sheilayee / project-5

Project Description

While adult mammalian hearts possess limited regenerative ability upon cardiac injuries, neonatal cardiac myocytes are known to regenerate via proliferation of pre-existing cardiomyocytes. The molecular mechanisms and transcriptional regulators that oversee this regenerative process remain unclear. O'Meara et al. (2015) sought to elucidate these transcriptional changes and determine if regenerating cardiac myocytes revert to a less differentiated state. This project seeks to reproduce some of those findings.

This repository contains the scripts used to process and analyze the data.

O’Meara, C. C., Wamstad, J. A., Gladstone, R. A., Fomovsky, G. M., Butty, V. L., Shrikumar, A., Gannon, J. B., Boyer, L. A., & Lee, R. T. (2015). Transcriptional Reversion of Cardiac Myocyte Fate During Mammalian Cardiac Regeneration. Circulation Research, 116(5), 804–815. https://doi.org/10.1161/CIRCRESAHA.116.304269

Contributor

Sheila Yee [email protected]

Repository Contents

run_tophat.qsub

Implements TopHat, a splice aligner, to align RNA-seq reads from P0_1 sample to the mouse reference genome (mm9). Other utilities - Bowtie2, Boost, Python, and SAMtools - are also implemented.

To run, submit as a job on the cluster:

qsub run_tophat.qsub

This outputs accepted_hits.bam.

Determine alignment statistics

In the directory where the TopHat results are located, run on the command line:

samtools flagstat P0_1_tophat/accepted_hits.bam 

Index BAM file

In the directory where the TopHat results are located, run on the command line:

samtools index accepted_hits.bam

This outputs accepted_hits.bam.bai.

rseqc.qsub

Uses RseQC to performs quality control analysis on RNA-seq data and alignment.

To run, submit as a job on the cluster:

qsub rseqc.qsub

This outputs gene_body.geneBodyCoverage.curves.pdf and inner_distance.inner_distance_plot.pdf plots.

run_cufflinks.qsub

Uses Cufflinks to determine FPKM values of reads mapped to genomic regions. Run Cufflinks on P0_1_tophat/accepted_hits.bam.

To run, submit as a job on the cluster:

qsub run_cufflinks.qsub

This outputs genes.fpkm_tracking file which contains FPKM values for all genes.

run_cuffdiff.qsub

Uses Cuffdiff to identify differentially expressed genes in the P0_1 sample.

To run, submit as a job on the cluster:

qsub run_cuffdiff.qsub

This outputs genes_exp.diff.

FPKM_histogram.R

Creates a histogram of FPKM values from the P0_1 sample. Requires genes.fpkm_tracking to run. Run using RStudio.

FPKM_sar_mit_cc_plots.R

Creates plot to visualize FPKM values of representative sarcomere, mitochondrial, and cell cycle genes of interest during in vivo maturation across all neonatal and adult stages. Recreates Figure 1D from O'Meara et al. Requires P0_1_cufflinks/genes.fpkm_tracking and genes.fpkm_tracking for the remaining 7 samples (provided by instructor) to run. Run using RStudio.

FPKM_heatmap.R

Creates a heatmap of the top 1000 and top 10 differentially expressed genes across all neonatal and adult stages (8 samples total). Recreates Figure 2A from O'Meara et al. Requires fpkm_matrix.csv (provided by instructor), gene_exp.diff, and genes.fpkm_tracking to run. Run using RStudio.