Methods to enable direct RNA sequencing without amplification

PROJECT SUMMARY The recent emergence of high throughput DNA sequencing technologies and their application to transcriptomics by sequencing complementary DNA cDNA have resulted in an even more complicated view of the transcriptome and its regulation demonstrating our limited knowledge of transcription DNA sequencing technologies have eliminated some of the technical challenges posed by hybridization based microarray strategies but several fundamental shortcomings still remain that prevent us from understanding the true nature of transcriptomes and from identifying the fold transcription changes that can distinguish a disease associated physiological process from a normal one To respond to this need SeqLL LLC Woburn MA is proposing a Phase I SBIR project to develop optimize and demonstrate the feasibility of a direct RNA sequencing technology to capture whole transcriptome information from the RNA isolated from a single cell This work builds on encouraging preliminary data demonstrating the feasibility of a direct RNA sequencing technology based on a single molecule sequencing platform however existing challenges must be overcome for this technology to be a viable and reliable commercial option including a reduced read length compared to DNA sequencing a lower yield compared to a similar input of DNA and is less reproducible in lower order species To achieve this goal the team proposes three specific aims Specific Aim To design synthesize and screen multi color reversible terminators optimized for the sequencing of RNA molecules This aim will use chemical synthesis methods to generate reversible terminator RT analogs that are well suited to direct RNA sequencing These RTs will be third generation based on innovative iterations of these molecules by SeqLL fluorescently labeled and have a variety of inhibitory moieties that will be tested for improved sequencing performance Specific Aim Optimize chemical and reagents for use with high throughput single molecule direct RNA sequencing to improve base stacking sequencing yield read length and accuracy This aim will optimize imaging buffer generate mechanisms for cleave capping and to test alternative modifications that should allow for significant improvements in direct RNA sequencing base stacking sequencing yield read length and accuracy Specific Aim Whole transcriptome sequencing of single cell RNA without amplification This aim will seek to efficiently perform whole transcriptome sequencing of RNA derived from a single cell without amplification Single cell preparations will be obtained from our collaborators at the Bernstein lab Harvard Medical School This work will report upon methods and the sequencing performance of using the optimized methods identified in Aims and to perform single cell direct RNA sequencing of the whole transcriptome PROJECT NARRATIVE The objective of this Phase I SBIR is to develop optimize and demonstrate the feasibility of a direct RNA sequencing technology to generate whole transcriptome information from the RNA isolated from a single cell The ability to sequence RNA directly and without the need for bias introducing and complicated amplification steps would allow researchers and clinicians to overcome the current technical challenges that prevent us from understanding the true nature of transcriptomes and from identifying the fold transcription changes that can distinguish a disease associated physiological process from a normal one particularly at the single celled level