# Running command-line BLAST¶

The goal of this tutorial is to run you through a demonstration of the command line, which you may not have seen or used much before.

Start up an m1.medium instance running Ubuntu 16.04 on Jetstream.

All of the commands below can copy/pasted.

## Updating the software on the machine¶

Copy and paste the following commands:

sudo apt-get update && sudo apt-get -y install python ncbi-blast+


(make sure to hit enter after the paste – sometimes the last line doesn’t paste completely.)

This updates the software list and installs the Python programming language and NCBI BLAST+.

## Running BLAST¶

First! We need some data. Let’s grab the mouse and zebrafish RefSeq protein data sets from NCBI, and put them in our home directory. If you’ve just logged in, you should be there already, but if you’re unsure, just run cd and hit enter. Now, we’ll use curl to download the files:

curl -O ftp://ftp.ncbi.nih.gov/refseq/M_musculus/mRNA_Prot/mouse.1.protein.faa.gz
curl -O ftp://ftp.ncbi.nih.gov/refseq/M_musculus/mRNA_Prot/mouse.2.protein.faa.gz
curl -O ftp://ftp.ncbi.nih.gov/refseq/M_musculus/mRNA_Prot/mouse.3.protein.faa.gz

curl -O ftp://ftp.ncbi.nih.gov/refseq/D_rerio/mRNA_Prot/zebrafish.1.protein.faa.gz


If you look at the files in the current directory, you should see four files, along with a directory called lost+found which is for system information:

ls -l


should show you:

total 29056
drwxr-xr-x 2 titus titus     4096 May  5 08:26 Desktop
drwxr-xr-x 2 titus titus     4096 Jun 14 18:03 Documents
-rw-rw-r-- 1 titus titus  3610407 Jun 14 18:11 mouse.1.protein.faa.gz
-rw-rw-r-- 1 titus titus  6080985 Jun 14 18:11 mouse.2.protein.faa.gz
-rw-rw-r-- 1 titus titus  7520591 Jun 14 18:11 mouse.3.protein.faa.gz
drwxr-xr-x 2 titus titus     4096 Jun 14 18:03 Music
drwxr-xr-x 2 titus titus     4096 Jun 14 18:03 Pictures
drwxr-xr-x 2 titus titus     4096 Jun 14 18:03 Public
drwxr-xr-x 2 titus titus     4096 Jun 14 18:03 Templates
drwxr-xr-x 2 titus titus     4096 Jun 14 18:03 Videos
-rw-rw-r-- 1 titus titus 12500932 Jun 14 18:11 zebrafish.1.protein.faa.gz


All four of the files are FASTA protein files (that’s what the .faa suggests) that are compressed with gzip (that’s what the .gz means).

Uncompress them:

gunzip *.faa.gz


and let’s look at the first few sequences in the file:

head mouse.1.protein.faa


These are protein sequences in FASTA format. FASTA format is something many of you have probably seen in one form or another – it’s pretty ubiquitous. It’s a text file, containing records; each record starts with a line beginning with a ‘>’, and then contains one or more lines of sequence text.

Let’s take those first two sequences and save them to a file. We’ll do this using output redirection with ‘>’, which says “take all the output and put it into this file here.”

head -11 mouse.1.protein.faa > mm-first.fa


So now, for example, you can do cat mm-first.fa to see the contents of that file (or less mm-first.fa).

Now let’s BLAST these two sequences against the entire zebrafish protein data set. First, we need to tell BLAST that the zebrafish sequences are (a) a database, and (b) a protein database. That’s done by calling ‘makeblastdb’:

makeblastdb -in zebrafish.1.protein.faa -dbtype prot


Next, we call BLAST to do the search:

blastp -query mm-first.fa -db zebrafish.1.protein.faa


This should run pretty quickly, but you’re going to get a lot of output!! To save it to a file instead of watching it go past on the screen, ask BLAST to save the output to a file that we’ll name mm-first.x.zebrafish.txt:

blastp -query mm-first.fa -db zebrafish.1.protein.faa -out mm-first.x.zebrafish.txt


and then you can ‘page’ through this file at your leisure by typing:

less mm-first.x.zebrafish.txt


(Type spacebar to move down, and ‘q’ to get out of paging mode.)

Let’s do some more sequences (this one will take a little longer to run):

head -500 mouse.1.protein.faa > mm-second.fa
blastp -query mm-second.fa -db zebrafish.1.protein.faa -out mm-second.x.zebrafish.txt


will compare the first 83 sequences. You can look at the output file with:

less mm-second.x.zebrafish.txt


(and again, type ‘q’ to get out of paging mode.)

Notes:

• you can execute multiple commands at a time;

• You might see a warning -

Selenocysteine (U) at position 310 replaced by X

what does this mean?

• why did it take longer to BLAST mm-second.fa than mm-first.fa?

Things to mention and discuss:

• blastp options and -help.
• command line options, more generally - why so many?
• automation rocks!