Introduction+to+Unix+for+NGS+Fall 2014

= Genomic Analysis at the UNIX Command Line =

Your instructor for today is Ravi Alla.

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.

@ravi ~$ **ssh ralla@poset.cgrl.berkeley.edu** Password: Last login: Tue Mar 18 18:10:47 2014 from 128.32.218.204

Navigation in the terminal
ralla@poset ~]$ **whoami** ralla

[ralla@poset ~]$ **id** uid=694(ralla) gid=500(cgrl) groups=500(cgrl)

[ralla@poset ~]$ **pwd** /global/home/ralla

[ralla@poset ~]$ ** cd **

[ralla@poset ~]$ **pwd** /global/home/ralla

[ralla@poset ~]$ **cd ..**

[ralla@poset home]$ ** pwd ** /global/home

[ralla@poset home]$ **cd ..**

[ralla@poset global]$ **pwd** /global

[ralla@poset global]$ **cd ..**

[ralla@poset /]$ **pwd** /

[ralla@poset /]$ **cd**

[ralla@poset ~]$ **pwd** /global/home/ralla

[ralla@poset ~]$ **cd /global/**

[ralla@poset global]$ ** pwd ** /global

[ralla@poset global]$ **cd ~**

[ralla@poset ~]$ **pwd** /global/home/ralla

Paths can be absolute as in /global/ or relative as in ../ or ./

Looking at files and directories
[ralla@poset ~]$ **ls** fasta_files galaxy-dist galaxy-python

[ralla@poset ~]$ **ls /global/** courses galaxy home scratch software

[ralla@poset ~]$ **ls ..** acarver cellison dportik galaxy jefdaj jrefsnid jzhaolfalcon mjohnson polina rharris sbranco smckay tpeixoto aihardin ckotwali drisso ganeshm jguevara jswenson kclemenslpu msyed postgres rsung scolmena ssinghal ylee alam cmarshal earmstrong gsluser jheller jtaylor kgoodmanmberg mvandam qshi rumachik shepp test1 zhouqi astrom dcotoras efarrer gwang jkarijol jvillaro kmackmchung pbrowne ralla rwelch shykin test2 ccattogl dkhendrix epurdom jchoi jmchoi jxu kmurikimganesh phsieh rghosh sbouzid smajumda test3

[ralla@poset ~]$ **ls -l** total 8 drwxr-xr-x 2 ralla cgrl 4096 Mar 11 14:45 fasta_files drwxr-xr-x 21 ralla cgrl 4096 Feb 26 10:13 galaxy-dist drwxr-xr-x 2 ralla cgrl 19 Jan 16 12:05 galaxy-python

[ralla@poset ~]$ **ls -al** total 88 drwx-- 11 ralla cgrl 4096 Mar 11 14:04. drwxr-xr-x 70 root root 4096 Feb 19 18:28 .. -rw-r--r-- 1 ralla cgrl 8891 Feb 19 15:58 .RData -rw--- 1 ralla cgrl 154 Feb 19 15:58 .Rhistory -rw--- 1 ralla cgrl 21333 Mar 18 18:24 .bash_history -rw-r--r-- 1 ralla cgrl 33 Jan 7 09:57 .bash_logout -rw-r--r-- 1 ralla cgrl 203 Jan 28 12:52 .bash_profile -rw-r--r-- 1 ralla cgrl 124 Jan 7 09:57 .bashrc -rw-r--r-- 1 ralla cgrl 515 Jan 7 09:57 .emacs drwx-- 2 ralla cgrl 6 Feb 19 16:22 .gconf drwx-- 2 ralla cgrl 24 Feb 19 16:22 .gconfd drwx-- 3 ralla cgrl 16 Feb 19 17:12 .local -rw--- 1 ralla cgrl 6 Jan 29 15:06 .psql_history drwxr-xr-x 7 ralla cgrl 4096 Jan 7 12:25 .python-eggs drwx-- 2 ralla cgrl 24 Jan 28 10:35 .ssh drwxr-xr-x 3 root root 61 Feb 19 17:51 .subversion -rw--- 1 ralla cgrl 7263 Feb 19 17:38 .viminfo drwxr-xr-x 2 ralla cgrl 4096 Mar 11 14:45 fasta_files drwxr-xr-x 21 ralla cgrl 4096 Feb 26 10:13 galaxy-dist drwxr-xr-x 2 ralla cgrl 19 Jan 16 12:05 galaxy-python

