UnixSpring2013

= Genomic Analysis at the UNIX Command Line =

Your instructor for today is Madhavan Ganesh.

IMPORTANT NOTES
The material below is intended for the in-class instruction. Others trying to learn this material offline can follow along on any Unix (like) machine, however, please be aware the file names and path names will be different on your machine. The material here is adapted to a large extent from prior workshops at CGRL and presented by other instructors.

Topics covered in this module:

 * Why familiarity with UNIX is necessary for genomic analyses
 * Navigating the UNIX environment
 * Basic UNIX utilities
 * Text editors
 * Basic data manipulation with UNIX

Introduction and Requirements
Welcome to the CGRL module for Genomic Analysis at the UNIX command line. This module is designed for scientists hoping to analyze genomic datasets, but who have no experience with the UNIX computing environment or common genomic analysis tools.

The module requires that you use a recent MacOS or Linux based system. Future versions of the module may accomodate Microsoft Windows users.

What are you going to learn?
After this morning's session, you will be able to navigate the UNIX environment, allowing you to create, manipulate, and store large data files efficiently. You will find that in many ways, the UNIX environment is designed for speed and simplicity in using large files in a way that is unavailable in more "user-friendly" environments like Microsoft Windows or Mac OS. We will conclude this morning's session with a set of exercises that demonstrate the power of the UNIX environment to generate a basic description of the //Saccharomyces cerevisiae// genome. Further, if time permits we will go through the process of downloading and installing a genomic utility (Bowtie) to familiarize users with the execution environment.

What are you not going to learn?
This module is intended to teach very basic skills for the UNIX environment, allowing students to pursue more instruction with other modules that will guide you through the complications and considerations for proper genomic analysis. Accordingly, we will try to demonstrate the very basics of using one genomic utility (Bowtie), but we will not venture into the algorithms, the considerations, or the statistics behind a proper analysis. We hope that you are interested enough after finishing this module to pursue those questions with our other instructors.

Schedule for today
We will go along with a interleaving of description of the topic and a few hands-on exercises that attendees are expected follow along. To derive benefit from this workshop please try to keep up with the topic being discussed. During the hands-on exercises we will try to make sure that every on is in sync.

Warning: Learning curve ahead!
If you're unfamiliar with a text-based computing environment (some of us are old enough to have started with one as a kid), things may seem a bit archaic at first. That's okay, because UNIX is more than a bit archaic. It's not your fault. So if things seem opaque or difficult to remember, don't fret, and please feel free to ask us questions. In addition, you may want to queue up a couple of websites for quick reference. For example, [|this Unix Quick Reference] might be of use if you want to search for key words to remind you which command does what.

In the longer term, you may appreciate the following text available to us via the UC Berkeley Library: Linux Pocket Guide

For the lecture portion of this course, I would actually recommend something a bit more old-fashioned. You might find it easiest to just have a pen and paper handy and jot down each command we go over along with a brief explanation of what it does. This will come in very useful during the exercises and you won't have to flip back and forth between your terminal screen and your web browser.

Using the UNIX shell
Your time in the UNIX environment will be spent in the **shell**, which is lingo for the Terminal window, if you're using Mac OS. The shell allows you to move and copy files, run programs, and more. We will also touch on the basic usage of the the text editor **emacs**.

Informative Interlude: Some notes on the formatting of the lessons for this course
Periodically, we may stop with an **informative interlude** outlined with a horizontal line above and below (like the one two lines up!). In this case, we're taking a quick break to discuss this and other aspects of the formatting.

For this and all further examples, a **$** represents your shell prompt, and **boldface** indicates the commands to type at the prompt. //Italics// will be used for output you should see when you take the described action.

This concludes our first informative interlude.

How do I open my terminal window?
In Mac OS, you can open a new terminal window by opening the Finder, selecting the Applications folder, then the Utilities sub-folder, and then double-clicking on the Terminal application. You may want to drag the Terminal icon to your Application Dock for ready access going forward.

Alternatively, you can just use Spotlight to search for "Terminal" each time, and open the application after it is listed in the Spotlight results list.

