variant calling
• Improved variant detection with Torrent Suite Software v3.0 and Variant Caller Plugin in low allele variant detection along with improved indel sensitivity

Additional Mutational Content for Advancing Cancer Research Studies
The Ion AmpliSeq™Cancer Hotspot Panel v2 is designed to amplify 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes. This includes the 739 COSMIC mutations from 46 genes in the first Ion AmpliSeq™Cancer Panel along with added hotspot mutations from significant cancer genes. While maintaining our compatibility with FFPE samples and covering the same targets as the first Ion AmpliSeq™Cancer Panel, we have included additional mutations by adding several amplicons for hotspots in EZH2, GNA11, GNAQ, and IDH2, and by adding slightly longer amplicons for other genes to help advance your cancer research. The Ion AmpliSeq™Cancer Hotspot Panel v2 should be used with the Ion AmpliSeq™Library Kit 2.0.

More Efficient and More Cost-effective Sequencing with Extremely Uniform Coverage
The improved primer design in the Ion AmpliSeq™Cancer Hotspot Panel v2 improves upon the coverage uniformity of the Ion AmpliSeq™technology. With coverage that is more uniform, a minimum depth across all amplicons can be achieved using much less sequencing throughput. Consequently, additional samples can be multiplexed using any of the Ion chips. This results in cost savings with more efficient sequencing while maintaining high confidence in variant calling. Additionally, the low strand bias in the primer sets of the Ion AmpliSeq™Cancer Hotspot Panel v2 provides further confidence in accurate variant calling.

Simplicity, Speed, and Scalability of Ion AmpliSeq™Technology
Sustaining the simplicity of the Ion AmpliSeq™technology, the Ion AmpliSeq™Cancer Hotspot Panel v2 enables cancer genetic studies from FFPE tissues with as little as 10 ng of input DNA for targeted library construction. The Ion AmpliSeq™Cancer Hotspot Panel v2 also maintains the revolutionary workflow using standard PCR equipment and a simple PCR reaction for ultra-high multiplex PCR-based target selection, with no need for extensive capital investment. Additionally, the Ion AmpliSeq™Cancer Hotspot Panel v2 continues to enable you to achieve the fastest time to targeted libraries in approximately 3.5 hours. Scalability and flexibility is also preserved with the Ion AmpliSeq™Cancer Hotspot Panel v2, allowing the ability to sequence one sample or multiplex barcoded samples using any of the Ion chips.

Improved Variant Detection with Torrent Suite Software v3.0
The intuitive Torrent Suite Software and Variant Caller Plugin enable you to go from extracted DNA to variant calls in as little as 10 hours. With improvements to algorithms, you will see more sensitive indel detection along with more sensitive SNP detection with lower frequency alleles. Ion Reporter™Software can then be used for annotation of variants in routine DNA assays for the advancement of clinical research.

Ion S5™ Precision ID Chef & Sequencing Kit (1 run per initialization) (Applied Biosystems™)

The Ion S5 Precision ID Chef & Sequencing Kit enables robust and automated template preparation and sequencing of Precision ID libraries using the Ion GeneStudio S5 worfklow (eight initializations and eight sequencing runs). This kit provides template preparation and chip loading reagents for libraries generated with commercially available Precision ID panels (GlobalFiler NGS STR Panel v2, Identity Panel, Ancestry Panel, mtDNA Whole Genome Panel, mtDNA Control Region Panel, or custom Ion AmpliSeq SNP panel). For four initializations and eight sequencing runs, see Cat. No. A33208.

The Ion S5 Precision ID Chef & Sequencing Kit for the Ion Chef and Ion GeneStudio S5 workflow:
• Provides template preparation and chip loading reagents for Precision ID libraries
• Simplifies your testing with ready-to-use, disposable reagent cartridges for sequencing
• Delivers fast and simple sequencing instrument set-up, with less than 15 minutes hands-on time
• Leverages our latest advancements in data quality and accuracy
• Maximizes flexibility through compatibility with both the Ion 520 and Ion 530 chips

The Ion GeneStudio S5 sequencers, along with the Ion Chef System, provide reproducible sequencing workflows with minimal hands-on time. Choose to pair the Ion 520 & Ion 530 Kit with the Ion 520 (up to 5M reads) or Ion 530 (up to 20M reads) Chip kit, allowing for flexibility of total throughput.

