Mastering Perl for Bioinformatics

ChapterÂ 1.Â Modular Programming with Perl

Perl modules are essential to any Perl programmer. They are a great way to organize code into logical collections of interacting parts. They collect useful Perl subroutines and provide them to other programs (and programmers) in an organized and convenient fashion.

This chapter begins with a discussion of the reasons for organizing Perl code into modules. Modules are comparable to subroutines: both organize Perl code in convenient, reusable âchunks.â

Later in this chapter, Iâll introduce a small module, GeneticCode.pm. This example shows how to create simple modules, and Iâll give examples of programs that use this module.

Iâll also demonstrate how to find, install, and use modules taken from the all-important CPAN collection. A familiarity with searching and using CPAN is an essential skill for Perl programmers; it will help you avoid lots of unnecessary work. With CPAN, you can easily find and use code written by excellent programmers and road-tested by the Perl community. Using proven code and writing less of your own, youâll save time, money, and headaches.

What Is a Module?

A Perl module is a library file that uses package declarations to create its own namespace. Perl modules provide an extra level of protection from name collisions beyond that provided by my and use strict. They also serve as the basic mechanism for defining object-oriented classes.

Why Perl Modules?

Building a medium- to large-sized program usually requires you to divide tasks into several smaller, more manageable, and more interactive pieces. (A rule of thumb is that each âpieceâ should be about one or two printed pages in length, but this is just a general guideline.) An analogy can be made to building a microarray machine, which requires that you construct separate interacting pieces such as housing, temperature sensors and controls, robot arms to position the pipettes, hydraulic injection devices, and computer guidance for all these systems.

Subroutines and Software Engineering

Subroutines divide a large programming job into more manageable pieces. Modern programming languages all provide subroutines, which are also called functions, coroutines, or macros in other programming languages.

A subroutine lets you write a piece of code that performs some part of a desired computation (e.g., determining the length of DNA sequence). This code is written once and then can be called frequently throughout the main program. Using subroutines speeds the time it takes to write the main program, makes it more reliable by avoiding duplicated sections (which can get out of sync and make the program longer), and makes the entire program easier to test. A useful subroutine can be used by other programs as well, saving you development time in the future. As long as the inputs and outputs to the subroutine remain the same, its internal workings can be altered and improved without worrying about how the changes will affect the rest of the program. This is known as encapsulation .

The benefits of subroutines that Iâve just outlined also apply to other approaches in software engineering. Perl modules are a technique within a larger umbrella of techniques known as software encapsulation and reuse. Software encapsulation and reuse are fundamental to object-oriented programming.

A related design principle is abstraction , which involves writing code that is usable in many different situations. Letâs say you write a subroutine that adds the fragment TTTTT to the end of a string of DNA. If you then want to add the fragment AAAAA to the end of a string of DNA, you have to write another subroutine. To avoid writing two subroutines, you can write one thatâs more abstract and adds to the end of a string of DNA whatever fragment you give it as an argument. Using the principle of abstraction, youâve saved yourself half the work.

Here is an example of a Perl subroutine that takes two strings of DNA as inputs and returns the second one appended to the end of the first:

sub DNAappend {
        my ($dna, $tail) = @_;

        return($dna . $tail);
}

This subroutine can be used as follows:

my $dna = 'ACCGGAGTTGACTCTCCGAATA';
my $polyT = 'TTTTTTTT';

print DNAappend($dna, $polyT);

If you wish, you can also define subroutines polyT and polyA like so:

sub polyT {
    my ($dna) = @_;

    return DNAappend($dna, 'TTTTTTTT');
}
sub polyA {
    my ($dna) = @_;

    return DNAappend($dna, 'AAAAAAAA');
}

At this point, you should think about how to divide a problem into interacting parts; that is, an optimal (or at least good) way to define a set of subroutines that can cooperate to solve a particular problem.

Modules and Libraries

In my projects, I gather subroutine definitions into separate files called libraries,^[1] or modules, which let me collect subroutine definitions for use in other programs. Then, instead of copying the subroutine definitions into the new program (and introducing the potential for inaccurate copies or for alternate versions proliferating), I can just insert the name of the library or module into a program, and all the subroutines are available in their original unaltered form. This is an example of software reuse in action.

To fully understand and use modules, you need to understand the simple concepts of namespaces and packages. From here on, think of a Perl module as any Perl library file that uses package declarations to create its own namespace. These simple concepts are examined in the next sections.

