HW7 - RNA-Seq Analysis

3 minute read

EXCEPTION: Due to the cluster outage (May 7-8th), the read count tables required for HW7 have been made available for download from this GEN242_Data repository created for this purpose. To work with this data set, clone this repository (same way as HW repos), and then cd into the rnaseq subdirectory located under GEN242/HW7_data/ of this repos, or set your RStudio session to this directory. Next open the provided HW07.Rmd file under HW7_data/rnaseq and follow the instructions given in this file. I contains quite a bit of additional helper code, well beyond what is given below.

A. Unstranded and strand-specific read counting

• Task 1: Rerun or reload the RNA-Seq workflow with the toy data sets up to the read quantification step here. Note, the toy data set gets automatically loaded when intializing a workflow environment (directory structure) with the genWorkenvir function (see tutorial here).

  In the read quantification step with summarizeOverlaps generate count tables for exons by genes (eByg) of the following three strand modes:

  1. Unstranded
  2. Strand-specific for positive (sense) strand
  3. Strand-specific for negative (antisense) strand

  The solution for generating the unstranded read counts is given below. Note, the upstream steps of the RNA-Seq workflow only need to be rerun to generate the proper inputs for the read counting. Thus, they are not required to be included in the homework results (see HW7.R below).

unstranded <- summarizeOverlaps(eByg, bfl, mode="Union", 
                                                ignore.strand=TRUE, 
                                                # preprocess.reads=invertStrand,
                                                inter.feature=FALSE, 
                                                singleEnd=FALSE)
unstranded <- assays(unstranded)$counts
unstranded[1:4,]

Before attempting to solve this homework task please read the vignette Counting reads with summarizeOverlaps (here) from the GenomicAlignments package that defines the summarizeOverlap function. In addition, the help file for ?summarizeOverlaps provides useful information.

• Task 2: Provide R code that demonstrates that the two strand-specific count tables sum up to very similar values as the unstranded count table.

• Task 3: Explain the utility (biological relevance) of the different strand counting modes used under Task 1. Include your explanation as comment text in your homework script (see HW7.R below).

Note, for Tasks 1-3 only the code and/or text needs to be included in the homework submission (no data/result files). For details see below.

B. Read counting for different feature types

• Task 4: Compute strand-specific count tables for the positive (sense) strand of the following feature types. The help files of ?exonsBy and ?transcripts provide useful information for solving these tasks.

  1. Genes
  2. Exons
  3. Exons by genes
  4. Introns by transcripts
  5. 5’-UTRs by transcripts

Note, for Tasks 4 only include the code and/or text in your homework submission (no data/result files).

C. DEG analysis

• Task 5: Perform the DEG analysis with edgeR as outlined under section 6 of the RNA-Seq workflow here. Use in one case for the DEG analysis the unstranded count table as input (from Task 1.1) and in another the sense strand count table (from Task 1.2). Compare the DEG result of the two methods in two separate 4-way Venn diagrams for the same sample comparisons used in the workflow example here.

  1. 4-way Venn diagram for unstranded count table
  2. 4-way Venn diagram for sense strand count table

Note, for Tasks 5 include both the code and the resulting images in your homework submission.

Homework submission

Please submit the homework results in one well structured and annotated R script to your private GitHub repository under Homework/HW7/HW7.R. Instead of an R script the homework can be submitted in form of an R Markdown (*Rmd) file.

Due date

This homework is due on Tue, May 14th at 6:00 PM.

Homework Solutions

See here.