In Linux environments, your terminal program will depend on the version of Linux you have installed. If you are using Linux and you don't know how to open a terminal, raise your hand and let us know now.

Accessing your CGRL account
The next step is to login to the CGRL system (poset.cgrl.berkeley.edu) with your user account and password. You can do this with the **ssh** command.

$ **ssh mganesh@poset.cgrl.berkeley.edu**

Whoa, where am I?
When you first log into a system, you should be placed in your **home directory**. This is a special directory on the system meant just for you.

You can use the **pwd** command to see the location of the directory you're in, whether it's your home directory or any other directory you end up navigating.

$ **pwd**

///global/home/mganesh//

And you can use the **cd** command to change to another directory.

$ **cd /global**

$ **pwd** ///global//

To return to our home directory, we can simply change back.

$ **cd /global/home/mganesh**

$ **pwd** ///global/home/mganesh//

Although there is a shortcut for returning to your home directory from any other directory, which is to simply issue the **cd** command with no directory argument:

$ **cd**

$ **pwd** ///global/home/mganesh//

Another useful shorthand for **cd** is to use two dots after the command. This will take you one directory "up" in the tree. For example, if we want to go to **/global/home** from our home directory, we can just issue:

$ **cd ..**

$ **pwd** ///global/home//

And we can go all the way the to the top, or **root**, of the file system tree if we issue this command again.

$ **cd ..** $ **cd ..**

$ **pwd** /

But being that far from home might make us nervous, so with one quick command we can go back to our home directory.

$ **cd**

$ **pwd** ///global/home/mganesh//

Whew. That's better.

An aside on directories...

Directories in UNIX are set up the same way as any other computer. Just as you would open up a window into your directories and click to open up folders, here you use **cd** to go through the directories. You are just typing the command instead of clicking!

And if we want to find out what files are available in each folder, we have the command **ls**.

$ **ls**

Of course, we have to have files if we want to see them.

$ **ls /global/home** //across dbachtro gsluser jtaylor lgibilis mdavis mroth radams sbranco ssinghal vwu// //aihardin dkhendrix isylvain kmuriki maugusti mfischer msyed rfletche sglassma tandre ylee// //cellison ganeshm jchoi lbrunet mchung mganesh mturner rvonmay silyas tmadsens zhouqi//


 * ls** has many options. Here are some of the more useful ones to know.

The **-l** argument shows the detailed list of the files. This is similar to the options you're likely familiar with in MacOS or Windows.

$ **ls -l**

The **-t** argument will sort the files by their modification time, so you can see which file is the newest (first) or oldest (last) in the directory list.

$ **ls -lt**

What does the **-r** argument do?

$ **ls -ltr**

The same directory short hand for the directory above us in **cd** can be used for **ls**:

$ **ls ..** //across dbachtro gsluser jtaylor lgibilis mdavis mroth radams sbranco ssinghal vwu// //aihardin dkhendrix isylvain kmuriki maugusti mfischer msyed rfletche sglassma tandre ylee// //cellison ganeshm jchoi lbrunet mchung mganesh mturner rvonmay silyas tmadsens zhouqi//

"To create is divine..."
We can make new directories with the command **mkdir**. (Before that make sure (how?) we are doing do so in our home directory. If not how do we get back there?)

$ **mkdir myFirstDirectory**

And then, of course, we can change to that directory.

$ **cd myFirstDirectory**

Text Editor Interlude
What if we want to create a new text file? The way to do this that is most similar to what you are probably used to doing in a Mac or Windows operating system is to use a text editor. Unix text editors tend to have quite unintuitive commands, as you will soon see. Among the more popular ones are **vi**, **emacs**, and **nano**. In this class we will use the **emacs** text editor and I would now like to create a new file called //myFirstFile//.

$ **emacs myFirstFile**

This command let you enter the emacs text editor and you can immediately begin adding text to the file you created.

