Abstract
High throughput RNA sequencing (RNA-Seq) is becoming increasingly utilized as the technology of choice to detect and quantify known and novel transcripts. Multiple next-generation sequencing (NGS) platforms are available that enable transcriptome profiling through RNA-Seq workflows. Demonstrations of the power of RNA-Seq to profile the well annotated transcriptome and also identify novel transcribed regions, gene fusions, and even identify novel classes of RNA are rapidly increasing in the field of RNA research. Our aim has been to develop library preparation methods and tools that aid in the reliable generation of libraries for next generation sequencing from total RNA. Reported here are results from the development of the Ambion® RNA-Seq Library Construction kit optimized for sequencing on the Illumina® next generation sequencing instruments. We show results from two protocols utilizing the same reagents that allow generation of RNA-Seq libraries targeting either the small RNA fraction of total RNA, or the whole transcriptome which includes transcripts larger than 100 base pairs. Results are reported from Illumina® Genome Analyzer II sequencing of both small RNA and transcriptome libraries with a focus on mapping to the miRBase and RefSeq references respectively. We also demonstrate the use of External RNA Control Consortium (ERCC) transcripts as spike-in controls for transcriptome libraries that aid in quality control of the library generation procedure and aid in downstream data analysis. The library construction technology embedded in the Ambion® RNA-Seq Library Construction kit enables researchers to analyze the transcriptome of their research samples in a precise, sensitive and robust manner while maintaining information regarding the genomic DNA strand to which the RNA transcript maps utilizing the Illumina® Genome Analyzer II sequencing platform. The workflow and results reported here demonstrate new commercially available options for library construction enabling small RNA and transcriptome profiling and novel discovery using next-generation sequencing technology.