HW7 - RNA-Seq Analysis
3 minute read
A. Unstranded and strand-specific read counting
Task 1: Rerun 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
genWorkenvirfunction (see tutorial here). In the read quantification step with
summarizeOverlapsgenerate count tables for exons by genes (
eByg) of the following three strand modes:
- Strand-specific for positive (sense) strand
- 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
summarizeOverlaps(eByg, bfl, mode="Union", ignore.strand=TRUE, # preprocess.reads=invertStrand, inter.feature=FALSE, singleEnd=FALSE)
Before attempting to solve this homework task please read the vignette
Counting reads with
GenomicAlignments package that defines the
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
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
?transcriptsprovide useful information for solving these tasks.
- Exons by genes
- Introns by transcripts
- 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
edgeRas 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.
- 4-way Venn diagram for unstranded count table
- 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.
Please submit the homework results in one well structured and annotated R
script to your private GitHub repository under
of an R script the homework can be submitted in form of an R Markdown (*Rmd) file.
This homework is due on Thu, May 11th at 6:00 PM.