Namespaces

A namespace is implemented as a table containing the names of the variables and subroutines in a program. The table itself is called a symbol table and is used by the running program to keep track of variable values and subroutine definitions as the program evolves. A namespace and a symbol table are essentially the same thing. A namespace exists under the hood for many programs, especially those in which only one default namespace is used.

Large programs often accidentally use the same variable name for different variables in different parts of the program. These identically named variables may unintentionally interact with each other and cause serious, hard-to-find errors. This situation is called namespace collision . Separate namespaces are one way to avoid namespace collision.

The package declaration described in the next section is one way to assign separate namespaces to different parts of your code. It gives strong protection against accidentally using a variable name thatâs used in another part of the program and having the two identically-named variables interact in unwanted ways.

Namespaces Compared with Scoping: my and use strict

The unintentional interaction between variables with the same name is enough of a problem that Perl provides more than one way to avoid it. You are probably already familiar with the use of my to restrict the scope of a variable to its enclosing block (between matching curly braces {}) and should be accustomed to using the directive use strict to require the use of my for all variables. use strict and my are a great way to protect your program from unintentional reuse of variable names. Make a habit of using my and working under use strict.

Packages

Packages are a different way to protect a programâs variables from interacting unintentionally. In Perl, you can easily assign separate namespaces to entire sections of your code, which helps prevent namespace collisions and lets you create modules.

Packages are very easy to use. A one-line package declaration puts a new namespace in effect. Hereâs a simple example:

$dna = 'AAAAAAAAAA';
package Mouse;
$dna = 'CCCCCCCCCC';
package Celegans;
$dna = 'GGGGGGGGGG';

In this snippet, there are three variables, each with the same name, $dna. However, they are in three different packages, so they appear in three different symbol tables and are managed separately by the running Perl program.

The first line of the code is an assignment of a poly-A DNA fragment to a variable $dna. Because no package is explicitly named, this $dna variable appears in the default namespace main.

The second line of code introduces a new namespace for variable and subroutine definitions by declaring package Mouse;. At this point, the main namespace is no longer active, and the Mouse namespace is brought into play. Note that the name of the namespace is capitalized; itâs a well-established convention you should follow. The only noncapitalized namespace you should use is the default main.

Now that the Mouse namespace is in effect, the third line of code, which declares a variable, $dna, is actually declaring a separate variable unrelated to the first. It contains a poly-C fragment of DNA.

Finally, the last two lines of code declare a new package called Celegans and a new variable, also called $dna, that stores a poly-G DNA fragment.

To use these three $dna variables, you need to explicitly state which packages you want the variables from, as the following code fragment demonstrates:

print "The DNA from the main package:\n\n";
print $main::dna, "\n\n";

print "The DNA from the Mouse package:\n\n";
print $Mouse::dna, "\n\n";

print "The DNA from the Celegans package:\n\n";
print $Celegans::dna, "\n\n";

This gives the following output:

The DNA from the main package:

AAAAAAAAAA

The DNA from the Mouse package:

CCCCCCCCCC

The DNA from the Celegans package:

GGGGGGGGGG

As you can see, the variable name can be specified as to a particular package by putting the package name and two colons before the variable name (but after the $, @, or % that specifies the type of variable). If you donât specify a package in this way, Perl assumes you want the current package, which may not necessarily be the main package, as the following example shows:

#
# Define the variables in the packages
#

$dna = 'AAAAAAAAAA';

package Mouse;

$dna = 'CCCCCCCCCC';

#
# Print the values of the variables
#

print "The DNA from the current package:\n\n";
print $dna, "\n\n";

print "The DNA from the Mouse package:\n\n";
print $Mouse::dna, "\n\n";

This produces the following output:

The DNA from the current package:
CCCCCCCCCC

The DNA from the Mouse package:
CCCCCCCCCC

Both print $dna and print $Mouse::dna reference the same variable. This is because the last package declaration was package Mouse;, so the print $dna statement prints the value of the variable $dna as defined in the current package, which is Mouse.

The rule is, once a package has been declared, it becomes the current package until the next package declaration or until the end of the file. (You can also declare packages within blocks, evals, or subroutine definitions, in which case the package stays in effect until the end of the block, eval, or subroutine definition.)

By far the most common use of package is to call it once near the top of a file and have it stay in effect for all the code in the file. This is how modules are defined, as the next section shows.

