Lab3_QA_Server_HW_Projects.Rmd

---
title: 'Bioinformatics for Big Omics Data: Lab #3'
author: "Brian High"
date: "January 15, 2015"
output:
  ioslides_presentation:
    fig_caption: yes
    fig_retina: 1
    keep_md: yes
    smaller: yes
---

## Lab Session #3

- Your instructor, Rafael, could not come to class today.
- Brian will fill in for him.
- Rafael requests that we focus on any questions you might have.
- The main priority is computing/IT questions or problems.
- But we can try to address any sort of questions or problems after that.
- We will also talk about the server, how to access it and why.
- A major reason to use the server is for your course projects.
- Let's be sure and use Canvas for discussions/questions, okay?


## Any Computing/IT Questions?

- Software installation and operation
    * RStudio
    * Markdown
    * Git
    * GitHub
    * ???
    
We can talk about R too, but let's first cover computing support issues.

## RStudio Server: Phage

As you know, we have a RStudio server devoted to use in this course:

- http://phage.deohs.washington.edu

It is loaded with lots of other Bioinformatics software.

- [BioLinux 8](http://environmentalomics.org/bio-linux/)
    * [Ubuntu 14](http://www.ubuntu.com/)

You can access it with SSH, SFTP, and [X2Go](http://wiki.x2go.org/doku.php) also.

- phage.deohs.washington.edu port 54322

## RStudio Server

Looks and works almost exactly like RStudio desktop.

- Has built-in graphical Git client
- Fast network connection
- Lots of RAM and CPU
- Has "shell" (Terminal) access for running commands like "git".

## Tip: Rendering Presentation Files

To get the presentations to render properly in HTML:

- Clone the [Biostat-578](https://github.com/raphg/Biostat-578) repo.
- Pre-install all R packages with `installallpkgs.R`
- Open a Rmd presentation file in RStudio.
- Put your cursor at top of the editor pane.
- Step through the code chunks with Ctrl-Alt-N (Cmd-Opt-N on a Mac).
- Make sure all code runs without error.
- Then render the HTML presentation (Knit HTML).

## Other uses for the Server: Term Projects

Since *Phage* has SSH, SFTP, and X2Go access, plus a ton of software, it will
be most useful for your [term projects](https://github.com/raphg/Biostat-578/blob/master/Final-project.md).

Examples from last year: Reproduce analysis from a research paper.

- [Count-based differential expression analysis of RNA sequencing data using R and Bioconductor](http://www.nature.com/nprot/journal/v8/n9/full/nprot.2013.099.html)
- [Bisulfighter: accurate detection of methylated cytosines and differentially methylated regions.](http://www.ncbi.nlm.nih.gov/pubmed/24423865)

Both of these projects required lots of RAM (over 30 GB), and one required as 
many CPU cores as we could allocate (32).


## Differential expression analysis (RNA-seq): Materials

<img src="http://www.nature.com/nprot/journal/v8/n9/images/nprot.2013.099-I4.gif" alt="materials" style="width: 425px;"/>

## Differential expression analysis (RNA-seq): Materials

- [R](http://www.r-project.org/)
- [Bowtie2](http://bowtie-bio.sourceforge.net/index.shtml)
- [Tophat2](http://tophat.cbcb.umd.edu/)
- [htseq-count](http://www-huber.embl.de/HTSeq/doc/overview.html)
- [samtools](http://samtools.sourceforge.net/)


## Differential expression analysis (RNA-seq): Materials

The following is quoted from the [article](http://www.nature.com/nprot/journal/v8/n9/full/nprot.2013.099.html#materials)^1^:

> *This protocol assumes users have a Unix-like operating system (i.e., Linux or MacOS X), with a bash shell or similar. All commands given here are meant to be run in a terminal window. Although it is possible to follow this protocol with a Microsoft Windows machine (e.g., using the Unix-like Cygwin; http://www.cygwin.com/), the additional steps required are **not discussed here**.*

1. Anders, S. et al. Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nat Protoc 8, 1765–1786 (2013).

## Detection of mCs and DMRs

Article: *[Bisulfighter: accurate detection of methylated cytosines and differentially methylated regions.](http://www.ncbi.nlm.nih.gov/pubmed/24423865)*

The following is quoted from the [Bisulfighter website](http://epigenome.cbrc.jp/bisulfighter):

> *Requirements*
> 
> *bsf-call (Last) requires a large free memory area larger than 30GB for mapping. bsf-call requires Python 2.7 or later. ComMet requires Boost C++ library to compile. There are pre-compiled executables available in Download page.*
>
> *Download*
> 
> *Please visit Github to get the Bisulfighter source code.*

**They do not actually offer binary downloads any more. You need to compile this software from source code. We can do any software compilation for you.**

## Using X2Go Client

- [Download it](http://wiki.x2go.org/doku.php/download:start)
- Install it (OSX uses will also need [XQuartz](http://xquartz.macosforge.org/landing/))
- Approve firewall access
    * "Allow Access" prompts in Windows or System Preferences in OSX
- Configure it:
    * Session name: phage.deohs.washington.edu
    * Host: phage.deohs.washington.edu
    * SSH Port: 54322
    * Session Type: XFCE (or use MATE if using OSX)
- Host Key verification: Click "Yes"
- Log in with your UW NetID credentials
- Log out when you are done. (Don't just close the window.)

## File Transfer and SSH (Terminal)

* host: phage.deohs.washington.edu
* port: 54322

To transfer files to and from Phage, use SFTP ...

- [FileZilla](https://filezilla-project.org/)

If you just need to access a command-line terminal, use SSH:

- OSX: Use Terminal and type "ssh -p 54322 yournetid@phage.washington.edu"
- Windows: use [PuTTY](http://www.chiark.greenend.org.uk/~sgtatham/putty/download.html)