Abstract
As Next Generation Sequencing matures, it is quickly moving into translational research applications where it has promise to be a useful tool for diagnosing and treating in a clinical setting. RNA profiling using NGS (RNA-seq) is one of the applications where this potential is currently being realized. RNA-seq experiments have traditionally started with a whole-transcriptome library preparation that produces a sequencing template from all RNA species in a sample. However, in many cases, only a handful of the genes present are necessary to make a clinically relevant diagnosis.
We have demonstrated new technology that allows for RNA-seq from a panel of directed amplicons using an AmpliSeq™ approach with Ion Torrent semiconductor sequencing. This approach offers many advantages over microarray or qPCR such as faster turnaround and data analysis, sample multiplexing, lower RNA inputs, and ability to use degraded or FFPE-derived samples. In addition, the technique simultaneously provides quantitative gene expression information and gene sequence at the single nucleotide level.
We have compiled three gene panels for testing the method including a cancer panel, apoptosis panel, and a panel derived from the Micro Array Quality Control (MAQC) consortium. Starting with 10ng of total RNA, cDNA is made, followed by amplification using primers designed for targeted genes. Resulting amplicons are prepared for sequencing using the AmpliSeqTM technology and sequenced on the Ion Torrent PGM. We demonstrate that the technique produces results that are technically reproducible, quantitative, and have excellent correlation with qPCR using TaqMan® assays. Employing barcodes, we have also tested multiple samples on a single chip thereby increasing the cost-effectiveness of the tool for clinical and research use.