The regulation of gene expression drives all biological processes. A thorough understanding of the mechanisms governing the patterns of gene expression is necessary to understand the fundamentals of biology and the complexities of cancers and other diseases. The future of research into human disease will be based in next generation sequencing (NGS) of the human genome and transcriptome (the set of all RNA molecules, including mRNA, rRNA, tRNA, and non-coding RNA produced in one or a population of cells);and will be dependent on accurate sequence representation from DNA and RNA libraries. The cost of DNA sequencing has dropped dramatically in recent years with the advent of NGS and this has revolutionized the way that gene expression is studied. In this new paradigm, the transcriptome is converted to complimentary DNA (cDNA) by a class of enzyme known as reverse transcriptase. Nucleotide sequence analysis reveals the genetic code for mRNAs as well as numerous post transcriptional process steps that can only be studied by RNA sequencing. This provides a comprehensive view of all expressed genes, as well as additional information not available through traditional approaches. NGS platforms are increasingly robust and reliable;however, the adaption of these technologies for transcriptome sequencing is less developed. A key step in transcriptome sequencing, the conversion of all RNA species (the transcripts) to DNA (the material analyzed in the NGS systems) is laden with technical difficulties and compromised by copying errors, rearrangements and biases during the conversion by low fidelity reverse transcriptases. This conversion process is time consuming and prone to operator error. Through a decade-long program to study the genetics of viruses and phage in hot springs, Lucigen has discovered and developed a unique enzyme called PyroScript RT that promises to improve the accuracy of RNA sequence analysis by two orders of magnitude compared to conventional enzymes and greatly reduce the frequency of rearrangements. The high temperature optimum of PyroScript RT also promises to reduce bias due to secondary structures. We intend to use this enzyme to develop and validate transcriptome library synthesis kits that incorporate a single, novel, high fidelity polymerase for reverse transcription of RNA and subsequent DNA amplification. To achieve this goal we will develop a simple protocol for cDNA library synthesis that is flexible for all of the major NGS platforms and based on the unique qualities of PyroScript RT that will reduce misincorporation artifacts and bias. The result of our proposed study will be a reagent that will allow both the small and large research laboratory to compete on equal grounds and advance the knowledge of transcriptome diversity through a more accurate and unbiased sequence database.
The human genome and transcriptome (transcribed RNA) contain information relevant to human disease. With the advent of next-generation sequencing and improved bioinformatics analysis techniques, we are able to more rapidly characterize disease samples and thus increase our understanding of the susceptibility and onset of inherited diseases, the somatic mutations that initiate cancers and the identity of pathogenic microbes that affect humans. New tools are needed to improve the accuracy, eliminate rearrangements and reduce the bias of transcriptomics research as well as simplified kits that provide easy access to next-generation sequencing to all researchers. The funding provided for this project will help us develop these tools and help speed the research on human diseases.