Analyzing the millions of disease research samples stored in biorepositories around the world is a primary unmet need for biomedical researchers. Sequencing these samples using next-generation sequencing (NGS) has been difficult, as typically only nanogram amounts of DNA can be isolated, far less than current commercial protocols require. In addition, NGS creates a flood of data, requiring expensive bioinformatics expertise to interpret. The latest set of Life Technologies Ion AmpliSeq™ and Ion Reporter™ products, when augmented with specific and sensitive confirmation using TaqMan® assays, offer turnkey solutions to overcome these challenges.
The introduction of the Ion PGM™ Sequencer democratized sequencing—it is a fast, simple, scalable, and affordable platform. The Ion AmpliSeq Cancer Panel, Comprehensive Cancer Panel, and Inherited Disease Panel further expand the capabilities of this platform by transforming oncology and disease research.
Ion AmpliSeq panels, whether predesigned and ready to use, or custom-built to probe specific genes of interest, deliver a simplified single-day workflow comprising target selection, amplification, sequencing, and analysis (Figure 1). Further, with the use of Ion Reporter Software, the integrated workflow also includes an annotated readout detailing the biological significance of observed gene mutations. The Ion AmpliSeq technology requires only tens of nanograms of input DNA. Orthogonal confirmation of variants observed with Ion AmpliSeq panels is accomplished by selecting from more than 4.5 million ready-to-use TaqMan assays or by designing a custom TaqMan assay.
TaqMan assays deliver high sensitivity and specificity for SNP genotyping, and they may be analyzed in standard or digital PCR mode if increased sensitivity is required to detect low-frequency or somatic mutations.
Targeted sequencing with Ion AmpliSeq technology is scalable in multiple ways: multiplexing of primer pairs in each tube, multiple Ion Chips with different throughputs, and up to 96 barcodes for sample multiplexing. The number of primer pairs per pool is scalable, allowing researchers to target anything from a single gene to hundreds of genes while maintaining a low number of pools per panel.
Ion AmpliSeq custom panels typically contain one to two pools with 24 to 3,072 pairs per pool. Currently available Ion AmpliSeq ready-to-use panels contain 190, ~3,500, or ~4,000 primer pairs per pool. For each experiment, you can use Ion Xpress™ Barcode Adaptors for multiplexing samples at equal or asymmetrical concentrations.
The utility of this solution is further elevated with the introduction of Ion AmpliSeq Designer for creating and ordering Ion AmpliSeq custom panels. Performance specifications of Ion AmpliSeq Designer include target design rate of >85%, coverage uniformity of >85%, and on-target bases of >80%. These three performance metrics were tracked during development of Ion AmpliSeq Designer and Ion AmpliSeq custom panels, and results exceeded specification values (Table).
Ion AmpliSeq Cancer Panels
The Ion AmpliSeq Cancer Panel is designed to match the needs of clinical and translational researchers. The rapid, low-input DNA (10 ng) protocol makes it possible to screen FFPE samples that are out of reach with other target selection methods. A single-day workflow allows rapid parallel assessment of hundreds of informative mutations.
Targets are amplified using 10 ng of DNA and the Ion AmpliSeq Cancer Panel primer pool in a single-tube multiplex PCR. After purification and phosphorylation of amplicons, standard library preparation follows.
With this workflow, you can go from DNA to complete library generation typically in just 3.5 hours, with 30 minutes of hands-on time for target selection of 190 amplicons. Template preparation follows with the automated Ion OneTouch™ System. Results are available for analysis after 90 minutes of sequencing run time on the Ion PGM™ system.
The entire Ion AmpliSeq workflow—from DNA to variant calls—is typically complete in 10 hours, with walk-away sequencing and data analysis. Variant calls from Torrent Suite Software are then viewed through a standard web browser.
In a recent publication, Yang and colleagues described the histological and genetic analysis of two tumor samples (initial and reoccurrence) from the same pediatric patient. Following negative screening of candidate genes using Sanger sequencing, the Ion AmpliSeq Cancer Panel was employed.
Ten nanograms of FFPE-derived DNA from each sample was used for sequencing, and a variant in the MET gene that caused an amino acid change (a variant not previously observed in this particular tumor type) was present at 5.5% in the recurrent sample and at a very low but detectable level in the initial sample, demonstrating the ability of the Ion AmpliSeq Cancer Panel to detect new variants at very low allele frequencies.
Ion Reporter Software
Ion Reporter Software is a cloud-hosted software tool for automated variant analysis that enables laboratories of any size to create reproducible, quality-controlled analysis pipelines and produce annotated and classified variant calls. These annotations are derived from public databases (e.g., dbSNP, COSMIC, Ensembl, RefSeq). Users can also add their own data and utilize this information to classify and filter the variants. The workflow is summarized in Figure 2 and consists of four basic activities: Import→Analyze →Report→Archive.
Ion Reporter Software automates all the steps from the sequencing reads to the annotated variants (Figure 2). Researchers are able to concentrate on the interpretation of the observed variants and decisions about their relevance.
In order to confirm the accuracy of the Ion AmpliSeq Cancer Panel, 62 of the variant positions targeted by the Ion AmpliSeq Cancer Panel were also assayed using TaqMan Mutation Detection Assays powered by castPCR™ technology.
The castPCR technology uses competitive allele-specific TaqMan PCR for mutation detection, with a limit of detection of 5 to 10 copies. These assays provide superior sensitivity (down to 0.1%) and high specificity to detect and quantitate very low amounts of mutant DNA in mixed sample populations.
Nine breast cancer sample pairs were tested, each sample consisting of an FFPE sample and a fresh frozen sample taken at the same time. The results from the Ion AmpliSeq Cancer Panel and TaqMan Mutation Detection Assays at these 62 variant positions were compared.
Of the 558 sites assayed, concordance was 99.5% between the two technologies for all mutations present at >1% frequency. To enable easy selection of TaqMan Genotyping Assays, the Torrent Variant Caller allows users to select called variants and automatically search for the matching TaqMan SNP Genotyping Assays (recommended for germline mutation) or the TaqMan Mutation Detection Assays (specifically designed for somatic mutation detection). This feature greatly facilitates the design of variant confirmation for sample-screening experiments.
The expanded Ion AmpliSeq ready-to-use panels, when coupled with Ion AmpliSeq Designer v1.2, the Ion AmpliSeq Library Kit 2.0, the new Ion Reporter Software, and custom or ready-to-use TaqMan probes for downstream validation, represent complete solutions for scientists researching the genetic basis of human diseases.