Category Archives: laboratory

Mar 30 2012
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laboratory, perl

Fifth lab: pick your primers

Today we’ll meet at 1.30 pm in aula F pr to finish together our “pick-primers.pl” program. During last lab most of you got Primer3 running with our custom parameters. Primer3 output consists in a list of possible primer pairs.

We want to blast each primer against reference contigs to check how many sequence similar to it are present in the genome. This is a pivotal test to have specific PCRs. To make this we design a subroutine called ‘blast’ that takes as input a sequence (primer) and blast it against the contigs and parses the output returning a list of hints. In particular we consider dangerous those aligment long as the query -2 bp. Thus if our primers is long 20 and the match is 15 we will discard it. If the match with another contig is 18 bases long, we will report this to the user.

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Mar 26 2012
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laboratory, perl

Lab 4: solution

Click “read more” to see today’s solution…

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Mar 26 2012
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laboratory, perl

Fourth lab: shell commands via Perl

Today we:

  • have a small lesson on shell commands with Perl
  • optimize last time’s script so that Primer3 execution will be faster in most cases
  • see some more example of regular expressions
Mar 21 2012
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laboratory

Third computer lab

Lesson

We start with a brief class lesson to view how to access the visual interface of Contig Browser (see video) and  start coding the pickprimers.pl program.
You can browse the contigs from the platform http://www.4ngs.com/alga_v1/ using your log-in as username and your UPPERCASE (without spaces) surname as password.

Get your gaps

In this list we have our mission: the gaps to close. You’ll see lines starting with # with four columns: the username assigned to the gap (e.g. geno-20), the PCR identifier (e.g. PCR22) and the two contigs to join. Following lines tell you the mutual orientation of the contigs as follows.

These are complex regions and rather than being side-to-side gaps they have (small) contigs between them.  Let’s see this example: contig02430 to contig01619. If you browse from one contig to the other you obtain:

In our jargon we say that contig00068 is uncomplemented (U) while the others are complemented (C). So the PCR has to be designed between contig00068U and contig00122C. Note that three contigs have no direct link,

Primer3: make it running

Review last lab and try the Primer3 part. I saved the pickprimers.pl program we made in class in /home/geno/tools/pickprimers.pl. Copy it to your home so that you’ll edit your own version, with this command:

cp /home/geno/tools/pickprimers.pl ~

Now consider a program that receive from the user four parameters: contig1, direction1, contig2, direction2.
Example:

pickprimers.pl contig00068 U contig00122 C

The program is able to give you access to both sequences (we did it togegher) so you have in $seq1 the first and in $seq2 the second.
Now arrange the program so that it will print the proper Primer3 file.

Now:

1) Create a $template variable with the right template

2) Optionally print a Primer3 compliant instruction

 

 

Mar 15 2012
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laboratory

Second computer lab

After the brief introduction in class you are now ready to try by yourself a couple of command line programs: BLAST and Primer3. After having worked with these tools, you’ll have some more time to play with Perl. Today we will

  • Practice again with the shell
  • Perform a BLAST via command line
  • Perl scripts
  • Primer3 test

Now our servers are set up and we can monitor your activity… so remember: read carefully, and type filenames exactly as we suggest.

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Mar 13 2012
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First computer lab

Welcome to the Computer Lab #1

From text editor to program execution

Today we start playing with our computer: we want to

  1. start using the shell terminal
  2. start using a programming editor
  3. preparing and executing simple Perl scripts
  4. Make Primer3 design a couple of primers

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Mar 13 2012
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laboratory

Auto-completing filenames

The shell if filled with shortcuts and tricks, but there a vital feature you can’t miss at all: the auto-completion (see what says Wikipedia about it). In a few words if you start typing a filename or command an you press [Tab] once it will be automatically completed. If more than a file exist that starts with the characters you typed, double [Tab] prints a list of possibilities.

This is much more than a trick: it’s a safe way to check that you are giving the right parameter to your scripts/programs.

If you mis-type a filename (or directory name) you’ll see the error only executing the command.

cd ~
cd Documanti
-bash: cd: Documanti: No such file or directory
 
cd ~
cd Docu[Tab]
cd Documenti (automatically completed)

 

 

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