Defining Modules

To begin, take a file of subroutine definitions and call it something like Newmodule.pm. Now, edit the file and give it a new first line:

package Newmodule;

and a new last line 1;. Youâve now created a Perl module.

To make a Celegans module, place subroutines in a file called Celegans.pm, and add a first line:

package Celegans;

Add a last line 1;, and youâve defined a Celegans module. This last line just ensures that the library returns a true value when itâs read in. Itâs annoying, but necessary.

Storing Modules

Where you store your .pm module files on your computer affects the name of the module, so letâs take a moment to sort out the most important points. For all the details, consult the perlmod and the perlmodlib parts of the Perl documentation at http://www.perldoc.org. You can also type perldoc perlmod or perldoc perlmodlib at a shell prompt or in a command window.

Once you start using multiple files for your program code, which happens if youâre defining and using modules, Perl needs to be able to find these various files; it provides a few different ways to do so.

The simplest method is to put all your program files, including your modules, in the same directory and run your programs from that directory. Hereâs how the module file Celegans.pm is loaded from another program:

use Celegans;

However, itâs often not so simple. Perl uses modules extensively; many are built-in when you install Perl, and many more are available from CPAN, as youâll see later. Some modules are used frequently, some rarely; many modules call other modules, which in turn call still other modules.

To organize the many modules a Perl program might need, you should place them in certain standard directories or in your own development directories. Perl needs to know where these directories are so that when a module is called in a program, it can search the directories, find the file that contains the module, and load it in.

When Perl was installed on your computer, a list of directories in which to find modules was configured. Every time a Perl program on your computer refers to a module, Perl looks in those directories. To see those directories, you only need to run a Perl program and examine the built-in array @INC , like so:

print join("\n", @INC), "\n";

On my Linux computer, I get the following output from that statement:

/usr/local/lib/perl5/5.8.0/i686-linux
/usr/local/lib/perl5/5.8.0
/usr/local/lib/perl5/site_perl/5.8.0/i686-linux
/usr/local/lib/perl5/site_perl/5.8.0
/usr/local/lib/perl5/site_perl/5.6.1
/usr/local/lib/perl5/site_perl/5.6.0
/usr/local/lib/perl5/site_perl
.

These are all locations in which the standard Perl modules live on my Linux computer. @INC is simply an array whose entries are directories on your computer. The way it looks depends on how your computer is configured and your operating system (for instance, Unix computers handle directories a bit differently than Windows).

Note that the last line of that list of directories is a solitary period. This is shorthand for âthe current directory,â that is, whatever directory you happen to be in when you run your Perl program. If this directory is on the list, and you run your program from that directory as well, Perl will find the .pm files.

When you develop Perl software that uses modules, you should put all the modules together in a certain directory. In order for Perl to find this directory, and load the modules, you need to add a line before the use MODULE directives, telling Perl to additionally search your own module directory for any modules requested in your program. For instance, if I put a module Iâm developing for my program into a file named Celegans.pm, and put the Celegans.pm file into my Linux directory /home/tisdall/MasteringPerlBio/development/lib, I need to add a use lib directive to my program, like so:

use lib "/home/tisdall/MasteringPerlBio/development/lib";

use Celegans;

Perl then adds my development module directory to the @INC array and searches there for the Celegans.pm module file. The following code demonstrates this:

use lib "/home/tisdall/MasteringPerlBio/development/lib";

print join("\n", @INC), "\n";

This produces the output:

/home/tisdall/MasteringPerlBio/development/lib
/usr/local/lib/perl5/5.8.0/i686-linux
/usr/local/lib/perl5/5.8.0
/usr/local/lib/perl5/site_perl/5.8.0/i686-linux
/usr/local/lib/perl5/site_perl/5.8.0
/usr/local/lib/perl5/site_perl/5.6.1
/usr/local/lib/perl5/site_perl/5.6.0
/usr/local/lib/perl5/site_perl
.

Thanks to the use lib directive, Perl can now find the Celegans.pm file in the @INC list of directories.

A problem with this approach to finding libraries is that the directory pathnames are hardcoded into each program. If you then want to move your own library directory somewhere else or move the programs to another computer where different pathnames are used, you need to change the pathnames in all the program files where they occur.