I will add this to my text file: //Hello, from inside emacs!//

Now for the tricky part. To save this text file, you have to hold down the **control** key and then press **x** followed by **s**. If you did this correctly, on the bottom left of your screen, you should see this:

//Wrote /global/home/mganesh/myFirstDirectory/myFirstFile//

And now we want to exit emacs to go back to our command line. Again, hold down the **control** key but this time press **x** followed by **c**

Fantastic! You survived the dreaded unix text editor. You will be relieved to learn that unix has a great set of built-in utilities which make it so that you rarely have to use a text editor. This makes sense given that genome sequences, for example, are essentially text files with millions of characters. You definitely aren't going to be editing those by hand!

In unix, there are many different ways to add text to a file as well as look at the current contents of a file.

The command **cat**, for example, dumps the entire contents of a file to your screen. Lets dump the contents of the file we just made:

$**cat myFirstFile** //Hello, from inside emacs!//

The command **echo** repeats whatever we tell it to (make sure to use single quotation marks).

$ **echo 'Hello, World!'** //Hello, World!//

And we can use it to change the contents of our new file if we **redirect** the output with the **>** arrow.

$ **echo 'Hello, World!' > myFirstFile**

Let's see what text is now contained inside myFirstFile.

//Hello, World!//
 * $ cat myFirstFile**

This is a good time to bring up a few salient points:

1. If you redirect output from the command line into a filename that doesn't exist, that file will automatically be created. 2. If you redirect output from the command line into a filename that already exists, that file will be overwritten (as you saw above). 3. WARNING: Unix is very trusting in the sense that, in many cases, it does whatever you tell it to do without warning you when you are about to overwrite a currently existing file. This applies for the redirection arrow as well as the copy and move commands we will learn next.

"...to reproduce is human."
Often, we will merely want to copy or move existing files.

To copy, we use the command **cp**.

$ **cp myFirstFile mySecondFile**

$ **ls -lt** //-rw-r--r-- 1 mganesh cgrl 14 Feb 11 05:32 mySecondFile// //-rw-r--r-- 1 mganesh cgrl 14 Feb 11 05:31 myFirstFile//

Or, maybe we just want to move (i.e. rename) the file with the **mv** command.

$ **mv myFirstFile myRenamedFile**

But no worries if you change your mind, you can always move it back.

$ **mv myRenamedFile myFirstFile**

Peeking inside files
For the following examples, we're going to need a bit more text in our file. Since we're genomicists, we may as well use a FASTA file.

code >seq1 This is the description of my first sequence. AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT CGACGTAGATGCTAGCTGACTCGATGC >seq2 This is a description of my second sequence. CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC CATCGTCAGTTACTGCATGCTCG >seq3 and so on... GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG >seq4 CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC GTAGATGCTAGCTGACTCGAT code

Let's start by copying this text by highlighting. Then we'll echo the entire block to a new file. We have to add the first and last quotation marks, the **>** arrow, and the name of our new file we're echoing the text to,**test.FASTA**

$ **echo '>seq1 This is the description of my first sequence.**
 * AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA**
 * TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT**
 * CGACGTAGATGCTAGCTGACTCGATGC**
 * >seq2 This is a description of my second sequence.**
 * CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG**
 * GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC**
 * CATCGTCAGTTACTGCATGCTCG**
 * >seq3 and so on...**
 * GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT**
 * CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT**
 * AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG**
 * >seq4**
 * CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG**
 * GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC**
 * GTAGATGCTAGCTGACTCGAT**
 * ' > test.FASTA**

Now we can check to make sure the file looks we want it to with the cat command.

//>seq1 This is the description of my first sequence.// //AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA// //TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT// //CGACGTAGATGCTAGCTGACTCGATGC// //>seq2 This is a description of my second sequence.// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //CATCGTCAGTTACTGCATGCTCG// //>seq3 and so on...// //GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT// //CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT// //AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG// //>seq4// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //GTAGATGCTAGCTGACTCGAT//
 * $ cat test.FASTA**

We've seen how to use the **cat** command to dump the contents of a file to the screen, but there is a slightly more sophisticated utility, **less**.