[ralla@poset ~]$ **ls -lt** total 8 drwxr-xr-x 2 ralla cgrl 4096 Mar 11 14:45 fasta_files drwxr-xr-x 21 ralla cgrl 4096 Feb 26 10:13 galaxy-dist drwxr-xr-x 2 ralla cgrl 19 Jan 16 12:05 galaxy-python

[ralla@poset ~]$ **ls -aF** ./ .RData.bash_history .bash_profile .emacs .gconfd/ .psql_history.ssh/ .viminfo galaxy-dist/ ../ .Rhistory.bash_logout .bashrc .gconf/ .local/ .python-eggs/.subversion/ fasta_files/ galaxy-python/

[ralla@poset ~]$ **ls -R fasta_files/** fasta_files/:

GA_repeats.txtSGD_features.tab cerevisiae_genome.fasta fasta_dir md5sum.txt

fasta_files/fasta_dir:

chrI.fa chrIII.fa chrIX.fa chrV.fa chrVII.fa chrX.fa chrXII.fa chrXIV.fa chrXVI.fa chrII.fa chrIV.fa chrM.fa chrVI.fa chrVIII.fa chrXI.fa chrXIII.fa chrXV.fa

You can use tab key to autocomplete after you type the first few letters of a file or a directory or even a command. Hitting tab twice will list all the possibilities. Beware this might fill up your screen very fast if there are a lot of possibilities.

You can also use the up and down arrow keys to cycled through your previously typed commands. This saves you a lot of re-typing and reduces errors.

Another useful command is the **history** command. This prints to screen all your previous commands and their line numbers.

[ralla@poset ~]$ **history** .... ....  1037 ls  1038 cd .. 1039 clear 1040 ls -R fasta_files/ 1041 ls -Sr 1042 ls -S 1043 ls -s 1044 ls .. 1045 pwd 1046 ls /global/ 1047 ls -l /global/ 1048 clear 1049 history

Including the last command which was **history** itself.

You can re run any command in the history by using the command's line number in history

[ralla@poset ~]$ **!1046** ls /global/ courses galaxy home scratch software


 * clear** is a command that clears your working space so you can have a clean looking terminal

**Creating files and directories**

[ralla@poset ~]$ **mkdir unix_playground**

[ralla@poset ~]$ **ls -l** total 4 drwxr-xr-x 3 ralla cgrl 113 Mar 18 18:35 fasta_files drwxr-xr-x 21 ralla cgrl 4096 Feb 26 10:13 galaxy-dist drwxr-xr-x 2 ralla cgrl 19 Jan 16 12:05 galaxy-python drwxr-xr-x 2 ralla cgrl 6 Mar 18 18:54 unix_playground

Notice the date and time the new directory was created.

[ralla@poset ~]$ **cd unix_playground/**

[ralla@poset unix_playground]$ **touch my_first_file**

[ralla@poset unix_playground]$ **ls -l** total 0 -rw-r--r-- 1 ralla cgrl 0 Mar 18 18:55 my_first_file

[ralla@poset unix_playground]$ **touch My_First_file**

[ralla@poset unix_playground]$ **ls -l** total 0 -rw-r--r-- 1 ralla cgrl 0 Mar 18 18:55 My_First_file -rw-r--r-- 1 ralla cgrl 0 Mar 18 18:55 my_first_file