If, for instance, you download several programs from this bookâs web site, and you donât want to edit each one to change pathnames, you can use the PERL5LIB environmental variable. To do so, put all the modules under the directory /my/perl/modules (for example). Now set the PERL5LIB variable:

PERL5LIB=$PERL5LIB:/my/perl/modules

You can also set it this way:

setenv PERL5LIB /my/perl/modules

If you have âtaintâ security checks enabled in your version of Perl, you still have to hardcode the pathname into the program. This, of course, behaves differently on different operating systems.

You can also specify an additional directory on the command line:

perl -I/my/perl/modules myprogram.pl

Thereâs one other detail about modules thatâs important. Youâll sometimes see modules in Perl programs with names such as Genomes::Modelorganisms::Celegans , in which the name is two or more words separated by two colons. This is how Perl looks into subdirectories of directories named in the @INC built-in array. In the example, Perl looks for a subdirectory named Genomes in one of the @INC directories; then for a subdirectory named Modelorganisms within the Genomes subdirectory; finally, for a file named Celegans.pm within the Modelorganisms subdirectory. That is, my module is in the file:

/home/tisdall/MasteringPerlBio/development/lib/Genomes/Modelorganisms/Celegans.pm

and itâs called in my Perl program like so:

use lib "/home/tisdall/MasteringPerlBio/development/lib";

use Genomes::Modelorganisms::Celegans;

There are more details you can learn about storing and finding modules on your computer, but these are the most useful facts. See the perlmod, perlrun, and perlmodlib sections of the Perl manual for more details if and when you need them.

Writing Your First Perl Module

Now that youâve been introduced to the basic ideas of modules, itâs time to actually examine a working example of a module.

In this section, weâll write a module called Geneticcode.pm , which implements the genetic code that maps DNA codons to amino acids and then translates a string of DNA sequence data to a protein fragment.

An Example: Geneticcode.pm

Letâs start by creating a file called Geneticcode.pm and using it to define the mapping of codons to amino acids in a hash variable called %genetic_code. Weâll also discuss a subroutine called codon2aa that uses the hash to translate its codon arguments into amino acid return values.

Here are the contents of the first module file Geneticcode.pm:

package Geneticcode;

use strict;
use warnings;

my(%genetic_code) = (
   
    'TCA' => 'S',    # Serine
    'TCC' => 'S',    # Serine
    'TCG' => 'S',    # Serine
    'TCT' => 'S',    # Serine
    'TTC' => 'F',    # Phenylalanine
    'TTT' => 'F',    # Phenylalanine
    'TTA' => 'L',    # Leucine
    'TTG' => 'L',    # Leucine
    'TAC' => 'Y',    # Tyrosine
    'TAT' => 'Y',    # Tyrosine
    'TAA' => '_',    # Stop
    'TAG' => '_',    # Stop
    'TGC' => 'C',    # Cysteine
    'TGT' => 'C',    # Cysteine
    'TGA' => '_',    # Stop
    'TGG' => 'W',    # Tryptophan
    'CTA' => 'L',    # Leucine
    'CTC' => 'L',    # Leucine
    'CTG' => 'L',    # Leucine
    'CTT' => 'L',    # Leucine
    'CCA' => 'P',    # Proline
    'CCC' => 'P',    # Proline
    'CCG' => 'P',    # Proline
    'CCT' => 'P',    # Proline
    'CAC' => 'H',    # Histidine
    'CAT' => 'H',    # Histidine
    'CAA' => 'Q',    # Glutamine
    'CAG' => 'Q',    # Glutamine
    'CGA' => 'R',    # Arginine
    'CGC' => 'R',    # Arginine
    'CGG' => 'R',    # Arginine
    'CGT' => 'R',    # Arginine
    'ATA' => 'I',    # Isoleucine
    'ATC' => 'I',    # Isoleucine
    'ATT' => 'I',    # Isoleucine
    'ATG' => 'M',    # Methionine
    'ACA' => 'T',    # Threonine
    'ACC' => 'T',    # Threonine
    'ACG' => 'T',    # Threonine
    'ACT' => 'T',    # Threonine
    'AAC' => 'N',    # Asparagine
    'AAT' => 'N',    # Asparagine
    'AAA' => 'K',    # Lysine
    'AAG' => 'K',    # Lysine
    'AGC' => 'S',    # Serine
    'AGT' => 'S',    # Serine
    'AGA' => 'R',    # Arginine
    'AGG' => 'R',    # Arginine
    'GTA' => 'V',    # Valine
    'GTC' => 'V',    # Valine
    'GTG' => 'V',    # Valine
    'GTT' => 'V',    # Valine
    'GCA' => 'A',    # Alanine
    'GCC' => 'A',    # Alanine
    'GCG' => 'A',    # Alanine
    'GCT' => 'A',    # Alanine
    'GAC' => 'D',    # Aspartic Acid
    'GAT' => 'D',    # Aspartic Acid
    'GAA' => 'E',    # Glutamic Acid
    'GAG' => 'E',    # Glutamic Acid
    'GGA' => 'G',    # Glycine
    'GGC' => 'G',    # Glycine
    'GGG' => 'G',    # Glycine
    'GGT' => 'G',    # Glycine
);


