Targeted RNA Sequencing by Ion Torrent Next-Generation Sequencing
Targeted, quantitative gene expression analysis
Researchers employ targeted sequencing as a rapid and cost-effective alternative to whole genome sequencing by focusing on selected sets of genes or genomic regions. Ion AmpliSeq™ technology delivers simple and fast library construction for affordable targeted sequencing of specific human genes.
Ion AmpliSeq™ RNA Panels enable scientists to choose from over 20,000 targeted genes to survey gene expression fold changes starting with as little as 500 pg of unfixed RNA or 5 ng of formalin-fixed, paraffin-embedded (FFPE) RNA. The Ion PGM™ or Ion Proton™ System, coupled with Ion AmpliSeq™ RNA Panels, Torrent Suite™ Software, and Partek® Flow® Software, enables a unique and complete workflow solution.
Researchers employ targeted sequencing as a rapid and cost-effective alternative to whole genome sequencing by focusing on selected sets of genes or genomic regions. The ability to efficiently and uniformly amplify just the genomic regions of interest and to keep the RNA input amount low are critical factors, especially in human disease research applications. Expression profiling is of fundamental interest to a wide range of biological research applications, including gene regulation analysis, correlating gene expression with phenotypic information, and cellular pathway analyses. Targeted RNA sequencing, using Ion AmpliSeq™ technology, is a sequencing-based gene expression profiling method that allows quantification of messenger RNA (mRNA) or targeted non-coding RNA (ncRNA) levels for up to 300 customer-defined genes in a single amplification reaction. Importantly, the Ion AmpliSeq™ targeted RNA workflow utilizes very low amounts of input total RNA: 500 pg of unfixed RNA or 5 ng of RNA isolated from FFPE samples. Further, targeted RNA sequencing can be used to detect fusion transcripts from chromosomal rearrangements that are critical initiating steps of tumorigenesis. This technology, coupled with the Ion PGM™ System, Torrent Suite™ Software, and the Coverage Analysis plugin, enables researchers to go from precious FFPE samples to quantitative gene expression profiling in a single day.
Design your own Ion AmpliSeq™ RNA custom panel
Creating and ordering custom panels is easy with the Ion AmpliSeq™ Designer—a free, online assay design tool that connects you directly to our assay design pipeline. Create and order custom RNA panels by simply selecting your targets of interest or entering your specific genomic content online, and we will design and create a customized Ion AmpliSeq™ panel just for you. Leveraging more than a decade of expertise powering the TaqMan® Custom Assay design pipeline, the Ion AmpliSeq™ Designer produces optimized primer designs in just hours. You now have the flexibility to analyze hundreds of genes of your choice, such as those implicated in a particular disease state or representing specific biochemical pathways. Ion AmpliSeq™ RNA Designer custom-designs a target selection of 12 to 1200 human amplicons in a single reaction, leveraging the experience of a community of >2,700 registered users with >18,000 panels submitted. High-quality amplicons are designed that account for GC content, and are filtered for repeats, SNPs, and target exons that represent the maximum number of transcripts for single genes that have multiple transcript isoforms documented in standard databases such as RefSeq.
In addition to custom panels, we offer these verified Ion AmpliSeq™ RNA ready-to-use panels:
Ion AmpliSeq™ Transcriptome Human Gene Expression Kit
The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit is a comprehensive tool for gene-level expression analysis. Targeting >20,000 human RefSeq transcripts in a single reaction with an formalin-fixed, paraffin-embedded (FFPE)-compatible workflow, the complete Ion AmpliSeq™ Transcriptome Solution leverages existing downstream microarray or next-generation sequencing (NGS) pipelines for fast and easy data analysis.
Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel
Developed in collaboration with the OncoNetwork Consortium, the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was designed to detect expression imbalance in fusion driver genes using only 10 ng of FFPE RNA. The panel is a single pool of primers targeting fusion transcripts included in the ALK, RET, ROS1, and NTRK genes.
Ion AmpliSeq™ RNA Cancer Panel
The Ion AmpliSeq™ RNA Cancer Panel was developed as an RNA complement to the Ion AmpliSeq™ Cancer Hotspot Panel v2. The panel is a single pool of primers targeting 50 oncogenes and tumor suppressor genes including the KRAS, BRAF, and EGFR genes.
Ion AmpliSeq™ RNA Apoptosis Panel
The Ion AmpliSeq™ RNA Apoptosis Panel is an analysis tool designed against 267 TaqMan® Assay-verified genes involved in the cellular apoptosis pathway. Included are genes associated with death receptor-mediated apoptosis, c-myc-mediated apoptosis, and p53-mediated apoptosis.
Targeted RNA sequencing workflow for the Ion PGM™ or Ion Proton™ System
Targeted RNA Sequencing Informatics Solutions
Torrent Suite™ Software coordinates all the experiment planning and data processing steps necessary to complete your targeted sequencing workflow. New product templates enable users to plan targeted runs simply and quickly. Raw data are processed on the Ion PGM™ or Ion Proton™ Sequencer and transferred to the Ion PGM™ or Ion Proton™ Torrent Server for base calling and variant calling. This entire process takes just a few hours, and further analysis can be performed using the Coverage Analysis plugin, which reports amplicon counts in spreadsheet file formats, allowing users to import data into other software packages for differential expression analysis. A more comprehensive analysis can be performed using Partek® Flow® software.
Confirm expression using TaqMan® Assays
Universal human reference (UHR) RNA and human brain reference (HBR) RNA control samples were used to compare fold-change for each of the 50 genes included in the Ion AmpliSeq™ RNA Cancer Panel against the fold change calculations of the same genes with TaqMan® Gene Expression Assays, resulting in a correlation of 0.989, as shown in Figure 1. These results show the accuracy of the gene expression data obtained with the Ion AmpliSeq™ RNA workflow. Find TaqMan® Assays corresponding to the same gene regions interrogated by Ion AmpliSeq™ RNA amplicons in the Ion Community.
* Based on recommendations of multiplexing 4-6 libraries generated using the Ion AmpliSeq™ RNA Apoptosis Panel on one 318™ chip.
**The content provided herein may relate to products that have not been officially released and is subject to change without notice.
For Research Use Only. Not for use in diagnostic procedures.