Oncomine™ Pan-Cancer Cell-Free Assay (Ion Torrent™)

The Oncomine Pan-Cancer Cell-Free Assay is part of a complete solution to detect multiple targets in tumor-derived DNA and RNA isolated from the plasma fraction of whole blood. The assay provides the reagents and a single pool of multiplex PCR primers for preparation of an amplicon library from cell-free total nucleic acid (cfTNA) obtained from the plasma fraction of a single 10-mL tube of whole blood. This library can then be used for highly multiplexed targeted next-generation sequencing (NGS).

Oncomine cell-free assay advantages:
• From a single tube of blood, generates an amplicon library from both DNA and RNA with a detection limit of 0.1% for SNVs
• Amplicon size optimized for short cfDNA, ensuring highest possible capture rate
• Tag Sequencing technology minimizes false positives by removing randomly incorporated errors
• Optimized targeted assay design allows highly multiplexed NGS, reducing sequencing costs per sample
• Two-day workflow from single 10-mL tube of blood to report; total time to targeted libraries is just four hours
• Enables cancer genetic studies from just 5 ng of input cfTNA
• Compatible with FFPE samples for possible concordance studies

The 52-gene panel includes:
• Hotspot genes (SNVs) and short indels: AKT1, ALK, AR, ARAF, BRAF, CHEK2, CTNNB1, DDR2,EGFR, ERBB2, ERBB3, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, RAF1, RET, ROS1, SF3B1, SMAD4, SMO
• Gene fusions: ALK, BRAF, ERG, ETV1, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK3, RET, ROS1
• MET exon 14 skipping
• Copy number genes (CNVs): CCND1, CCND2, CCND3, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, MET, MYC
• Tumor suppressor genes: APC, FBXW7, PTEN, TP53

These genes have been identified as frequently mutated in multiple cancer types, including: bladder, brain and CNS, breast, cervical, colorectal, endometrial, esophageal, gastric, head and neck, kidney, liver, lung, melanoma, ovarian, pancreatic, prostate, sarcoma, and thyroid.

Detection limits:
• SNVs/short indels: a limit of detection (LOD) down to 0.1% allele frequency (AF) can be achieved with a sensitivity of >80% and specificity of >98%*
• TP53 whole-target SNVs/indels: 0.5% AF (looking at all bases within amplicons)
• Fusions and MET exon skipping: LOD down to 1% can be achieved
• CNV targets: detection as low as 1.4-fold change can be achieved

The MagMAX Cell-Free Total Nucleic Acid Isolation Kit (A36716) is highly recommended for isolation of both DNA and RNA from the plasma fraction of whole blood.

Scalability and flexibility are achieved using the Tag Sequencing Barcode Set 1-24 or 25-48 (Cat. Nos. A31830 & A31847 respectively) for multiplexing barcoded samples on the Ion S5 chips.

Analysis of SNVs and short indels can be achieved using Torrent Suite Software 5.2 or higher. In order to analyze SNVs, short indels, fusions, and CNVs, Ion Reporter Software 5.6 (cloud or server based) is required.

Technology
cfDNA and cfRNA are found at extremely low concentrations in the plasma fraction of whole blood. Because of this low prevalence, a tag sequencing technology is utilized in this assay. The technology attaches unique molecular tags to the gene-specific primers. After amplification, the tagged molecules are grouped based on the tags. Groups containing the same mutant variant 80% of the time or greater will be called positive. Using this Tag technology, groups that contain random errors generated through the library construction/sequencing process are removed.