#
# codon2aa
#
# A subroutine to translate a DNA 3-character codon to an amino acid
#   Version 3, using hash lookup

sub codon2aa {
        my($codon) = @_;

        $codon = uc $codon;
 
        if(exists $genetic_code{$codon}) {
                return $genetic_code{$codon};
        }else{
                die "Bad codon '$codon'!!\n";
        }
}

1;

Now, letâs examine the code. First, the module declares its package with a name (Geneticcode) that is the same as the file it is in (Geneticcode.pm), but minus the file extension .pm.

The directives:

use strict;
use warnings;

will appear in all the code. The use strict directive enforces the use of the my directive for all variables. The use warnings directive produces useful messages about potential problems in your code. (It is possible to turn both directives off when requiredâto avoid annoying warnings in your program output, for instance. See the perldiag, perllexwarn, and perlmodlib sections of the Perl manual.)

Finally, there is a subroutine definition for codon2aa . As an argument, this subroutine takes a codon represented as a string of three DNA bases and returns the amino acid code corresponding to the codon. It accomplishes this by a simple lookup in the hash %genetic_code and returns the result from the subroutine using the return built-in function.

The codon2aa subroutine calls die and exits the program when it encounters an undefined codon. See the exercises at the end of this chapter for a discussion of the pros and cons of this behavior.

In my earlier book, I defined the hash %genetic_code within the subroutine codon2aa. That meant that every time the subroutine was called, the hash would have to be initialized, which took a bit of time. In this version, the hash only has to be initialized once, when the program is first called, which results in a significant speedup. The definition of the hash is outside of the subroutine definition, but in the namespace of the Geneticcode package. The hash is initialized when the Geneticcode.pm module is loaded by this statement:

use Geneticcode;

Every subsequent call to the codon2aa subroutine simply accesses the hash without having to initialize it each time.

Hereâs an example that uses the new Geneticcode module, which is saved in a file called testGeneticcode and run by typing perl testGeneticcode:

use strict;
use warnings;

use lib "/home/tisdall/MasteringPerlBio/development/lib";

use Geneticcode;

my $dna = 'AACCTTCCTTCCGGAAGAGAG';

# Initialize variables
my $protein = '';

# Translate each three-base codon to an amino acid, and append to a protein 
for(my $i=0; $i < (length($dna) - 2) ; $i += 3) {
        $protein .= Geneticcode::codon2aa( substr($dna,$i,3) );
}

print $protein, "\n";

Recall that the Perl built-in function substr can extract a portion of a string. In this case, substr extracts from $dna the three characters beginning at the position given in the counter variable $i; this three-character codon is then passed as the argument to the subroutine codon2aa. This program produces the output:

NLPSGRE

Expanding Geneticcode.pm

Now, letâs expand our Geneticcode module example. This new version of the module includes a few short helper subroutines. The interest here lies in how the subroutines interact with each other in the moduleâs namespace, and how to access the code within the module from a Perl program that uses the module.

Modules are a great way to organize code into logical collections of interacting parts. When you create modules, you need to decide how to organize your code into the appropriate collection of modules. Here, we have some subroutines that translate codons into amino acids; others read sequence data from files and print it to the screen. This is a fairly obvious division of functionality, so letâs create two modules for this code. Weâll expand the Geneticcode module; letâs also create a SequenceIO module. Of course, the new module will be created in a file called SequenceIO.pm , and that file will be placed in a directory that Perl can findâin this case, the same directory in which weâve placed the Geneticcode module.

Hereâs the code for Geneticcode.pm:

package Geneticcode;

use strict;
use warnings;

my(%genetic_code) = (
    
    'TCA' => 'S',    # Serine
    'TCC' => 'S',    # Serine
    'TCG' => 'S',    # Serine
    'TCT' => 'S',    # Serine
    'TTC' => 'F',    # Phenylalanine

     ... as before ...