//> seq1 This is the description of my first sequence.// //AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA// //TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT// //CGACGTAGATGCTAGCTGACTCGATGC// //> seq2 This is a description of my second sequence.// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //CATCGTCAGTTACTGCATGCTCG// //> seq3 and so on...// //GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT// //CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT// //AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG// //> seq4// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //GTAGATGCTAGCTGACTCGAT// //test.FASTA (END)//
 * $ less test.FASTA**

Here are some useful navigational tips for **less**:
 * You can use the arrow keys to move up or down a line in the text.
 * The spacebar will advance an entire page.
 * You can search for a word by typing a slash (e.g. **/)** followed by the search word.
 * To quit, type **q**.
 * To see the full help screen, type **h**.

Informative Interlude: UNIX command names tend to be overly clever
As you've seen with the basic commands thus far, the names are generally descriptive abbreviations of the program's function. For example, **mkdir** is for making a directory, **ls** is for listing the contents of a directory, etc. However, programmers, especially UNIX programmers, tend to get increasingly clever as things progress. Unaware of the fact that this practice makes things opaque, the typically programmer cries out for attention by making program names self-referentially clever. **less** is a good example of this. In the olden days, the most basic ways to view a text file could not divide files into individual pages, thus a multipage document would scroll off the screen before the first page could be read. As a solution, a program called **more** was written, which paused at the bottom of each page and prompted the user to press the spacebar for "more." The program name here is reasonably descriptive, but **more** had some noticeable feature deficiencies: you could neither advance the text one line at a time nor navigate backward in the document without reloading the whole file. The program written to accommodate these features is **less**. The cleverness of the name is revealed by the paradoxical adage " [|less is more] ."

By default, the **head** command prints the top 10 lines of the input file. To print a different number, say 5, lines:

$ **head test.FASTA** //> seq1 This is the description of my first sequence.// //AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA// //TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT// //CGACGTAGATGCTAGCTGACTCGATGC// //> seq2 This is a description of my second sequence.// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //CATCGTCAGTTACTGCATGCTCG// //> seq3 and so on...// //GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT//

$ **head -5 test.FASTA** //> seq1 This is the description of my first sequence.// //AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA// //TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT// //CGACGTAGATGCTAGCTGACTCGATGC// //> seq2 This is a description of my second sequence.//

And if you should want to see the end of the file, you can use the **tail** command.

$ **tail** **test.FASTA** //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //CATCGTCAGTTACTGCATGCTCG// //> seq3 and so on...// //GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT// //CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT// //AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG// //> seq4// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //GTAGATGCTAGCTGACTCGAT//

As I alluded to earlier, the **cat** command stands for **concatenate**, and though we were using it to view the contents of a single file, it was originally intended to do just what its name suggests: put multiple files together.

$ **cat** **mySecondFile test.FASTA** //Hello, World!// //> seq1 This is the description of my first sequence.// //AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA// //TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT// //CGACGTAGATGCTAGCTGACTCGATGC// //> seq2 This is a description of my second sequence.// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //CATCGTCAGTTACTGCATGCTCG// //> seq3 and so on...// //GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT// //CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT// //AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG// //> seq4// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //GTAGATGCTAGCTGACTCGAT//

More Utilities
At this point, you may be thinking to yourself, "Hrmmm.... This is all well and good, Mr. Computerpants, but it sure seems like a lot of trouble to go through just to read and write some sequences to a text file. I could have done this at home!" And if so, I say it's hard to blame you. But that's because we're just getting to the good part.

Maybe you were wondering, "Gosh, I wish I could search one of these sequence files for a particular sequence without having to open up a Word document or spreadsheet!" Never fear, **grep ** is here.


 * grep** searches for the a string of text in a file and prints out all lines where it finds the specified text.

$ **grep seq test.FASTA** //> seq1 This is the description of my first sequence.// //> seq2 This is a description of my second sequence.// //> seq3 and so on...// //> seq4//

If you wanted to see how many lines contain your query, you can give **grep** the **-c** argument.

$ **grep -c seq test.FASTA** //4//

The **-v** argument inverts the search (i.e. prints lines that //do not// contain your search string).

$ **grep -v seq test.FASTA** //AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA// //TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT// //CGACGTAGATGCTAGCTGACTCGATGC// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //CATCGTCAGTTACTGCATGCTCG// //GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT// //CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT// //AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //GTAGATGCTAGCTGACTCGAT//

Some of the basic functions you would use a complicated speadsheet for can be acheive with the utility **sort**, which true to its name, sorts the line in a file.

