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Richard Fekete, Kelli Bramlett, Yongming Sun, Jeff Schageman, Luming Qu, Ross Hershorn, Charmaine San Jose Hinahon, Brian Sanderson, Angie Cheng, Bob Setterquist Life Technologies, United States of America |
Abstract
RNA-Seq technology has become widely utilized as a tool to understand the transcriptome of a given experimental system. This method utilizes next generation sequencing platforms to sequence a cDNA library in order to gain information about the RNA content and transcriptional status of a sample of interest. Profiling the transcriptome of a system in this way has become an invaluable tool in many genomic studies.
More information is now available through the use of the newly launched Proton™ system utilizing the same simplified chemistry first introduced with the Personal Genome Machine (PGM™). The increased sequencing depth of the Proton™ instrument and the Ion PI™ chip now lends this simplified sequencing technology to true whole transcriptome evaluation including sequencing analysis of polyadenylated RNAs, long non-coding RNAs, and non-adenylated transcripts. We describe the differential gene expression profiling of two well-studied RNAs, Universal Human Reference RNA (UHRR) and Human Brain Reference RNA (HBRR), with the External RNA Control Consortium transcripts (ERCCs) spiked into the RNAs. This study demonstrating over 50M mapped reads from each Proton™ transcriptome sequencing run, comparable gene expression profiles to an orthologous technology, and good sensititivity and dynamic range as shown with the ERCC controls, solidifies the new Proton™ sequencing system as a optimal platform for complex transcriptome analysis.
Less information is more appropriate when only a handful of the genes present are necessary to make a decision such as a clinically relevant diagnosis. We have demonstrated new technology that allows the expression analysis from a panel of selected genes by sequencing directed amplicons using an AmpliSeqTM 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 developed two ready-to-use gene panels targeting cancer and apoptotic pathways. Gene specific amplicons are prepared for sequencing using the AmpliSeqTM technology and sequenced using Ion Torrent sequencing technology. We demonstrate that the technique produces results that are technically reproducible, quantitative, and have excellent correlation with qPCR using TaqMan® gene expression assays. Employing barcodes, we have also multiplexed AmpliSeq™ RNA libraries thereby increasing the cost-effectiveness of the tool for higher throughput laboratory settings.
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