    'GAG' => 'E',    # Glutamic Acid
    'GGA' => 'G',    # Glycine
    'GGC' => 'G',    # Glycine
    'GGG' => 'G',    # Glycine
    'GGT' => 'G',    # Glycine
);


#
# codon2aa
#
# A subroutine to translate a DNA 3-character codon to an amino acid
#   Version 3, using hash lookup

sub codon2aa {
    my($codon) = @_;

    $codon = uc $codon;
 
    if(exists $genetic_code{$codon}) {
        return $genetic_code{$codon};
    }else{

            die "Bad codon '$codon'!!\n";
    }
}

#
# dna2peptide 
#
# A subroutine to translate DNA sequence into a peptide

sub dna2peptide {

    my($dna) = @_;

    # Initialize variables
    my $protein = '';

    # Translate each three-base codon to an amino acid, and append to a protein 
    for(my $i=0; $i < (length($dna) - 2) ; $i += 3) {
        $protein .= codon2aa( substr($dna,$i,3) );
    }

    return $protein;
}

# translate_frame
#
# A subroutine to translate a frame of DNA

sub translate_frame {

    my($seq, $start, $end) = @_;
    my $protein;

    # To make the subroutine easier to use, you won't need to specify
    #  the end point-it will just go to the end of the sequence
    #  by default.
    unless($end) {
        $end = length($seq);
    }

    # Finally, calculate and return the translation
        return dna2peptide ( substr ( $seq, $start - 1, $end -$start + 1) );
}

1;

Now, we have in one module the code that accomplishes a translation from the genetic code. However, we also need to read sequence in from FASTA sequence files, and print out sequence (the translated protein) to the screen. Because these needs are likely to recur in many programs, it makes sense to make a separate module for just the file reading, sequence extraction, and sequence printing operations. (This may even be too much in one module; maybe there should be separate modules for each need? See the exercises at the end of the chapter.)

Hereâs the code for the second module SequenceIO.pm , which handles reading from a file, extracting FASTA sequence data, and printing sequence data:

package SequenceIO;

use strict;
use warnings;

# get_file_data
#
# A subroutine to get data from a file given its filename

sub get_file_data {

    my($filename) = @_;

    # Initialize variables
    my @filedata = (  );

    open(GET_FILE_DATA, $filename) or die "Cannot open file '$filename':$!\n\n";

    @filedata = <GET_FILE_DATA>;

    close GET_FILE_DATA;

    return @filedata;
}

# extract_sequence_from_fasta_data
#
# A subroutine to extract FASTA sequence data from an array

sub extract_sequence_from_fasta_data {

    my(@fasta_file_data) = @_;

    # Declare and initialize variables
    my $sequence = '';

    foreach my $line (@fasta_file_data) {

        # discard blank line
        if ($line =~ /^\s*$/) {
            next;

        # discard comment line
        } elsif($line =~ /^\s*#/) {
            next;

        # discard fasta header line
        } elsif($line =~ /^>/) {
            next;

        # keep line, add to sequence string
        } else {
            $sequence .= $line;
        }
    }

    # remove non-sequence data (in this case, whitespace) from $sequence string
    $sequence =~ s/\s//g;

    return $sequence;
}

# print_sequence
#
# A subroutine to format and print sequence data 

sub print_sequence {

    my($sequence, $length) = @_;

    # Print sequence in lines of $length
    for ( my $pos = 0 ; $pos < length($sequence) ; $pos += $length ) {
        print substr($sequence, $pos, $length), "\n";
    }
}

1;

Before we discuss the code, letâs see a small program that uses it:

# Translate a DNA sequence into one of the six reading frames

use strict;
use warnings;

use lib "/home/tisdall/MasteringPerlBio/development/lib";
use Geneticcode;
use SequenceIO;

# Initialize variables
my @file_data = (  );
my $dna = '';
my $revcom = '';
my $protein = '';

# Read in the contents of the file "sample.dna"
@file_data = SequenceIO::get_file_data("sample.dna");

# Extract the sequence data from the contents of the file "sample.dna"
$dna = SequenceIO::extract_sequence_from_fasta_data(@file_data);

# Translate the DNA to protein in one of the six reading frames
#   and print the protein in lines 70 characters long

print "\n -------Reading Frame 1--------\n\n";