//> seq1 This is the description of my first sequence.// //> seq2 This is a description of my second sequence.// //> seq3 and so on...// //> seq4// //AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA// //AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG// //CATCGTCAGTTACTGCATGCTCG// //CGACGTAGATGCTAGCTGACTCGATGC// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT// //GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //GTAGATGCTAGCTGACTCGAT// //TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT//
 * $ sort test.FASTA**

The next utilities we will cover only with a description, but keep them in mind, because it will help you in the exercises later this morning.

Imagine that you have a spreadsheet worth of data, and that you only want to consider the third column of the spreadsheet. Instead of opening Excel and copying and pasting your column to a new file, you could use the **cut** utility.

$ **cut -f 3 filename**

This command will return only the third column from a tab-delimited file.

Wildcard Matching with the asterisk ( * )
You may want to use a utility on multiple files, which is possible using the * wildcard character. The simplest example of this is using **ls** to list multiple files.

$ **ls** //myFirstFile mySecondFile test.FASTA//

$ **ls *File** //myFirstFile mySecondFile//

$ **ls myF*File** //myFirstFile//

In these examples, we can see that the asterisk will match any set of intervening characters.

Controlling Information Flow with the Pipe
UNIX has several features that have no good analogous function in other operating systems, and one such commonly used feature is called the Unix **pipe**. Often, you will want to use several utilities serially in one process. The **pipe ** allows you to send the output of one program as the input to the next program automatically.

The **pipe** character is the **|** located above the backslash key.

If we wanted to search the a list of files, we could send the output of **ls** to **grep** with a **pipe**.

$ **ls -lt | grep my** //-rw-r--r-- 1 mganesh cgrl 14 Feb 11 05:32 mySecondFile// //-rw-r--r-- 1 mganesh cgrl 14 Feb 11 05:31 myFirstFile//

Here, we see the two files in our directory listing that contain the search term "my" returned by **grep.**

**Redirection**
Another built-in UNIX tool is called **redirection**, and we've already seen it once today (I just didn't bother to explain it earlier!).

When we copied and pasted our sequences to **test.FASTA**, we used **redirection** with the **>** arrow.

$ **cat myFirstFile test.FASTA mySecondFile > myCombinedFile**

$ **cat myCombinedFile** //Hello, World!// //> seq1 This is the description of my first sequence.// //AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA// //TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT// //CGACGTAGATGCTAGCTGACTCGATGC// //> seq2 This is a description of my second sequence.// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //CATCGTCAGTTACTGCATGCTCG// //> seq3 and so on...// //GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT// //CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT// //AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG// //> seq4// //CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG// //GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC// //GTAGATGCTAGCTGACTCGAT// //Hello, World!//

We can see from this example that the **redirection** arrow tells UNIX to send the output of the command to a file, instead of the screen.

Downloading Files
Instead of using your web browser to download files from a data resource like NCBI, in UNIX, we have efficient command line utilities for retrieving files. There are many tools for this purpose, but we'll use one common one, **wget** to retrieve files containing the sequences of the yeast genome.

First, let's make a new directory, called **fasta_files.**

$ **mkdir fasta_files**

And then we'll change to the new directory.

$ **cd fasta_files**

Now we can use the **wget** command to retrieve the files from the UC Santa Cruz sequence archive.

$ **wget** [|ftp://hgdownload.soe.ucsc.edu/goldenPath/sacCer3/chromosomes/*] //--2013-02-11 05:50:07-- ftp://hgdownload.soe.ucsc.edu/goldenPath/sacCer3/chromosomes/*// //=> `.listing'// //Resolving hgdownload.soe.ucsc.edu... 128.114.119.163// //Connecting to hgdownload.soe.ucsc.edu|128.114.119.163|:21... connected.// //Logging in as anonymous ... Logged in!// //==> SYST ... done. ==> PWD ... done.// //==> TYPE I ... done. ==> CWD /goldenPath/sacCer3/chromosomes ... done.// //==> PASV ... done. ==> LIST ... done.// //[ <=> ] 1,412 --.-K/s in 0.001s// //2013-02-11 05:50:07 (998 KB/s) - `.listing' saved [1412]// //Removed `.listing'.//

