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David Ruff Life Technologies, United States of America |
Abstract
Ion AmpliSeqTM technology is a simple, efficient and rapid process for enriching hundreds to thousands of targets for next generation sequencing. This technology is based on highly multiplexed single tube PCR amplification using either gDNA or cDNA templates. A great advantage for this approach is the small sample requirement for amplification reactions. As little as 1-10 ng of FFPE or whole genomic DNA can be readily amplified for Ion Personal Genome Machine® (PGMTM) sequencer runs. This chemistry is scaleable to very high levels of multiplex. The Ion AmpliSeqTM Comprehensive Cancer Gene Panel has been designed to include 4 pools of ~4,000 amplicons per tube. This panel covers the coding exon regions for 409 cancer genes. Another panel of ~10,000 amplicons distributed into 3 tubes covers 325 inherited disease gene exon coding regions. Moreover, the upper boundaries of plexity have yet to be reached – successful feasibility has been demonstrated at the 26,500-plex level for individual AmpliSeq reactions. Ion AmpliSeqTM technology is seamlessly applied to discover somatic biomarker mutations in cancer research samples. For example, we embarked on a collaborative effort to identify somatic biomarkers that may drive neoplastic evolution in esophageal cancer. The Ion AmpliSeqTM Custom Design pipeline was employed to layout 1395 primer sets for amplicons to cover the coding exons of 20 frequently mutated genes. Our study interrogated 40 tumor gDNA samples along with their matched normal counterparts and identified 21 novel somatic mutations. An emerging application for translational research is to access, detect and sequence circulating cell-free (ccf) nucleic acid biomarkers in human blood samples. We have a developed a workflow to purify and sequence ccf gDNA. Furthermore, we can also detect and quantify ccf mRNA using a multiplex preamplification scheme. And finally, employing a novel mRNA-expression capture method, we report a breakthrough approach that enables detection and analysis of newly synthesized mRNA species. This tool provides an avenue to identify and quantify real-time transcriptional biomarker activity in cellular responses. In summary, highly multiplexed PCR technology powers the Ion AmpliSeqTM platform and this combination promises to bring forth a next generation landscape that will redefine the boundaries of biomarker discovery.
For Research Use Only. Not for Use in Diagnostic Procedures.
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