$protein = Geneticcode::translate_frame($dna, 1);

SequenceIO::print_sequence($protein, 70);

exit;

Hereâs the input file:

> sample dna  (This is a typical fasta header.)
agatggcggcgctgaggggtcttgggggctctaggccggccacctactgg
tttgcagcggagacgacgcatggggcctgcgcaataggagtacgctgcct
gggaggcgtgactagaagcggaagtagttgtgggcgcctttgcaaccgcc
tgggacgccgccgagtggtctgtgcaggttcgcgggtcgctggcgggggt
cgtgagggagtgcgccgggagcggagatatggagggagatggttcagacc
cagagcctccagatgccggggaggacagcaagtccgagaatggggagaat
gcgcccatctactgcatctgccgcaaaccggacatcaactgcttcatgat
cgggtgtgacaactgcaatgagtggttccatggggactgcatccggatca
ctgagaagatggccaaggccatccgggagtggtactgtcgggagtgcaga
gagaaagaccccaagctagagattcgctatcggcacaagaagtcacggga
gcgggatggcaatgagcgggacagcagtgagccccgggatgagggtggag
ggcgcaagaggcctgtccctgatccagacctgcagcgccgggcagggtca
gggacaggggttggggccatgcttgctcggggctctgcttcgccccacaa
atcctctccgcagcccttggtggccacacccagccagcatcaccagcagc
agcagcagcagatcaaacggtcagcccgcatgtgtggtgagtgtgaggca
tgtcggcgcactgaggactgtggtcactgtgatttctgtcgggacatgaa
gaagttcgggggccccaacaagatccggcagaagtgccggctgcgccagt
gccagctgcgggcccgggaatcgtacaagtacttcccttcctcgctctca
ccagtgacgccctcagagtccctgccaaggccccgccggccactgcccac
ccaacagcagccacagccatcacagaagttagggcgcatccgtgaagatg
agggggcagtggcgtcatcaacagtcaaggagcctcctgaggctacagcc
acacctgagccactctcagatgaggaccta

Hereâs the output of the program:

 -------Reading Frame 1--------

 RWRR_GVLGALGRPPTGLQRRRRMGPAQ_EYAAWEA_LEAEVVVGAFATAWDAAEWSVQVRGSLAGVVRE
 CAGSGDMEGDGSDPEPPDAGEDSKSENGENAPIYCICRKPDINCFMIGCDNCNEWFHGDCIRITEKMAKA
 IREWYCRECREKDPKLEIRYRHKKSRERDGNERDSSEPRDEGGGRKRPVPDPDLQRRAGSGTGVGAMLAR
 GSASPHKSSPQPLVATPSQHHQQQQQQIKRSARMCGECEACRRTEDCGHCDFCRDMKKFGGPNKIRQKCR
 LRQCQLRARESYKYFPSSLSPVTPSESLPRPRRPLPTQQQPQPSQKLGRIREDEGAVASSTVKEPPEATA
 TPEPLSDEDL

A few comments are in order. First, the subroutines for translating codons are in the Geneticcode module. They include the hash %genetic_code and the subroutines codon2aa, dna2peptide, and translate_frame, which are involved with translating DNA data to peptides. The subroutines for reading sequence data in from files, and for formatting and printing it to the screen, are in the SequenceIO module. They are the subroutines get_file_data, extract_sequence_from_fasta_data, and print_sequence.

Now, we have two modules and code that exercises them; letâs look at some more facets of using modules.

Using Modules

So far, the benefit of modules may seem questionable. You may be wondering what the advantage is over simple libraries (without package declarations), since the main result seems to be the necessity to refer to subroutines in the modules with longer names!

Exporting Names

Thereâs a way to avoid lengthy module names and still use the short ones if you place a call to the special Exporter module in the module code and modify the use MODULE declaration in the calling code.

Going back to the first example Geneticcode.pm module, recall it began with this line:

package Geneticcode;

and included the definition for the hash genetic_code and the subroutine codon2aa.