Unlike other technologies with LODs of 1-5%, the Oncomine Pan-Cancer Cell-Free Assay has a flexible detection limit down to 0.1% for SNVs or 1 mutant copy in a background of 1,000 wild-type copies. To achieve 0.1% LOD, 20 ng of input cfTNA is required. Lower amounts of cfTNA can be used, but the %LOD will be higher depending on the input amount.

Liquid biopsies offer several advantages over conventional solid tumor biopsies:
• Liquid biopsy samples are less invasive enabling them to be taken at multiple time points to monitor progression of the cancer
• Lower cost compared to traditional tissue biopsies
• Faster turnaround time from sample to results
• Better represent tumor heterogeneity

*Sensitivity and specificity for each variant type were determined using a collection of contrived positive samples and cfTNA isolated from normal healthy donors.

Ion 530™ Chip Kit (Ion Torrent™)

The Ion 530 Chip Kit contains 8 barcoded chips for sample tracking and sequencing with the Ion S5 and Ion S5 XL Sequencing Systems. The Ion 530 Chip electronically detects polymerase-driven base incorporation without the use of fluorescence. By eliminating the use of an optical detection system, this advance in next-generation sequencing technology allows for rapid sequencing times of less than 2.5 hours for 200 bp sequencing.

The Ion 530 Chip offers:

• Sample tracking functionality from an integrated barcode
• A rapid run time of only 2.5–4 hours, generating 15–20 million reads
• Read lengths up to 600 bp to support a variety of research applications, including targeted gene sequencing and microbial sequencing
• Easy handling and loading of templated products for sequencing
• Compatibility with current library preparation methods

Notes:
This kit is only compatible with the Ion S5 and Ion S5 XL systems.
Use this kit with the Ion 510 & Ion 520 & Ion 530 Kit-Chef, the Ion 520 & Ion 530 ExT Kit-Chef, and the Ion 520 & Ion 530 Kit-OT2.

dGTP BigDye™ Terminator v1.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems™)

dGTP BigDye® Terminator Cycle Sequencing Kit with the AmpliTaq® DNA Polymerase, FS. This Ready Reaction Kit uses the same dye set as the BigDye® v1.0 kits.

The dGTP BigDye® Terminator Cycle Sequencing Kit's fluorescent dideoxy terminator chemistry is optimized for sequencing through GT- and G-rich templates.

• Ensure reliable results with difficult regions and finishing applications.
• Sequence templates with challenging sequence contexts.
• Sequence problematic templates that standard chemistries cannot.

Sequence Challenging Templates
The dGTP BigDye® Terminator Kit is ideal for working with GT- and G-rich and other difficult-to-sequence templates. The dGTP BigDye® Terminator Kit replaces dITP with dGTP and it enables you to extend through those difficult-to-sequence regions with confidence, avoiding early signal loss in these samples.

Optimized for GT-Rich Templates
Obtain higher success rate even with your difficult GT-rich templates. By using the same chemistries as the standard BigDye® Terminator Kits (only replacing dITP with dGTP) the dGTP BigDye® Terminator Kit provides proven performance, optimized for GT-rich and other difficult-to-sequence templates.

Easily Integrate into Your Workflow
dGTP BigDye® Terminator Kits require no new software or instrument recalibration, so you can easily integrate it into your current workflow with minimal changes to your protocols.

For Research Use Only. Not for use in diagnostics procedures.

Ion Xpress™ Plus Fragment Library Kit for AB Library Builder™ System (Ion Torrent™)

The Ion Xpress™ Plus Fragment Library Kit for AB Library Builder™ System provides a rapid, flexible, and high-throughput solution for library construction upstream of the semiconductor sequencing workflow. This ready-to-use reagent cartridge simplifies next-generation sequencing by enabling automated DNA fragment library preparation for the Personal Genome Machine® and soon the Proton™ sequencing systems.

This kit contains sample preparation reagents for enzymatic shearing and library construction of 13 DNA libraries for semiconductor sequencing. Use of this reagent cartridge requires the addition of library adapters (sold separately).

