General outline of the RNA-Seq experiment and analysis workflow.

<p>RNA was extracted from PD leukocytes pre-DBS, post-DBS on stimulation and following one hour of electrical stimulation cessation (off-Stim). Libraries were constructed for SOLiD whole transcriptome sequencing following ribosomal RNA removal, fragmentation and gel purification. Primary analysis of the 50 nucleotide long reads was followed by mapping of the outcome reads to the human genome through BioScope cloud computation. Tertiary data analysis workflow was developed through a novel RNA-Seq module of AltAnalyze (version 2.0). The analysis steps included: sample-specific database construction followed by detection of known and novel junctions and exons. Four different algorithms were used for measurement of splicing changes from single features (junctions/exons) and feature pairs. These included: linear regression, splicing-index, ASPIRE and FIRMA. Subsequent annotation of the splicing events at the transcript level enabled detection of various annotations including 3′ and 5′ end variations, cassette exons and mutually exclusive exons. Additionally, detection of both promoter region and splicing alternations as well as intron retention events was enabled, and a novel module for poly-A site prediction was incorporated. Enrichment for protein binding domains and miRNA target sites was conducted using z-score and the corresponding databases. Additionally, detection of all the lncRNAs expressed in these samples expressed lncRNAs was conducted through alignment to GENCODE 7 database, and followed by differential expression analysis (EdgeR) and predictions of secondary structure and potential miRNA complementarity predictions.