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.xlarge Amazon EC2 instance.

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 /mnt, which is the scratch disk space for Amazon machines

sudo chmod a+rwxt /mnt
cd /mnt

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 28904
drwx------ 2 root   root      16384 Nov  2  2014 lost+found
-rw-rw-r-- 1 ubuntu ubuntu  8132346 Aug 10 21:44 mouse.1.protein.faa.gz
-rw-rw-r-- 1 ubuntu ubuntu  8091255 Aug 10 21:44 mouse.2.protein.faa.gz
-rw-rw-r-- 1 ubuntu ubuntu   565224 Aug 10 21:44 mouse.3.protein.faa.gz
-rw-rw-r-- 1 ubuntu ubuntu 12735506 Aug 10 21:44 zebrafish.1.protein.faa.gz

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

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 just 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 of output!! To save it to a file instead of watching it go past on the screen, do:

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:

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 should 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??
automation rocks!

Reminder: shut down your instance!

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