[ralla@poset unix_playground]$ **touch my_first_file**

[ralla@poset unix_playground]$ **ls -l** total 0 -rw-r--r-- 1 ralla cgrl 0 Mar 18 18:55 My_First_file -rw-r--r-- 1 ralla cgrl 0 Mar 18 18:56 my_first_file

Notice how touch recreated a new file with an updated time stamp. This is the behavior if file already exists, so be careful not to overwrite an existing file. A few important things to learn from this, file and directory names are case sensitive in linux. Always name your files and directories something short and informative and use extensions (such as .fasta or .fastq to let you know what kind of file it is). Avoid using spaces in your file and directory names. We will see later why.

Modifying files and directories
[ralla@poset unix_playground]$ **rm My_First_file**

[ralla@poset unix_playground]$ **ls -l**

total 0

-rw-r--r-- 1 ralla cgrl 0 Mar 18 18:56 my_first_file

[ralla@poset unix_playground]$ **mkdir temp1 temp2**

[ralla@poset unix_playground]$ **ls -l** total 0 -rw-r--r-- 1 ralla cgrl 0 Mar 18 18:56 my_first_file drwxr-xr-x 2 ralla cgrl 6 Mar 18 19:05 temp1 drwxr-xr-x 2 ralla cgrl 6 Mar 18 19:05 temp2

[ralla@poset unix_playground]$ **rmdir temp1/**

[ralla@poset unix_playground]$ **ls -l** total 0 -rw-r--r-- 1 ralla cgrl 0 Mar 18 18:56 my_first_file drwxr-xr-x 2 ralla cgrl 6 Mar 18 19:05 temp2

[ralla@poset unix_playground]$ **cd temp2**

[ralla@poset temp2]$ **touch test.txt**

[ralla@poset temp2]$ **cd ..**

[ralla@poset unix_playground]$ **rmdir temp2/** rmdir: temp2/: Directory not empty

[ralla@poset unix_playground]$ **rm -r temp2/**

[ralla@poset unix_playground]$ **ls -l** total 0 -rw-r--r-- 1 ralla cgrl 0 Mar 18 18:56 my_first_file

[ralla@poset unix_playground]$ **mv my_first_file my_first_move.txt**

[ralla@poset unix_playground]$ **ls -l** total 0 -rw-r--r-- 1 ralla cgrl 0 Mar 18 18:56 my_first_move.txt

[ralla@poset unix_playground]$ **mkdir move_dir**

[ralla@poset unix_playground]$ **mv my_first_move.txt move_dir/**

[ralla@poset unix_playground]$ **ls move_dir/** my_first_move.txt

[ralla@poset unix_playground]$ **cp move_dir/my_first_move.txt my_first_copy.txt**

[ralla@poset unix_playground]$ **ls -l** total 0 drwxr-xr-x 2 ralla cgrl 30 Mar 18 19:08 move_dir -rw-r--r-- 1 ralla cgrl 0 Mar 18 19:09 my_first_copy.txt

Be careful with **mv** and **cp** they will overwrite existing copies of files without warning. It is a good idea to use **rm,** **mv** and **cp** with **-i** option to alert before making changes.

Adding text to a file, the dreaded terminal text editor.
[ralla@poset unix_playground]$ **nano my_first_fasta.fasta**

Copy the following and paste into the txt file and then ctrl+o to write and ctrl+x to exit

>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

Peeking inside text based files
[ralla@poset unix_playground]$ **cat my_first_fasta.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 CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG >seq4 CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC GTAGATGCTAGCTGACTCGAT

[ralla@poset unix_playground]$ ** less my_first_fasta.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 CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG >seq4 CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC GTAGATGCTAGCTGACTCGAT my_first_fasta.fasta (END)

Press **q** to exit out of this

[ralla@poset unix_playground]$ **head my_first_fasta.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

[ralla@poset unix_playground]$ **tail my_first_fasta.fasta** GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC CATCGTCAGTTACTGCATGCTCG >seq3 and so on... GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG >seq4 CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC GTAGATGCTAGCTGACTCGAT

[ralla@poset unix_playground]$ **head -3 my_first_fasta.fasta** >seq1 This is the description of my first sequence. AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT

[ralla@poset unix_playground]$ **tail -3 my_first_fasta.fasta** CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC GTAGATGCTAGCTGACTCGAT

If you need help about any command or its options use **man .** This is very useful when you learning command line. We will be using this in the coming sections.

A few more handy utilities
[ralla@poset unix_playground]$ **nano second_file.txt**

[ralla@poset unix_playground]$ **cat second_file.txt my_first_fasta.fasta** This is my second file! I am getting good at this. >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

[ralla@poset unix_playground]$ **wc my_first_fasta.fasta** 16 35 598 my_first_fasta.fasta

[ralla@poset unix_playground]$ **wc -l my_first_fasta.fasta** 16 my_first_fasta.fasta

[ralla@poset unix_playground]$ **wc -c my_first_fasta.fasta** 598 my_first_fasta.fasta

Download this Right click and copy link and go to unix_playground and use **wget** command

[ralla@poset unix_playground]$ **wget http://cgrlucb.wikispaces.com/file/view/gene_exp.diff** --2014-03-18 19:41:41-- http://cgrlucb.wikispaces.com/file/view/gene_exp.diff Resolving cgrlucb.wikispaces.com... 208.43.192.33, 75.126.104.177 Connecting to cgrlucb.wikispaces.com|208.43.192.33|:80... connected. HTTP request sent, awaiting response... 200 OK Length: 4173755 (4.0M) [text/plain] Saving to: `gene_exp.diff'

100%[=========================================================================================================================>] 4,173,755 4.11M/s in 1.0s

2014-03-18 19:41:42 (4.11 MB/s) - `gene_exp.diff' saved [4173755/4173755]

[ralla@poset unix_playground]$ **ls** gene_exp.diff my_first_fasta.fasta second_file.txt

Cut can be used to extract columns from a delimited file.

[ralla@poset unix_playground]$ **cut -f 3 gene_exp.diff** **...** **...**  TTTY14 CD24 BCORP1 KDM5D TTTY10 RBMY2EP TTTY13 RBMY1B RBMY1B PRY RBMY1J TTTY5 TTTY6,TTTY6B DAZ1,DAZ4 TTTY3B CDY1 CSPG4P1Y GOLGA2P2Y DAZ3,DAZ4 BPY2C TTTY4B TTTY17B

How would you extract columns if the file was a csv??

Sort can be used to sort based on any given column

[ralla@poset unix_playground]$ **sort -k3 gene_exp.diff** LOC_001608XLOC_001608ZYG11Bchr1:53192130-53293013MockOGTOK05790.27infnan0.136850.42102no XLOC_000409XLOC_000409ZYG11Bchr1:53192130-53293013MockOGTOK11.941513.21850.1465790.1444550.82990.998471no XLOC_031804XLOC_031804ZYXchr7:143078359-143220540MockOGTOK5.088096.067640.254010.1167770.716950.996395no XLOC_014317XLOC_014317ZZEF1chr17:3907738-4046253MockOGTOK2.743513.341760.2845830.1992790.658150.977286no XLOC_001676XLOC_001676ZZZ3chr1:78030189-78148343MockOGTOK42.826824.3581-0.814114-0.6450210.29120.682889no test_idgene_idgenelocussample_1sample_2statusvalue_1value_2log2(fold_change)test_statp_valueq_valuesignificant

How would you sort this in a reverse order? Use **man sort**

Sort the gene_exp.diff file by column 4 with -n option and without -n option, what is the difference?

What does the **uniq** command do?

Redirection and piping
Let us get the first 100 lines of gene_exp.diff and save it to a new file. How do we do this?

[ralla@poset unix_playground]$ **head -100 gene_exp.diff > gene_exp_first_100_lines.diff**

[ralla@poset unix_playground]$ **wc -l gene_exp.diff gene_exp_first_100_lines.diff**

37605 gene_exp.diff

100 gene_exp_first_100_lines.diff

37705 total

This **>** sign is used to redirect the output of **head** into a new file called gene_exp_first_100_lines.diff This will overwrite whatever is in the recipient file name. To append use >> sign

[ralla@poset unix_playground]$ **echo "I am adding a new line" > gene_exp_first_100_lines.diff**

[ralla@poset unix_playground]$ **head gene_exp_first_100_lines.diff** I am adding a new line

[ralla@poset unix_playground]$ ** head -100 gene_exp.diff > gene_exp_first_100_lines.diff **

[ralla@poset unix_playground]$ ** echo "I am adding a new line" >> gene_exp_first_100_lines.diff **

[ralla@poset unix_playground]$ **tail -5 gene_exp_first_100_lines.diff** XLOC_000096XLOC_000096PLOD1chr1:11994723-12035599MockOGTOK4.9559657.11413.526610.8052650.082050.288638no XLOC_000097XLOC_000097MFN2chr1:12040237-12073572MockOGTOK22.837323.35650.03243250.01837010.97060.998471no XLOC_000098XLOC_000098MIIPchr1:12079298-12092106MockOGTOK10.270610.80530.07321580.06297720.8970.998471no XLOC_000099XLOC_000099TNFRSF8chr1:12123433-12204264MockOGTOK0.1032310.4866612.237040.6853430.02570.224628no I am adding a new line

Let's say we want to get the gene name column (column 3) and then sort the output. How would we do this??

This is where we use pipes, pipes take the output of one command and use it as input for the command after the pipe. Useful for chaining commands which have one output and accept one input

[ralla@poset unix_playground]$ **cut -f 3 gene_exp_first_100_lines.diff | sort | head -5** - -  -  ACTRT2 AGRN

How would you use pipes to give me a list of only unique gene names in this file?

Wildcards
A lot of commands can take multiple inputs, like **touch, mkdir, rm, cp, mv, wc** etc So if we are able to pass multiple files (or dir) to these commands, they will work on all of them. We could do this file by file or we could use an expression to match these multiple files. This is where wildcards come in. The following are a few wildcards we will talk about

. is a wildcard that can match zero or more characters of any kind ? is a wildcard that can match 1 occurrence of any character [ABC] can match for any of the characters occurring inside them, A or B or C ! negates match expressions [!ABC], any character except A or B or C {A,B,C} expands all the elements within the braces {1..9} expands a range of elements within the braces

[ralla@poset unix_playground]$ **mkdir wildcards**

[ralla@poset unix_playground]$ **cd wildcards/**

[ralla@poset wildcards]$ **touch file{1..6}.txt**

[ralla@poset wildcards]$ **ls** file1.txt file2.txt file3.txt file4.txt file5.txt file6.txt

[ralla@poset wildcards]$ **touch file_{a..e}.log**

[ralla@poset wildcards]$ **ls**

file1.txt file2.txt file3.txt file4.txt file5.txt file6.txt file_a.log file_b.log file_c.log file_d.log file_e.log

[ralla@poset wildcards]$ **ls *.txt**

file1.txt file2.txt file3.txt file4.txt file5.txt file6.txt

[ralla@poset wildcards]$ **ls file***

file1.txt file2.txt file3.txt file4.txt file5.txt file6.txt file_a.log file_b.log file_c.log file_d.log file_e.log

[ralla@poset wildcards]$ **ls *[23].txt**

????

[ralla@poset wildcards]$ **ls ?[46].txt**

[ralla@poset wildcards]$ **ls file?.***

????

[ralla@poset wildcards]$ **ls file{4,_?}*** ???

[ralla@poset wildcards]$ **rm *[!46].txt** [ralla@poset wildcards]$ **ls**

???

[ralla@poset wildcards]$ **touch [!46].txt** [ralla@poset wildcards]$ **ls** [!46].txt file4.txt file6.txt file_a.log file_b.log file_c.log file_d.log file_e.log

How do you remove this file [!46].txt? Hint: You would have to escape the [ and ! and ] to make them not special.

Remove all the files in this directory. And then navigate out of the directory and remove the directory named wildcards

Searching for files and their contents
Look up grep and find.

Find is useful for finding file names that match certain criteria

[ralla@poset ~]$ **find unix_playground/ -type f** unix_playground/my_first_fasta.fasta unix_playground/second_file.txt unix_playground/gene_exp.diff unix_playground/gene_exp_first_100_lines.diff

[ralla@poset ~]$ **find unix_playground/ -type d** unix_playground/

[ralla@poset ~]$ **find unix_playground/ -name "*.txt"** unix_playground/second_file.txt

[ralla@poset ~]$ **find unix_playground/ -name "*.fasta"** unix_playground/my_first_fasta.fasta

[ralla@poset ~]$ find unix_playground/ -name "*.fasta" -type d ???

[ralla@poset ~]$ **wc -l $(find unix_playground/ -name "*" -type f)** 16 unix_playground/my_first_fasta.fasta 1 unix_playground/second_file.txt 37605 unix_playground/gene_exp.diff 101 unix_playground/gene_exp_first_100_lines.diff 37723 total

Here we are using the output of find by placing its output in parenthesis with a $ and passing it to wc. This is another way of passing arguments to commands.

[ralla@poset unix_playground]$ **grep '>' my_first_fasta.fasta** >seq1 This is the description of my first sequence. >seq2 This is a description of my second sequence. >seq3 and so on... >seq4

Do not forget the ' ' around the > or you will overwrite your fasta file

[ralla@poset unix_playground]$ **grep -n '>' my_first_fasta.fasta** 1:>seq1 This is the description of my first sequence. 5:>seq2 This is a description of my second sequence. 9:>seq3 and so on... 13:>seq4

[ralla@poset unix_playground]$ **grep -no '>' my_first_fasta.fasta** 1:> 5:>  9:>  13:>

[ralla@poset unix_playground]$ **grep -nv '>' my_first_fasta.fasta** 2:AGTACGTAGTAGCTGCTGCTACGTGCGCTAGCTAGTACGTCAA 3:TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT 4:CGACGTAGATGCTAGCTGACTCGATGC 6:CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG 7:GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC 8:CATCGTCAGTTACTGCATGCTCG 10:GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT 11:CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT 12:AGTACGTAGTAGCTGTAGCTGATCGTACGTAGCTGATCGTACG 14:CGATCGATCGTACGTCGACTGATCGTAGCTACGTCGTACGTAG 15:GTACGTGATCGTACGTCGACTGATCGTAGCTATCGTAGCTACC 16:GTAGATGCTAGCTGACTCGAT

[ralla@poset unix_playground]$ **grep -n TMEM201 gene_exp.diff** 77:XLOC_000076XLOC_000076TMEM201chr1:9648931-9674935MockOGTOK1.615331.53688-0.0718273-0.05297790.900450.998471no

How would you get the header file in the gene_exp.diff using grep?

