Analysis of Differential Gene Expression in a Myotonic Dystrophy Tissue Cultue Model

Matthew TannerBerglund Lab, Institute of Molecular Biology

CIS 407Dec. 2nd, 2013

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.

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.

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

RNA-Seq Library Preparation

Pease & Sooknanan, 2012.

Illumina Raw Output

Four lines for each sequence:Coordinates of readSequence+ASCII quality score for each base call (Phred-33)

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.

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.

Integrative Genome Viewer

Integrative Genome Viewer

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

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

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?

A closer look

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low

med

high

Gene ontologies found in this subset

Next steps?

• Analyze this and other clustered subsets by:– Sequence analysis (motifs)– GC/AT content– Gene ontology enrichment