Blue:RNA-Seq Analysis:06202013
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All processing using Galaxy
Step 1: Read processing
- Work Flow:
- Fastq groomer (version 1.0.4)
- TXT file (Illumina 1.3-1.7 to Sanger)
- Fastq Trimmer (version 1.0.0)
- Remove the 15nt of TSO.r04 from the 5' end
- Clip (version 1.0.1)
- TSO sequence (CACACTCTTTCCCTACACGACG)
- Keeping min 20bp and only non-clipped sequences
- Clip (Version 1.0.1)
- Rev Comp of T20 (AAAAAAAAAAAAAAAAAAAAAGATCGGAAGAGCGGTTCAGCAGGAATGC)
- Keeping min 15bp of both clipped and non-clipped sequences
- Computing quality statistics (version 1.0.0)
Step 2: Mapping
- Tophat for Illumina (version 1.5.0)
- using hg19 reference genome
- using known junctions from gene annotation model: hg19_genes.gtf
- Merged multiple bam files for the same sample using Merge BAM Files (version 1.1.2)
Step 3: Transcript Assembly and Differential Gene Expression
- Ran Cufflinks (version 0.0.5)using the reference annotation (hg19_genes.gtf)
- Ran Cuffmerge (version 0.0.5) using all sample GTF input files against the reference annotation hg19_genes.gtf
- Ran Cuffdiff (version 0.0.5) using transcripts generated from Cuffmerge and replicate analysis of mCherry high against mCherry High/CD184+CD44- and human Brain control
Step 4: Heatmaps
- Gene Pattern
- Used Fpkm_trackingToGct (version 5) to convert FPKM_tracking file generated from cuffdiff into a Gct file
- Generated heatmaps from Gct file useing GENE-E - manually selecting genes for multiple subsets
Neurogeneis:
Cell Cycle:
File:06202013 Cell Cycle HeatMap.jpg
Metabolic Genes:
File:06202013 Metabolic HeatMap.jpg
Terminally Differentiated Markers: