Fully Automated RNA-Seq Sample Preparation
Engineering
Biomolecular Engineering - Genome Technology Center
RNA-Seq is an emerging technology for transcriptome studies using next-generation sequencing technologies compared to conventional microarray methods. RNA-Seq focuses on mRNA transcripts to determine gene expression patterns of specific cells. Constructing a genome-wide picture of all active genes in a cell allows for deeper understanding of what constitutes a particular cell type. Sample preparation for RNA-Seq is a time and labor-intensive process that requires a large amount of manual handling and attention-to-detail to sensitive RNA samples. To address the sample preparation issues of RNA-Seq, automation can increase throughput and reproducibility, minimize contamination, and reduce human error and process time through a hands-free closed system. The automated process that I developed, starts with cDNA synthesis using the Nugen Ovation RNA-Seq V2 kit followed by ligation of adapters to prepare libraries for sequencing. My automated cDNA synthesis protocol processes 8 samples in 5.5 hours with only 30 minutes of preparation time compared to 9 hours of manual preparation time. Starting with 100ng of total RNA, I can produce, 2ug of cDNA, which when sequenced in the Illumina HiSeq 2000 produces 6.2-9.8 million reads and 60% mapped back to the genome.