This library kit offers:

Simplicity and convenience of automated library preparation upstream of semiconductor sequencing
Superior coverage uniformity for diverse sample types using our proprietary Ion Shear™ Plus chemistry
Lower input requirement providing the ability to make amplification-free libraries from as little as 50 ng of starting DNA
Rapid and flexible workflow producing Ion fragment libraries containing user-selectable and automatically size-selected insert sizes of 100–400 bp in as little as 2 hours
Significant cost savings by removing the need for ancillary shearing devices and size-selection gels
Scalable methodology to meet high-throughput sequencing requirements

With its plug-n-play modular reagent design, this revolutionary kit automates all steps of library creation, including both DNA fragmentation and library size selection, in as little as 2 hours with limited set-up time and minimal subsequent user-intervention. DNA fragmentation is automated with enhanced, proprietary Ion Shear™ DNA fragmentation chemistry to achieve user-selectable inserts of 100–400 bp in length. Size selection of the libraries is performed automatically without the expense or effort of gel-based methods. Alternatively, libraries can be manually size-selected using conventional methods after the automated run. Starting with 50–1000 ng of input DNA, the kit yields significantly higher amounts of library than current manual methods, eliminating the need for PCR amplification (i.e., "amplification-free" libraries) for most sequencing needs.

A Single-Day Workflow: The Next Stage in a Sequencing Revolution
With the ability to prepare up to 13 libraries in a little over 2 hours, the Ion Xpress™ Plus Fragment Library Kit for AB Library Builder™ System is a key step towards automation of the Ion sequencing workflow, making comprehensive analyses of your sample(s) of interest more convenient than ever before. Furthermore, Ion Xpress™ Plus fragment libraries generated using the AB Library Builder™ system exhibit the superb coverage uniformity typically seen with proprietary Ion Shear™ DNA fragmentation chemistry and higher recovery rates than other library construction methodologies. So you can be confident of generating the best data in the shortest time, without the hassle or expense of manual shearing and size selection.

Ion Torrent Sequencing Made Simpler, Faster, and More Affordable
Ion Shear™ Plus DNA fragmentation technology eliminates the need for costly physical shearing devices for most applications and requires as little as 50 ng of sample, accelerating your path to semiconductor sequencing. This flexible technology also provides users the ability to adjust fragmentation and size-selection sizes to accommodate a range of sequencing read lengths depending on their project requirements. This automated library preparation solution provides fast and scalable workflows for sequencing projects requiring low- to high-throughput processing capabilities.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

Ion AmpliSeq™ Mouse TCR Beta SR Assay, RNA (Ion Torrent™)

The Ion AmpliSeq Mouse TCR Beta SR Assay is a robust, targeted next-generation sequencing (NGS) assay designed to accurately identify and measure the clonal expansion of T lymphocytes by targeting the complementarity-determining region 3 (CDR3) of the T-cell receptor (TCR) gene locus from total RNA input. The assay can be used for basic and translational research to identify T-cell clones since the nucleotide sequence of the CDR3 region is unique to each T cell and codes for the part of the TCR beta chain that is involved in antigen recognition. For DNA samples please see Cat. No. A45488.

Benefits of the Ion AmpliSeq Mouse TCR Beta SR Assay, RNA, include:
Compatibility with a vast array of research sample types, including FFPE tissue, fresh-frozen (FF) tissue, whole blood, PBLs, and PBMCs
High sensitivity and low limit of detection (LoD) for rare clone identification through dual-barcode indexing
Efficient workflow with 48 hour sample-to-results time
Flexible input requirements ranging from 25 ng to 1 µg
Streamlined and user-friendly informatics solution with with multi-sample analysis functionality

The Ion AmpliSeq Mouse TCR Beta SR Kit, RNA, provides a single pool of multiplex PCR primers and library reagents to generate 90-bp amplicons that can be sequenced on all chip types supported by the Ion GeneStudio S5 sequencing systems, allowing you to pick the best multiplexing configuration for your unique sample batching needs and throughput requirements. The entire workflow from library preparation to analysis of samples can be accomplished in two days using the Ion Chef templating system and Ion GeneStudio S5 system.