//--2013-02-11 05:50:07-- ftp://hgdownload.soe.ucsc.edu/goldenPath/sacCer3/chromosomes/README.txt// //=> `README.txt'//

//==> CWD not required.// //==> PASV ... done. ==> RETR README.txt ... done.// //Length: 2135 (2.1K)// //100%[======================================>] 2,135 --.-K/s in 0s// //...//

Now, when we look at what's happened to our directory, we'll see a list of files:

$ **ls** //README.txt chrIV.fa.gz chrVI.fa.gz chrXI.fa.gz chrXV.fa.gz// //chrI.fa.gz chrIX.fa.gz chrVII.fa.gz chrXII.fa.gz chrXVI.fa.gz// //chrII.fa.gz chrM.fa.gz chrVIII.fa.gz chrXIII.fa.gz md5sum.txt// //chrIII.fa.gz chrV.fa.gz chrX.fa.gz chrXIV.fa.gz//

All but two of these files contain the sequence from a chromosome. The **README.txt** file contains information about the files, and the **md5sum.txt** contains information that can be used to verify the integrity of the files (but we won't be concerned with this).

You'll notice that all of the other files end with a double file extension, **.fa.gz**. File extensions tell you what to expect to be in the file, and in this case they're telling you that the file contains fasta sequence (the .fa part), and that the file has been compressed with the **gzip** utility, which saves disk space. **gzip** is only of several utilities available for compression. Others you may encounter include **tar** and **bzip**, though there are more.

In order to use the files, however, we need to uncompress them with the utility **gunzip**. We'll do this once here, and the again in this morning's exercises.

$ **gunzip chrM.fa.gz**

$ **ls** //README.txt chrIV.fa.gz chrVI.fa.gz chrXI.fa.gz chrXV.fa.gz// //chrI.fa.gz chrIX.fa.gz chrVII.fa.gz chrXII.fa.gz chrXVI.fa.gz// //chrII.fa.gz chrM.fa chrVIII.fa.gz chrXIII.fa.gz md5sum.txt// //chrIII.fa.gz chrV.fa.gz chrX.fa.gz chrXIV.fa.gz//

Notice that the only change is the name of the file we uncompressed, **chrM.fa.gz** has become **chrM.fa**

And if we take a peak at the beginning of the file with **head**, we can see that indeed looks like a fasta file:

$ **head chrM.fa** //>chrM// //TTCATAATTAATTTTTTATATATATATTATATTATAATATTAATTTATAT// //TATAAAAATAATATTTATTATTAAAATATTTATTCTCCTTTCGGGGTTCC// //GGCTCCCGTGGCCGGGCCCCGGAATTATTAATTAATAATAAATTATTATT// //AATAATTATTTATTATTTTATCATTAAAATATATAAATAAAAAATATTAA// //AAAGATAAAAAAAATAATGTTTATTCTTTATATAAATTATATATATATAT// //ATAATTAATTAATTAATTAATTAATTAATAATAAAAATATAATTATAAAT// //AATATAAATATTATTCTTTATTAATAAATATATATTTATATATTATAAAA// //GTATCTTAATTAATAAAAATAAACATTTAATAATATGAATTATATATTAT// //TATTATTATTAATAAAATTATTAATAATAATCAATATGAAATTAATAAAA//

The UNIX Path and Environment Variables
There are a lot of things going on behind the scenes of your UNIX shell. For the most part, Mac OS and modern Linux distributions will shield you from these mechanics, but we want to make you aware of two important concepts: the **UNIX Path** and your **environment variables**.

If we issue the **env** command, we will get something similar to the following output.

