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The goal of this tutorial is to run you through a demonstration of thecommand line, which you may not have seen or used much before.
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Prepare for this tutorial by working throughStart up an EC2 instance, but follow the instructionsto start up Starting up a custom operating system instead; useAMI ami-7606d01e.
All of the commands below can and should be copy/pasted rather thanre-typed.
Note: on Windows using TeraTerm, you can select the commands inthe Web browser, then go to TeraTerm and click your right mousebutton to paste. On Mac OS X using Terminal, you can select thecommands in the Web browser, use Command-C to copy, and then gothe terminal and use Command-V to paste.
Switching to root¶
Start by making sure you’re the superuser, root:
Updating the software on the machine¶
Copy and paste the following two commands
(make sure to hit enter after the paste – sometimes the last line doesn’tpaste completely.)
If you started up a custom operating system, then this should finishquickly; if instead you started up Ubuntu 14.04 blank, then this willtake a minute or two.
Install BLAST¶
Here, we’re using curl to download the BLAST distribution from NCBI;then we’re using ‘tar’ to unpack it into the current directory; andthen we’re copying the program files into the directory/usr/local/bin, where we can run them from anywhere.
OK – now you can run BLAST from anywhere!
Again, this is basically what “installing software” means – it justmeans copying around files so that they can be run, and (in some cases)setting up resources so that the software knows where specific datafiles are.
Running BLAST¶
Try typing:
You’ll get a long laundry list of output, with all sorts of options andarguments. Let’s play with some of them.
First! We need some data. Let’s grab the mouse and zebrafish RefSeqprotein data sets from NCBI, and put them in /mnt, which is thescratch disk space for Amazon machines
If you look at the files in the current directory, you should see bothfiles, along with a directory called lost+found which is for systeminformation:
should show you:
Both of these files are FASTA protein files (that’s what the .faa suggests)that are compressed by gzip (that’s what the .gz suggests).
Uncompress them
and let’s look at the first few sequences:
These are protein sequences in FASTA format. FASTA format is somethingmany of you have probably seen in one form or another – it’s prettyubiquitous. It’s just a text file, containing records; each recordstarts with a line beginning with a ‘>’, and then contains one or morelines of sequence text.
Let’s take those first two sequences and save them to a file. We’lldo this using output redirection with ‘>’, which says “takeall the output and put it into this file here.”
So now, for example, you can do ‘cat mm-first.fa’ to see the contents ofthat file (or ‘less mm-first.fa’).
Now let’s BLAST these two sequences against the entire zebrafishprotein data set. First, we need to tell BLAST that the zebrafishsequences are (a) a database, and (b) a protein database. That’s doneby calling ‘formatdb’
Next, we call BLAST to do the search
This should run pretty quickly, but you’re going to get a LOT of output!!What’s going on? A few things –
- if you BLAST a sequence against a large database, odds are it will turnup a lot of spurious matches. By default, blastall uses an e-value cutoffof 10, which is very relaxed.
- blastall also reports the first 100 matches, which is usually more thanyou want.
- a lot of proteins also have trace similarity to other proteins!
For all of these reasons, generally you only want the first few BLASTmatches, and/or the ones with a “good” e-value. We do that by adding‘-b 2 -v 2’ (which says, report only two matches and alignments); andby adding ‘-e 1e-6’, which says, report only matches with an e-valueof 1e-6 or better
Now you should get a lot less text! (And indeed you do..) Let’s put it anoutput file, ‘out.txt’
The contents of the output file should look exactly like the output beforeyou saved it into the file – check it out:
Converting BLAST output into CSV¶
Suppose we wanted to do something with all this BLAST output. Generally,that’s the case - you want to retrieve all matches, or do a reciprocalBLAST, or something.
As with most programs that run on UNIX, the text output is in somespecific format. If the program is popular enough, there will be oneor more parsers written for that format – these are just utilitieswritten to help you retrieve whatever information you are interestedin from the output.
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Let’s conclude this tutorial by converting the BLAST output in out.txtinto a spreadsheet format, using a Python script. (We’re not doing thisjust to confuse you; this is really how we do things around here.)
First, we need to get the script. We’ll do that using the ‘git’ program
We’ll discuss ‘git’ more later; for now, just think of it as a wayto get ahold of a particular set of files. In this case, we’ve placedthe files in /root/ngs-scripts/, and you’re looking to run thescript blast/blast-to-csv.py using Python
This outputs a spread-sheet like list of names and e-values. To save thisto a file, do:
The end file, ‘out.csv’, should soon be in your Dropbox on your localcomputer. If you have Excel installed, try double clicking on it.
And that’s the kind of basic workflow we’ll be teaching you:
- Download program
- Download data
- Run program on data
- Look at results
..but in many cases more complicated :).
Note that there’s no limit on the number of sequences you BLAST, etc.It’s just sheer compute speed and disk space that you need to worryabout, and if you look at the files, it turns out they’re not that big –so it’s mostly your time and energy.
This will also maybe help you understand why UNIX programs are sopowerful – each program comes with several, or several dozen, littlecommand line “flags” (parameters), that help control how it does itswork; then the output is fed into another such program, etc. The possibilitiesare literally combinatorial.
We’re running a Python program ‘blast-to-csv.py’ above – if you’reinterested in what the Python program does, take a look at the sourcecode:
Summing up¶
Command-line BLAST lets you do BLAST searches of any sequences you have,quickly and easily. It’s probably the single most useful skill abiologist can learn if they’re doing anything genomics-y ;).
Its main computational drawback is that it’s not fast enough to dealwith some of the truly massive databases we now have, but that’sgenerally not a problem for individual users. That’s because they justrun it and “walk away” until it’s done!
The main practical issues you will confront in making use of BLAST:
- getting your sequence(s) into the right place.
- formatting the database.
- configuring the BLAST parameters properly.
- doing what you want after BLAST!
Other questions to ponder:
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- if we’re using a pre-configured operating system, why did we have toinstall BLAST?