The Ion AmpliSeq Mouse TCR Beta SR RNA Assay, with its high sensitivity and specificity, supports key applications in the field of immunology, immuno-oncology, hemato-oncology, and vaccine research. The high-sensitivity dual-barcode indexing, flexible input requirements (100 ng–1 µg), high-depth sequencing, and high-throughput capability make this assay ideal for testing a variety of hypotheses for basic or translational biomarker research. A small fragment size requirement from the input materials for library creation and high multiplexing on the Ion 530 Chip of the Ion GeneStudio S5 sequencers enables researchers to focus on testing a variety of use cases in mouse models including the role of T cells in generating immune response to immunotherapies such as checkpoint blockade inhibitors, cancer vaccines, or chimeric antigen receptor (CAR) T-cell therapies.

Learn more about the assay ›

Note: Information about data analysis using Ion Reporter Software v5.12 can be found in the User Guide below.

Ion AmpliSeq™ Library Kit Plus (Ion Torrent™)

The Ion AmpliSeq Library Kit Plus is designed for rapid preparation of amplicon libraries using Ion AmpliSeq panels. Ion AmpliSeq technology enables scalable, multiplex polymerase chain reactions (PCR) from 12- to 24,000-plex in a single well. Next-generation sequencing (NGS) libraries can be prepared using as little as 1 ng of starting material.

Key product features:
• Scalable and efficient library preparation
• Easy and robust protocol
• Barcoded sample multiplexing

Scalable and efficient library preparation
Using as little as 1 ng of starting material, highly multiplexed PCR amplification of thousands of genomic target regions yields superior coverage uniformity and specificity, without the need of a specialized microfluidics PCR platform. The primers contain proprietary modifications that enable removal of primer sequences during library preparation, thereby resulting in efficient target assessment during sequencing.

Easy and robust protocol
The Ion AmpliSeq Library Kit Plus uses a plate-based format for easier sample handling and tracking, and for compatibility with automation and high-throughput laboratories. The protocol is simple to use and enables high yield and uniformity, reliably and reproducibly. The resulting libraries are ready for downstream template preparation using the automated Ion Chef System (which also provides the option of automated library preparation using the Ion AmpliSeq Kit for Chef DL8) or the Ion OneTouch 2 System followed by sequencing. The intuitive Torrent Suite Software and optional Ion Reporter Software enables automation of data analysis.

Barcoded sample multiplexing
The Ion AmpliSeq Library Kit Plus includes reagents for generating amplicons using Ion AmpliSeq primers and preparing libraries from the resulting amplicons. The kit enables the preparation of barcoded libraries using Ion Xpress Barcode Adapters 1-96 kits or IonCode Barcode Adapters 1-384 kits. Barcoded libraries can be combined and loaded onto a single Ion chip to minimize the sequencing run time and cost, and to allow for accurate sample-to-sample comparisons.

Ion 540™ Chip Kit (Ion Torrent™)

The Ion 540™ Chip Kit contains 8 barcoded chips for sample tracking and sequencing with the Ion S5™ and Ion S5™ XL Sequencing Systems. The Ion 540 Chip electronically detects polymerase-driven base incorporation without the use of fluorescence. By eliminating the use of an optical detection system, this advance in next-generation sequencing technology allows for rapid sequencing times of as little as 2.5 hours for 200 bp sequencing.

The Ion 540 Chip Kit offers:

• Sample tracking functionality from an integrated barcode
• A rapid run time of as little as 2.5 hours for 200 bp sequencing, generating 60–80 million reads
• Support for a variety of research applications, including sequencing of up to three human exomes, transcriptome profiling, and targeted resequencing of gene panels
• Easy handling and loading of templated products for sequencing
• Compatibility with current library preparation methods

Notes:
This kit is only compatible with the Ion S5 and Ion S5 XL systems.
Use this kit with the Ion 540 Kit-Chef and the Ion 540 Kit-OT2.

BigDye™ Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems™)

This BigDye® Terminator v1.1 Cycle Sequencing Kit is designed for specialty applications that require optimal basecalling adjacent to the primer and for sequencing short PCR product templates with rapid electrophoresis run modules.