$ **env** HOSTNAME=poset.cgrl.berkeley.edu TERM=xterm-color SHELL=/bin/bash HISTSIZE=1000 SSH_CLIENT=169.229.195.27 55475 22 SSH_TTY=/dev/pts/0 USER=cellison LS_COLORS=no=00:fi=00:di=01;34:ln=01;36:pi=40;33:so=01;35:bd=40;33;01:cd=40;33;01:or=01;05;37;41:mi=01;05;37;41:ex=01;32:*.cmd=01;32:*.exe=01;32:*.com=01;32:*.btm=01;32:*.bat=01;32:*.sh=01;32:*.csh=01;32:*.tar=01;31:*.tgz=01;31:*.arj=01;31:*.taz=01;31:*.lzh=01;31:*.zip=01;31:*.z=01;31:*.Z=01;31:*.gz=01;31:*.bz2=01;31:*.bz=01;31:*.tz=01;31:*.rpm=01;31:*.cpio=01;31:*.jpg=01;35:*.gif=01;35:*.bmp=01;35:*.xbm=01;35:*.xpm=01;35:*.png=01;35:*.tif=01;35: MAIL=/var/spool/mail/cellison PATH=/usr/kerberos/bin:/usr/local/bin:/bin:/usr/bin:/opt/dell/srvadmin/bin:/global/home/mganesh/bin INPUTRC=/etc/inputrc PWD=/global/home/cellison/myFirstDirectory/fasta_files LANG=C MODULEPATH=/usr/Modules/modulefiles:/usr/cports/modulefiles/redhat-5.x86_64:/etc/modulefiles:/global/software/centos-5.x86_64/modules/modfiles LOADEDMODULES= SHLVL=1 HOME=/global/home/mganesh LOGNAME=mganesh SSH_CONNECTION=169.229.195.27 55475 169.229.192.160 22 MODULESHOME=/usr/Modules LESSOPEN=|/usr/bin/lesspipe.sh %s G_BROKEN_FILENAMES=1 module= { eval `/usr/Modules/bin/modulecmd bash $*` } _=/bin/env OLDPWD=/global/home/mganesh/myFirstDirectory

What we see here are the values assigned to many **environment variables**. These are variables defined in your **shell** to make it easier for UNIX to do what you ask. For example, you can see that the variable **USER** is defined as **mganesh**. UNIX knows what my name is :-) And that is actually required for me to do anything useful as a user. Similarly, you can see **HOME** is defined as **/global/home/mganesh**. This is what tells UNIX where my home directory is located. If we change this variable to another directory, and then I issue the **cd** command to take me home, it will take me to the newly specified directory instead of my original home directory.

Sometimes this list can be very long and difficult to read, but if we know what we're looking for, we can use other utilities to help us.

$ **env | grep home** //HOME=/global/home/mganesh// //MODULESHOME=/usr/Modules//

This afternoon we will modify an important **environment variable** called **PATH**. The **PATH** variable tells UNIX a list of directories that it should put on your favorites list. That is, a list of directories that you don't have to specify the whole name of in order to use the files within them. For example, when we issue the command **ls**, a the program that runs is actually located in a directory called **/bin/**, so the program that we're actually running is **/bin/ls**. However, since **/bin/** is in our **PATH** by default, UNIX knows that when we type **ls**, we really meant **/bin/ls**.

In fact, if we use the **env** command, we can see the list of directories that are in our **PATH**, and they are separated by colons.

$ **env | grep PATH** //PATH=/usr/kerberos/bin:/usr/local/bin:/bin:/usr/bin:/opt/dell/srvadmin/bin:/global/home/mganesh/bin// //MODULEPATH=/usr/Modules/modulefiles:/usr/cports/modulefiles/redhat-5.x86_64:/etc/modulefiles:/global/software/centos-5.x86_64/modules/modfiles//

Here we see 6 directories in my **PATH**, separated by colons in the list.

Help, I'm stuck!
Most commands have many useful flags beyond what we've shown you. For information on a particular command, look at the manual pages with **man**.

$ **man ls**

code LS(1)                           User Commands                           LS(1)

NAME ls - list directory contents

SYNOPSIS ls [OPTION]... [FILE]...

DESCRIPTION List information  about the FILEs (the current directory by default). Sort entries alpha- betically if none of -cftuvSUX nor --sort.

Mandatory arguments to long options are mandatory for short options too.

-a, --all do not ignore entries starting with.

-A, --almost-all do not list implied. and ..

