Transcriptome Sequencing by Ion Torrent Next-Generation Sequencing

Gene transcription is an intricate and dynamic process involving a variety of RNA species, including short RNAs, polyadenylated RNAs, and nonadenylated transcripts.

The key to successful transcriptome sequencing is RNA enrichment, so that you get the quality and quantity of RNA needed to build a library. The type of enrichment you select for your research depends on your application and the amount of RNA available.

Strand specificity

RNA is transcribed in a strand-specific manner from genomic DNA. Retaining strand information helps you elucidate regulation, the potential role of sense and antisense transcription, and loci with complex patterns of overlapping transcription.

Proprietary Invitrogen RNA library construction technology attaches adapters in a directional manner that preserves strand information in the libraries, enabling mapped reads to be aligned relative to the chromosomal strand.

The Ion Total RNA-Seq Kit v2 provides a fast, simple workflow with as little as 100 ng of total RNA input, while maintaining strand orientation and minimizing bias and error. The Ribominus Eukaryote Kit v2 provides more than 90% rRNA depletion increasing the sequencing power of the Ion Proton System. The Invitrogen ERCC RNA Spike-In Mix comprises a set of unlabeled, polyadenylated transcripts designed to be added to an RNA analysis experiment after sample isolation in order to measure dynamic range, the lower limit of detection, and fold-change response

The Ion Chef System^* will provide high-throughput automated template preparation and chip loading for users of all expertise. Lower-throughput users can choose the Ion OneTouch 2 System, available now, for fully automated template preparation.

Sequencing RNA transcripts enables you to look at the presence of RNA in a cell at a given moment in time. By providing greater coverage of RNA transcripts, RNA-Seq enables you to see more than what is seen with microarrays. The Ion Proton System provides you the coverage you need to sequence transcriptomes in your lab, on your budget.

The Ion Proton System

• Scalable—flexible semiconductor chip design allows you to scale your research, so you don’t have to wait to run your samples
• Fast—go from RNA sample to data analysis in less than 24 hours
• Accurate—exceeds microarray sensitivity with wide dynamic range for detecting differentially expressed genes, with a high correlation to qPCR data
• Affordable—the Ion Proton System provides you with a cost-effective system for RNA-Seq

Transcriptome sequencing coverage required

The depth of coverage required for a particular transcriptome experiment depends on the purpose of the project. For example, if your project involves looking at only expression levels of miRNA species, that may require shorter read lengths and only a modest depth of sequencing. If you are looking for detection of rare transcripts, a higher number of reads would be advised.

The chip-based Ion Proton System is designed to be flexible to handle the depth you need for your research, whether you want to run only a few panels or are looking for whole-transcriptome sequencing results. This flexibility, combined with fast sequencing run times and compatibility with intuitive software, gets you from sample to results on your budget and schedule.

Using smart informatics

The simplicity of using Ion Torrent sequencing instruments is enhanced by the easy-to-use, preinstalled Torrent Suite Software. This data analysis software suite provides tools that take you from raw data, including optimized signal processing, basecalling, and sequence alignment, to answers.

Torrent Suite Software automatically provides sequence reads in exportable BAM format, which can be uploaded to third-party software such as Partek Flow software for comprehensive downstream analysis. Learn more about Torrent Suite Software.

Go from transcriptome sequence data to biological insights with Partek Flow Software

The transcriptome pipelines in Partek Flow software, built specifically for Ion Torrent data, simplify data analysis. Partek Flow software provides:

• Streamlined integration with the bioinformatics workflow
• Point-and-click interface
• Interactive visualizations
• Read alignment
• Transcript-level measurements
• Multi-sample differential gene expression analysis

With powerful statistical and visualization tools, an intuitive user interface, and pipelines built for the way biologists think, Partek Flow software takes you from sequence data to biological interpretation of research results quickly and easily.

Learn more about Partek Flow Software
Request a free trial of Partek Flow Software