You can use **egrep** which is a more powerful form of grep and you can specify wildcards and regular expression to search for patterns. Regular expressions are a whole other beast and we won't be talking about them today. The internet is your friend in this case.

as an example we could use egrep to find fasta files which end in 'T' for example

[ralla@poset unix_playground]$ **egrep -n 'T$' my_first_fasta.fasta** 3:TCGTACGTCGACTGATCGTAGCTACGTCGTACGTAGGTACGTT 10:GATCGTACGTGTAGCTGCTGTAGCTGATCGTACGGTAGCAGCT 11:CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT 16:GTAGATGCTAGCTGACTCGAT

We could find entries that begin with C and end with T

[ralla@poset unix_playground]$ **egrep -n '^C.+T$' my_first_fasta.fasta**

11:CGTCGACTGATCGTAGCTGATCGTACGTCGACTGATCGTAGCT

Exercise 1
make a new directory called fasta_files and download files from [] and place them in the new directory

Then do the following:

a) Unzip the downloaded file (HINT: use gunzip, look at its documentation for help) b) Change into your ** fasta_files ** directory, and unzip the remainder of the fasta files using only a one-line command. c) 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) Get size of total genome. (HINT: The size of the genome can be determined by counting the number of bases).


 * Exercise 2 **

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.


 * Exercise 3 **

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

b) Use your new found knowledge of grep and egrep, 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

c) 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.

Shell Scripting
We can write scripts that run in the bash shell to automate a lot of our commands. We can use this to run a set of commands repeatedly over a bunch of different files. We could use wild cards but not always. For example to get lines 15-20 of every fasta file in fasta_files directory.

[ralla@poset fasta_files]$ ** head -20 chrI.fa | tail -5 **

CATTTATATACACTTATGTCAATATTACAGAAAAATCCCCACAAAAATCA CCTAAACATAAAAATATTCTACTTTTCAACAATAATACATAAACATATTG GCTTGTGGTAGCAACACTATCATGGTATCACTAACGTAAAAGTTCCTCAA TATTGCAATTTGCTTGAACGGATGCTATTTCAGAATATTTCGTACTTACA CAGGCCATACATTAGAATAATATGTCACATCACTGTCGTAACACTCTTTA

Lets make a bash script to do this for all the files, building our code one step at a time

[ralla@poset fasta_files]$ nano subset.sh

And paste into the file and write and exit.
 * head -20 chrI.fa | tail -5 **

[ralla@poset fasta_files]$ **bash subset.sh**

CATTTATATACACTTATGTCAATATTACAGAAAAATCCCCACAAAAATCA CCTAAACATAAAAATATTCTACTTTTCAACAATAATACATAAACATATTG GCTTGTGGTAGCAACACTATCATGGTATCACTAACGTAAAAGTTCCTCAA TATTGCAATTTGCTTGAACGGATGCTATTTCAGAATATTTCGTACTTACA CAGGCCATACATTAGAATAATATGTCACATCACTGTCGTAACACTCTTTA

Great our first bash script runs as expected. But we have hard coded it to print only lines 15-20 for chrI.fa. What if instead we wanted to pass it any fasta file?

Change your subset.sh to the following

Save the file and rerun
 * head -20 $1 | tail -5 **

[ralla@poset fasta_files]$ **bash subset.sh chrVI.fa** CCGAAATCCAAGAAACTGTAAGACATTCATATTCTCGGAAGTATTGGGAA ATTGTGCTTTCAGTTTCTTTCTCTCTAGCAAAACCATTTGACTCCCTTTC CGCTTATACGACTCTTTGTTAATGTCGGTGACTGGATGGAATCTATTATC CTCAGCATTGCCATCTTTATTGGCGTCCTCCTTGGCACTAGCGTTGGTGC TGGCAGTGGTAGTAGCATTAGTCCTGACGTTGATGCTGGCAGTGGTAGTC

$1 is the variable that stores the first argument we passed to the script. We could use $2 for a second variable and $3 for a third variable. Let's try that change your subset.sh to the following


 * head -${2} $1 | tail -${3} **

Now when you run it again we can give it arguments for subsetting any number of lines.