--author with -l, print the author of each file

-b, --escape print octal escapes for nongraphic characters

--block-size=SIZE use SIZE-byte blocks

-B, --ignore-backups do not list implied entries ending with ~

-c    with -lt: sort by, and show, ctime (time of last modification of file status  infor-              mation) with -l: show ctime and sort by name otherwise: sort by ctime

-C    list entries by columns

--color[=WHEN] control whether  color  is  used  to  distinguish file types. WHEN may be 'never', 'always', or 'auto'

-d, --directory list directory entries instead of contents, and do not dereference symbolic links

-D, --dired generate output designed for Emacs' dired mode

-f    do not sort, enable -aU, disable -lst

-F, --classify append indicator (one of */=>@|) to entries ... code

The **man** reader is essentially **less**, so you already know how to move up and down, and page by page!

**Exercises**

 * 1) Cerevisiae chromosomes**

a) Change into your **fasta_files** directory, and unzip the remainder of the fasta files using only a one-line command.

b) Combine the sequence files to make a single whole genome file called "cerevisiae_genome.fasta"

d) Count the chromosomes in the whole genome file using commands from the lecture. (HINT: Each of the original FASTA files contains a single chromosome).

e) Look up the command **wc** and find out what it does. Get size of total genome. (HINT: The size of the genome can be determined by counting the number of bases).


 * 2) Cerevisiae genomic features**

a) Get the list of cerevisiae chromosome features from this address:

[]

Columns within SGD_features.tab:

1. Primary Stanford Gene Database ID (SGDID) (mandatory) 2. Feature type (mandatory) 3. Feature qualifier (optional) 4. Feature name (optional) 5. Standard gene name (optional) 6. Alias (optional, multiples separated by |) 7. Parent feature name (optional) 8. Secondary SGDID (optional, multiples separated by |) 9. Chromosome (optional) 10. Start_coordinate (optional) 11. Stop_coordinate (optional) 12. Strand (optional) 13. Genetic position (optional) 14. Coordinate version (optional) 15. Sequence version (optional) 16. Description (optional)

b) Next, count the total number of ORFs in the features file.

c) Now count the ORFs listed as Verified, and separately count those listed as Dubious. If you do the math and add up these two groups, why don't they make up all the ORF matches from **grep**?

d) Use the **cut** utility with **grep** to count the number of ORFs more accurately.

e) Use a combination of the the commands **cut, sort,** and **uniq** generate a list of all the genomic features (column 2) in this file. Don't forget that you can use the **man**pages to learn how to use new tools.

Is there a way to tell grep to ONLY print the part of the line that matches the pattern you give it?
 * 3) In depth with grep**

What does **egrep** allow you to do?

Use your new found knowledge of grep from the previous two questions, combined with some of the other unix utilities you learned about earlier, to produce a table that lists every length of GA microsatellite repeat in the yeast genome, along with the number of occurrences of each. You can do this with a single command, piping output between the different unix utilities.

Example: 5000 GA 3000 GAGA 1000 GAGAGA 100 GAGAGAGA 10 GAGAGAGAGA ... ...

HINT #1: You will need to use an extra argument with the uniq command, one that we haven't covered yet. HINT #2: Regular expressions provide the flexibility to match patterns rather than a specific string. You can use //special characters// to construct a regular expression, for example, the plus sign means //match one or more times//, so the pattern **'H+' ** will match the ** H ** in ** H **AT but also the ** HHHHHHHH ** in A** HHHHHHHH **!!!!!!! The period means//match any character// so, from the previous example, **'H.+' ** would match all of ** HAT ** as well as ** HHHHHHHH!!!!!!! ** . You can also group characters together with parentheses. For example, **'(I..)+' ** would match **ISSISSIPP ** in **MISSISSIPPI. **Remember to enclose your pattern in 'tick marks' just like with echo. CAVEAT: Assume that it is safe to ignore the fact that a repeat could possibly span multiple lines in your FASTA file

4) Save your table as a new file. Open the file in emacs and add column headers and/or anything else you can think of to make it pretty. Save the file and exit.

Solutions

Part 2 if we have time. You can try this own your own as well