The sequencing core will receive tissue samples from Research Projects 1-3 and use next-generation sequence to produce data that will be analyzed by Research Project 4. Two types of tissue samples will be processed by the core. The Sequencing Core will receive spleens from Research Projects 1-3 that will be used for genotyping. Genotype data will be produced using genotyping by sequencing (GBS), which is an innovative technology that takes advantage of the rapid advances in next-generation sequencing. This core will extract DNA from the spleens, make GBS libraries, multiplex those libraries, and sequence them using an Illumina 2400 equipment. The raw sequencing reads will be sent to Research Project 4 for further analysis.
The Sequencing Core will also perform RNA sequencing (RNAseq) of 5 key brain regions: nucleus accumbens core (AcbC), lateral habenula (LHb), prelimbic cortex (PC), infralimic cortex (IL) and orbitofrontal cortex (OFC). Total RNA from these brain regions will be supplied by Research Project 2, which will use microdissection to accurately dissect these regions from HS rats. This core will preparing libraries, multiplex them at a density of 12-15 per lane and sequence from a single end, 100 bp, using an Illumina 2400, to obtain 25-30 million reads per library. The raw reads will be passed to Research Project 4 for further analysis. The PI’s laboratory (Dr. Palmer) has extensive experience with all necessary techniques. This core will have access to numerous next-generation sequencing machines that are already in place at the UCSD as well as next-generation sequencing machines from other companies (e.g. 454, Pacific Biosystems, etc.). Because sequencing technology will continue to evolve rapidly, we anticipate that some of the technical details that we describe will change over the proposed funding period; since those changes are difficult to predict we have focused on describing technologies that are currently in use in Dr. Palmer’s lab. Note that we are not proposing to purchase any major equipment (e.g. we are not proposing to buy a nextgeneration sequencing machine) as part of this core. This allows us to deliver the necessary services while minimizing the costs paid by the center.
This core may also provide GBS services to outside investigators on a cost recovery basis. Because the analysis of GBS data requires large numbers of samples, such a service would only be appropriate for investigators who are interested in genotyping at least several hundred samples.