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:

File:06202013 HeatMap 2.jpg


Cell Cycle:

File:06202013 Cell Cycle HeatMap.jpg


Metabolic Genes:

File:06202013 Metabolic HeatMap.jpg


Terminally Differentiated Markers:

File:06202013 Term Diff HeatMap.jpg