analysis of differential gene expression in a myotonic dystrophy tissue cultue model
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Analysis of Differential Gene Expression in a Myotonic Dystrophy Tissue Cultue Model. Matthew Tanner Berglund Lab, Institute of Molecular Biology CIS 407 Dec. 2 nd , 2013. Myotonic Dystrophy 1 (DM1). Symptoms - PowerPoint PPT PresentationTRANSCRIPT
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Analysis of Differential Gene Expression in a Myotonic Dystrophy Tissue Cultue Model
Matthew TannerBerglund Lab, Institute of Molecular Biology
CIS 407Dec. 2nd, 2013
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Myotonic Dystrophy 1 (DM1)
Genetic PathologyDM1 caused by CTG trinucleotide repeat expansion in 3’ UTR of DMPK gene.
Unaffected: less than 34 repeatsDM1: 50 to >1000 repeats
Severity of disease increases and age of onset decreases with increasing repeat length.
Symptoms
Multisystemic – affects skeletal and smooth muscle (myotonia, atrophy), eyes (cataracts), heart, and endocrine system.
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Potential for small molecules to alleviate molecular symptoms of DM1
Coonrod, et al. (2013)
PentamidineAnti-fungal, anti-protozoan drug. Binds minor groove of DNA.
Wilson et al., 2008.
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Tissue Culture Model and ExperimentTransfect into HeLa cells. Drug 6 hours afterwards.
24 hours after transfection: harvest whole-cell RNA.
DMPK minigene containing 960 CTG
repeats. Induces DM1 disease state.
DMPK960
Illumina highthroughput sequencing library preparation (several steps)
Illumina Hi-Seq 2000 massively parallel sequencer
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RNA-Seq Library Preparation
Pease & Sooknanan, 2012.
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Illumina Raw Output
Four lines for each sequence:Coordinates of readSequence+ASCII quality score for each base call (Phred-33)
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Alignment and Analysis with Tuxedo Suite
Pass $SAMPLE variable in at command line: qsub tophat.sh –N align0A –V SAMPLE=“0A”
Tophat 2.0 – align FASTQ reads that were cleaned up with Stacks’ process_shortreads to human genome.
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Alignment and Analysis with Tuxedo SuiteTophat 2.0 – align FASTQ reads that were cleaned up with Stacks’ process_shortreads to human genome.
Cufflinks – take mapped reads (accepted_hits.bam) and generate transcript model of reads.
Cuffmerge – take individual transcript models (transcripts.gtf)and merge into master transcriptome.
Cuffdiff – take mapped reads from individual treatments (accepted_hits.bam) and, with aid of master transcriptome, compare each sample pair-wise.
Take various Cuffdiff output files (differential gene expression, splicing, promoter usage, isoforms, etc.) and analyze with original scripts or explore with existing programs.
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Integrative Genome Viewer
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Integrative Genome Viewer
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log10 (FPKM + 1) of genes at each dosage that are associated the p53 network.
FPKM: fragments per kilobase of exon model per million mapped fragments
Visualization of differential gene expression
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log10 (FPKM + 1) of genes at each dosage that are associated with the gene ontology “Regulation of RNA splicing” (GO:0043484)
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Visualization of differential gene expression
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How about all the (significantly differentially expressed) genes?
log2(foldchange) of 1210 genes with q < 0.05 between none and low pentamidine dosage
What are these genes?
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A closer look
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Gene ontologies found in this subset
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Next steps?
• Analyze this and other clustered subsets by:– Sequence analysis (motifs)– GC/AT content– Gene ontology enrichment