• Improve the quality of your results for a wide range of sequencing applications
• Sequence challenging templates and sequences more successfully
• Get longer, higher-quality reads with more uniform peak heights and optimal signal balance
• Enhance your productivity and reduce costs

Tackle Tough Sequences
With the BigDye® Terminator v1.1 Cycle Sequencing Kit you get a robust, highly flexible chemistry for a wide range of applications, including de novo sequencing and resequencing.

Get More Uniform Peak Heights, Improved Accuracy
With BigDye® Terminator v1.1 kit's superior chemistry, you generate data with uniform peak heights and optimized signal balance-which results in longer, higher-quality reads. You also get more accurate base assignments for heterozygote and mutation detection.

For Research Use Only. Not for use in diagnostics procedures.

Oncomine Lung Cell-Free Total Nucleic Acid Research Assay (Ion Torrent™)

The Oncomine Lung cfTNA Research Assay is part of a complete solution to detect lung tumor-derived cell-free DNA and RNA (cell-free total nucleic acid; cfTNA) isolated from the plasma fraction of whole blood. It provides the reagents for library construction and a single pool of multiplex PCR primers for preparation of amplicon libraries from cfTNA obtained from the plasma fraction of a single 10 mL tube of whole blood.

Liquid biopsies offer several advantages over conventional solid tumor biopsies:
• Less invasive, enabling them to be taken at multiple time points to monitor progression of the cancer
• Lower cost
• Faster turnaround time from sample to results
• Better represent tumor heterogeneity

The Oncomine Lung cfTNA Research Assay enables the analysis of:
• Hotspot genes (SNVs) and short indels: ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, ROS1, and TP53 (~168 hotspots covered)
• Gene fusions: ALK, RET, ROS1
• MET exon 14 skipping
• Copy number gene (CNV): MET

These genes have been identified as frequently mutated in non-small cell lung cancer (NSCLC). Through the use of Tag Sequencing technology, low limits of detection (LOD) can be achieved for different variant types*:
• For SNVs/short indels, an LOD of 0.1% can be achieved with sensitivity of ~90% and specificity of >99%
• For fusions & MET exon skipping, an LOD of 1% can be achieved with sensitivity of >90% and specificity of >99%
• For MET CNV target, detection as low as 1.2-fold amplification can be achieved with sensitivity of >90% and specificity of >99%

The entire workflow from isolation of cfTNA using the MagMAX Cell-Free Total Nucleic Acid Isolation Kit to analysis of samples can be accomplished in just two days using the Ion S5 XL sequencing system.

Technology
cfDNA and cfRNA are found at extremely low concentrations in the plasma fraction of whole blood. Because of this low prevalence, a tag sequencing technology is utilized in this assay. The technology attaches unique molecular tags to the gene-specific primers (figure below). After amplification, the tagged molecules are grouped based on the tags. Groups containing the same mutant variant 80% of the time or greater will be called positive. Using the Tag technology, groups that contain random errors generated through the library construction/sequencing process are removed.

Unlike other technologies with LODs of 1-5%, the Oncomine Lung cfTNA Research Assay has a flexible detection limit down to 0.1% for SNVs or one mutant copy in a background of 1,000 wild-type copies. To achieve 0.1% LOD, 20 ng of input cfDNA is required. Lower amounts of cfTNA can be used, but the %LOD will be higher depending on the input amount.

Advantages:
• Optimized amplicon design for short cfDNA ensures highest possible capture rate
• Tag Sequencing technology minimizes false positives by removing randomly incorporated errors
• Optimized targeted assay design allows highly multiplexed next-generation sequencing (NGS), reducing
sequencing costs per sample
• Efficient 2-day workflow

Analysis of SNVs and short indels can be achieved using Torrent Suite Software 5.2 or higher. In order to analyze SNVs, short indels, fusion, and CNVs, Ion Reporter 5.6 (cloud- or server-based) is required.

