Proposed Improvements

5-v. Proposed improvements

Update ftp links

Update all wget commands to download from new ftp folder locations instead of subfolders in GMS project

Update the AMI to use bash instead of dash as default shell

The STAR aligner currently throws errors when run on an ubuntu system because /bin/sh is linked to /bin/dash and not to /bin/bash.

ls -l /bin/sh
sudo mv /bin/sh /bin/sh.orig
sudo ln -s /bin/bash /bin/sh

Install 'tree' command into the AMI

Pre-install the tree command in the Amazon AMI so that it is ready for students to use

Install 'pip' command into the AMI

In order for htseq-count to use bam files directly it needs pysam. This can be installed with pip but that is not available by default.

Then, On AMI install and test htseq-count with bam files:

sudo apt-get install python-pip
sudo pip install pysam

An alternative install procedure that has been tested and worked is as follows. The above procedure is preferred and should be tried first.

cd ~/bin/
wget https://pysam.googlecode.com/files/pysam-0.7.5.tar.gz
tar -zxvf pysam-0.7.5.tar.gz
cd pysam-0.7.5/
python setup.py build
sudo python setup.py install

Create a trimming section

Create a wiki section and exercise that summarizes read trimming concepts. Start with some raw data, including aligned reads. Align these reads without any trimming and assess alignment statistics using Picard, FastQC, etc. Now take these same reads and perform both adaptor trimming and quality trimming. Re-align the trimmed reads and assess the effect of trimming on alignment metrics.

Add RSeQC to tutorial

Add installation and running of RSeQC This could possible replace Samstat which never works very well

Add integrated assignment content to the wiki

Get the integrated wiki from CBW and incorporate that here.

Create a batch effect section

We should add a section about batch effects. Both detecting the presence of batch effects as well as correcting for them during analysis.

Improve Cloud exercise

Have students actually spin up their own instances using IAM accounts (with one set up per student before the course). They would then be provided with their IAM user name, the appropriate AWS account #, and a default password. This account would have limited permissions to start EC2 instances but not much else.

Add a fusion detection section

We previously had a fusion detection module but it was difficult to complete in time frames appropriate for a workshop. Further optimization is required. Another challenge is the lack of well engineered fusion detection software. This publication State-of-the-art fusion-finder algorithms sensitivity and specificity does a decent job of summarizing the current options available. Another caveat of this topic is that is mostly of interest to cancer researchers so it might only be included where there are sufficient students with this interest.

Improve alignment QC section

In particular we should add use of Picard CollectRnaSeqMetrics (https://broadinstitute.github.io/picard/command-line-overview.html) and RNA-SeQC (http://www.broadinstitute.org/cancer/cga/rna-seqc). It would also be good to include use of splicing metrics calculated from the TopHat junctions files. A standalone version of the TGI tool that does this would need to be created for this purpose.

Identify more interesting data sets to use for the alternative splicing module

http://www.ncbi.nlm.nih.gov/gds/?term=rna-seq+splicing

• GSE63953
• GSE63375
• GSE63569
• GSE45119
• GSE48263
• GSE44402

Gray lab breast cancer cell line dataset:

• http://www.ncbi.nlm.nih.gov/pubmed/24176112
• https://www.biostars.org/p/111040/ (biostars tutorial on downloading data)
• https://github.com/genome/gms/wiki/Guide-to-Importing-and-Analyzing-External-Data (another guide on downloading and reformatting this data)

Update the tutorial to take into account recent developments in RNA-seq analysis methods, best practices, and new tools

• HISAT
• StringTie
• Ballbown

| Previous Section | This Section | Next Section | |:------------------------------------------------------------:|:--------------------------:|:-------------------------------------------:| | Integrated Assignment | Proposed Improvements | AWS Setup |