[ralla@poset fasta_files]$ **bash subset.sh chrVI.fa 50 10**

TTCTTCCAAATTTTATGAACATGCCCTAAAGGCACCTCGGATTTCTCCTT GATTAGATTAAACATCCGTGTTGGATAGCTGGATAGACCTCTGCTGAGAT CTTCCGACCGTTTGAGCTCGTTGATGTCCATCGACTTCTTGGCCAGTCCC GTAAGCCCAATACGCTGCAACGCAGCATCAGCTACAGCCTCAGGGGCTGT GCCGCTCAAAAAGATTGCTTTCTCAAAAGCGTCAAAATCAAGGTTAGTTA TGCCCCCAAATTGCGACTGCCGGTAGACCTCCGTTTCAAAGTTGTGAAAC TCATCTACAATGAGGTAACCCAATTTTACGTTGTTGGTCCTAAAGGTGCA CTCAACAATATTCTCCCACGCAGCTATCCTGTCTGTGAAATTAGTGCTAG CAAGATCATCGTAGATCCCCACGTATAAATCAGTAACGCCATCGTAACCT TCTTCAATAAAGTTTCTTACAGGGGCCACATTCAAGCAACCGCGTCGGCC

Great how about if we wanted to pass this script all the files in the folder, we wouldn't know how many variables to use inside our script, and we would have to loop through our list of variables. A full list of arguments passed is captured in a special variable $*. Let us loop through all elements of this special variable and print out lines 15-20

Change subset.sh to this for fasta in $* do echo "lines 15-20 of" $fasta head -20 $fasta | tail -5 done

[ralla@poset fasta_files]$ ** bash subset.sh chr* **

This is an example of for loops in a shell script, we are looping over each element of $* and assigning to a variable fasta and we are accessing that fasta file through $fasta and getting lines 15-20 and printing out. We could pipe the output and save it. There are other loops you can run in bash script as well. For example you could run Let us make it such that we don't have to type bash every time.

Add the following lines to the top of your shell script
 * 1) !/bin/bash

This tells the terminal to use bash to execute the script. But now we also need to make the script executable

[ralla@poset fasta_files]$ **ls -l subset.sh**

-rw-r--r-- 1 ralla cgrl 79 Mar 18 21:43 subset.sh

Notice how the permissions are not setup with an x to execute this script. So we will change permissions to allow execution

[ralla@poset fasta_files]$ **chmod +x subset.sh**

[ralla@poset fasta_files]$ **ls -l subset.sh**

-rwxr-xr-x 1 ralla cgrl 79 Mar 18 21:43 subset.sh

[ralla@poset fasta_files]$ **./subset.sh chr***

We still need to type ./ because terminal is not searching the current directory for executables, for example when you type **ls or cd or mv** these are actually present in the PATH.

[ralla@poset fasta_files]$ **which ls**

/bin/ls

So /bin/ must be in the path because we are able to type ls. To check this we could do

[ralla@poset fasta_files]$ **echo $PATH**

/usr/kerberos/bin:/usr/local/bin:**/bin:**/usr/bin:/opt/dell/srvadmin/bin:/global/home/ralla/bin

Indeed it is. So to get our subset.sh to run like ls or any other commands that are in the PATH, we need to add our current directory to the PATH, how do we do this?

[ralla@poset fasta_files]$ **pwd**

/global/home/ralla/unix_playground/fasta_files

[ralla@poset fasta_files]$ **export PATH=/global/home/ralla/unix_playground/fasta_files:$PATH**

[ralla@poset fasta_files]$ **echo $PATH**

**/global/home/ralla/unix_playground/fasta_files:**/usr/kerberos/bin:/usr/local/bin:/bin:/usr/bin:/opt/dell/srvadmin/bin:/global/home/ralla/bin

[ralla@poset fasta_files]$ **subset.sh chr***

See how my pwd is now in the path so I can start typing sub and use tab to file in subset.sh

Exercise bash scripting
Rename all the fasta files to so chr is now chromosome for example chrV.fa is now chromosomeV.fa

HINT: ${variable//} will return a variable with search_term replaced with replace_term

Software installation (if time permits)
UnixSpring2013_Part2