Simplicity, speed, and scalability of TagSequencing technology
The Oncomine Lung cfTNA Research Assay enables cancer genetic studies from just 5 ng of input cfTNA for targeted library construction. The Oncomine Lung cfTNA Research Assay is compatible with FFPE samples for possible concordance studies. Total time to targeted libraries is just four hours. Scalability and flexibility are achieved using the Tag Sequencing Barcode Set 1-24 or 25-48 (Cat. Nos. A31830, A31847) for multiplexing barcoded samples on Ion S5 chips.

*Sensitivity and specificity for each variant type were determined using a collection of contrived positive samples and cfTNA isolated from normal healthy donors.

Oncomine™ Breast cfDNA Assay (Ion Torrent™)

The Oncomine™ Breast cfDNA Assay is part of a complete solution to detect breast tumor-derived DNA (ctDNA) in cell-free DNA (cfDNA). It provides the reagents for library construction and a single pool of multiplex PCR primers for preparation of amplicon libraries from cfDNA obtained from the plasma fraction of a single tube of whole blood. The assay enables the analysis of single nucleotide variants and short indels that are frequently mutated in breast cancer. Ten genes with >150 hotspots are covered (Genes: AKT1, EGFR, ERBB2, ERBB3, ESR1, FBXW7, KRAS, PIK3CA, SF3B1, TP53). Through the use of tag sequencing technology, a limit of detection (LOD) down to 0.1% can be achieved.

Note: Target and hotspot BED files and User Guide document for cfDNA Variant Analysis with Torrent Suite Software 5.2 can be found below under "Documents".

• Optimized short amplicon design for fragmented cfDNA (~170 bp in length)
• Detection of both single nucleotide variants (SNVs) and hotspot indels down to 0.1% LOD
• Very high sensitivity and specificity, enabling extremely low levels of detection with minimal false positives
• 12 samples can be run on an Ion 530™ Chip, reducing overall sequencing costs
• Efficient workflow, from a single tube of blood to results in just 2 days

Liquid biopsies offer several advantages over conventional solid tumor biopsies:
• Liquid biopsies are less invasive, enabling samples to be taken at multiple time points to monitor progression
• Lower cost compared to traditional tissue biopsies
• Faster turnaround time from sample to results
• More tumor heterogeneity captured

The entire workflow (figure below) from isolation of cfDNA using the MagMAX™ Cell-Free DNA Isolation Kit to analysis of samples can be accomplished in just 2 days using the Ion S5™ XL sequencing system.

Technology
ctDNA is found at extremely low concentrations in the plasma fraction of whole blood. Because of this low prevalence, a tag sequencing technology is utilized in this assay. The technology attaches a unique molecular tag to the gene-specific primers. The amplified products are then grouped into families containing the same tags. Families that contain the same mutant variant will be called with optimized Variant Caller settings for the Oncology-Liquid Biopsy application. Families that contain random errors, typically generated through the library construction/sequencing process, are identified and removed from variant calling.

Unlike other technologies with LODs of 1-5%, the Oncomine Breast cfDNA Assay has a flexible detection limit down to 0.1% or 1 mutant copy in a background of 1,000 wild-type copies. To achieve 0.1% LOD, 20 ng of input cfDNA is required. Lower amounts of cfDNA can be used (down to 1 ng), but the LOD will be higher depending on the input amount.

Simplicity, speed, and scalability of tag-sequencing technology
The Oncomine Breast cfDNA Assay enables cancer genetic studies from just 1 ng of input cfDNA for targeted library construction. The cfDNA assay uses standard PCR equipment and two simple PCR reactions, one to attach the unique molecular tags and the second to amplify the library, for high multiplex PCR-based target selection with no need for extensive capital investment. Additionally, the Oncomine Breast cfDNA Assay is compatible with FFPE samples for possible concordance studies. Total time to targeted libraries is just 3.5 hours. Scalability and flexibility are achieved using the Tag Sequencing Barcode Set 1-24 (Cat. No. A31830) or Taq Sequencing Barcode Set 25-48 (Cat. No. A31847) for multiplexing barcoded samples on Ion S5 chips.

