As a powerful model system, X. tropicalis has been heavily used to study physiology, development, cell and molecular biology, and tissue regeneration. Whole transcriptome profiling certainly presents a key step to unravel gene regulatory networks and pathways involved in these basic life processes. As such, there are urgent needs to develop standardized next-generation sequencing (NGS)-based transcriptome profiling tools and resources for the community to investigate these biological processes easily, rapidly, accurately and thoroughly. Our long-term goal of this research is to standardize transcriptome profiling tools and resources to help the community further advance Xenopus genomics and genetics in the post genome sequencing era. Our overall objective for this R21 application is to develop a user friendly NGS-based transcriptome profiling system for data collection, processing and analysis. Now is the perfect time to pursue the proposed research because the genome assembly of X. tropicalis is greatly improved in the current build (v7.1) and GenBank databases have over 36,000 mRNA sequences plus 1,271,480 ESTs (expressed sequence tags) available for further gene/transcript annotation and validation. In the proposed project, we will pursue two specific aims: 1) develop a gene/transcript-specific target resource for transcriptome analysis in X. tropicalis and 2) develop a gene/transcript-specific target profiling method for transcriptome analysis in X. tropicalis.
In Aim 1, we will manually annotate gene/transcript mRNA sequences of X. tropicalis for their full-length cDNA statuses and then collect the targets from the 3'most cut sites to poly(A) tails based on in silico digestion with four enzymes, which will be used for target resource construction for transcriptome profiling.
In Aim 2, we will develop a NGS- based transcriptome profiling method, called whole transcriptome target sequencing (WTTS). The technique involves mRNA extraction, reverse transcription, enzyme digestion, bead collection, adaptor ligation, PCR amplification and NGS for production of targets described in Aim 1 for transcriptome profiling. Basically, the innovative feature of our proposed process is to simply use known genes/transcripts (Aim #1) to directly reveal known transcriptomes (Aim #2). The contribution of this R21 is expected to have a list of genes/transcripts and the WTTS tool ready for the Xenopus community, which will further empower the species as a model for biomedical research. This contribution is significant because understanding transcriptome dynamics and patterns will certainly provide new insights into mechanisms in cell cycle and mitosis, nuclear reprogramming and stem cell biology, organogenesis and tissue remodeling during metamorphosis and regeneration, and thus, deliver an essential model for the study of human diseases. Our proposed research is directly relevant to the objectives of PAR-12-249 in the area of development of new genomic resources that are of high priority for the Xenopus community. We will be committed to the free distribution and efficient use of NIH-funded data and materials generated in the proposed project to the research community.
It is becoming increasingly clear that whole transcriptome profiling presents a necessary step to unravel gene regulatory networks and pathways involved in physiology, development, cell and molecular biology, and tissue regeneration. We plan to develop standardized next-generation transcriptome profiling tools for X. tropicalis that will permit the community to investigate these biological processes easily, rapidly, accurately and thoroughly. The gene/transcript resources and tools developed in the proposed project would strengthen Xenopus as a model for understanding molecular mechanisms involved in cellular reprogramming, organogenesis, regeneration, and growth and development, thus providing insights into a multitude of human diseases and their potential treatments.