Learn more about Oncomine cfDNA Assays >

Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Ion Torrent™)

The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit enables the simultaneous measurement of the expression levels of over 20,000 human RefSeq genes in a single assay. Providing the ability to work directly with low input levels of total RNA derived from challenging samples such as formalin-fixed paraffin-embedded (FFPE) tissue or other samples with degraded RNA, the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit is ideal for those interested in obtaining comprehensive, highly sensitive, and easily obtained gene expression measurements from a wide range of RNA sources. For automated library production, see the Ion AmpliSeq Transcriptome Human Gene Expression Panel, Chef-ready Kit.

The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit offers:
• Flexible, straightforward library preparation – start with as little as 10 ng of total RNA from FFPE samples, cells, or other sources
• Representation of over 20,000 human RefSeq genes
• Wider dynamic range and improved detection sensitivity compared to conventional microarray analysis
• A complementary software plug-in for automated sequencing analysis that provides gene quantitation and reporting as well as quality control metrics
• Compatibility with Ion Xpress™ Barcode adapters allowing for multiplexed sequencing

Wider dynamic range for enhanced performance
With a wider dynamic range and improved gene detection compared to conventional microarray analysis, the Ion AmpliSeq Transcriptome™ Human Gene Expression Kit provides a clear solution to researchers needing enhanced performance for their gene expression needs. A simple workflow allows for sample-to-gene quantitation in less than 2 days, helping to reduce workload and increase throughput.

Straightforward library preparation and multiplex sequencing
Starting with as little as 10 ng of total RNA, libraries can be prepared in approximately 7 hours with only one hour of hands-on time. Total RNA is first processed with the included SuperScript® VILO™ cDNA Synthesis kit, and the resulting cDNA is amplified using Ion AmpliSeq™ technology to accurately maintain expression levels of all genes.

The resulting libraries are ready for downstream template preparation using the Ion OneTouch™ 2 System or Ion Chef™ System and sequencing on the Ion Proton™ sequencing system. Libraries can be barcoded for multiplexing, allowing for flexibility, helping to reduce cost, and increase throughput, with the recommended 16 libraries per Ion 550 Chip and 8 libraries per Ion 540 or Ion PI chip. Other multiplexing options are also possible, depending on the experimental design and desired level of sensitivity.

Automatic data analysis with Torrent Suite™ plug-in
Using the complementary analysis plug-in available with Torrent Suite™ software, gene quantitation information is automatically compiled, minimizing the requirement for costly computational resources and bioinformatics expertise to process raw sequencing data into meaningful information. An easy to interpret, highly portable table is produced containing all genes interrogated and their respective expression levels, output that may be used with a variety of existing software applications for downstream analysis of microarray or next-generation sequencing data.

Learn more about the Ion AmpliSeq Transcriptome Human Gene Expression Kit ›

Note: Libraries made from this protocol yield fragments suitable for 200-base read sequencing.

Collibri™ 3´ mRNA Library Prep Kit for Illumina™ Systems (Invitrogen™)

The Invitrogen Collibri 3’ mRNA Library Prep Kit is designed for robust construction of cDNA libraries for strand-specific RNA sequencing on Illumina next-generation sequencing (NGS) platforms. It supports library preparation from polyadenylated RNA in ~4.5 hours. The protocol generates one fragment per transcript, resulting in accurate gene expression values. Sequences obtained are close to the 3’ ends of the transcripts, so the kit is suitable for varying-quality RNA samples, including formalin-fixed paraffin-embedded (FFPE) samples. The majority of inserts are greater than 75 bp in size, corresponding to final library fragment sizes ≥200 bp. Multiplexing of libraries can be carried out using up to 96 single-indexed primers.

For convenience, the Collibri 3’ mRNA Library Prep Kit provides color-coded components for visual tracking of library preparation progress. Inert dyes in the reagents provide visual confirmation of additions and mixing, but do not interfere with enzymatic reactions and do not compromise library prep and sequencing results.