If you add these lines to the beginning of the file, you can export the symbol names of variables or subroutines in the module into the namespace of the calling program. You can then use the convenient short names for things (e.g., codon2aa instead of Geneticcode::codon2aa). Hereâs a short example of how it works (try typing perldoc Exporter to see the whole story):

package Geneticcode;

require Exporter;
@ISA = qw(Exporter);

@EXPORT_OK = qw(...);         # symbols to export on request

Hereâs how to export the name codon2aa from the module only when explicitly requested:

@EXPORT_OK = qw(codon2aa);    # symbols to export on request

The calling program then has to explicitly request the codon2aa symbol like so:

use Geneticcode qw(codon2aa);

If you use this approach, the calling program can just say:

codon2aa($codon);

instead of:

Geneticcode::codon2aa($codon);

The Exporter module thatâs included in the standard Perl distribution has several other optional behaviors, but the way just shown is the safest and most useful. As youâll see, the object-oriented programming style of using modules doesnât use the Export facility, but it is a useful thing to have in your bag of tricks. For more information about exporting (such as why exporting is also known as âpolluting your namespaceâ), see the Perl documentation for the Exporter module (by typing perldoc Exporter at a command line or by going to the http://www.perldoc.com web page).

CPAN Modules

The Comprehensive Perl Archive Network (CPAN, http://www.cpan.org) is an impressively large collection of Perl code (mostly Perl modules). CPAN is easily accessible and searchable on the Web, and you can use its modules for a variety of programming tasks.

By now you should have the basic idea of how modules are defined and used, so letâs take some time to explore CPAN to see what goodies are available.

There are two important points about CPAN. First, a large number of the things you might want your programs to do have already been programmed and are easily obtained in downloadable modules. You just have to go find them at CPAN, install them on your computer, and call them from your program. Weâll take a look at an example of exactly that in this section.

Second, all code on CPAN is free of charge and available for use by a very unrestrictive copyright declaration. Sound good? Keep reading.

CPAN includes convenient ways to search for useful modules, and thereâs a CPAN.pm module built-in with Perl that makes downloading and installing modules quite easy (when things work well, which they usually do). If you canât find CPAN.pm, you should consider updating your current version.

You can find more information by typing the following at the command line:

perldoc CPAN

You can also check the Frequently Asked Questions (FAQ) available at the CPAN web site.

Whatâs Available at CPAN?

The CPAN web site offers several âviewsâ of the CPAN collection of modules and several alternate ways of searching (by module name, category, full text search of the module documentation, etc.). Here is the top-level organization of the modules by overall category:

Development Support
Operating System Interfaces
Networking Devices IPC
Data Type Utilities
Database Interfaces
User Interfaces
Language Interfaces
File Names Systems Locking
String Lang Text Proc
Opt Arg Param Proc
Internationalization Locale
Security and Encryption
World Wide Web HTML HTTP CGI
Server and Daemon Utilities
Archiving and Compression
Images Pixmaps Bitmaps
Mail and Usenet News
Control Flow Utilities
File Handle Input Output
Microsoft Windows Modules
Miscellaneous Modules
Commercial Software Interfaces
Not In Modulelist

Searching CPAN

CPANâs main web page has a few ways to search the contents. Letâs say you need to perform some statistics and are looking for code thatâs already available. Weâll go through the steps necessary to search for the code, download and install it, and use the module in a program.

At the main CPAN page, look for âsearchingâ and click on search.cpan.org. If you search for âstatisticsâ in all locations, youâll get over 300 hits, so you should restrict your search to modules with the pull-down menu. Youâll get 25 hits (more by the time you read this); hereâs what youâll see:

1.  Statistics::Candidates
Statistics-MaxEntropy-0.9 - 26 Nov 1998 - Hugo WL ter Doest

2. Statistics::ChiSquare
How random is your data?
Statistics-ChiSquare-0.3 - 23 Nov 2001 - Jon Orwant

3. Statistics::Contingency
Calculate precision, recall, F1, accuracy, etc.
Statistics-Contingency-0.03 - 09 Aug 2002 - Ken Williams

4. Statistics::DEA
Discontiguous Exponential Averaging
Statistics-DEA-0.04 - 17 Aug 2002 - Jarkko Hietaniemi

5. Statistics::Descriptive
Module of basic descriptive statistical functions.
Statistics-Descriptive-2.4 - 26 Apr 1999 - Colin Kuskie

6. Statistics::Distributions
Perl module for calculating critical values of common statistical distributions
Statistics-Distributions-0.07 - 22 Jun 2001 - Michael Kospach

7. Statistics::Frequency
simple counting of elements
Statistics-Frequency-0.02 - 24 Apr 2002 - Jarkko Hietaniemi

8. Statistics::GaussHelmert
General weighted least squares estimation
Statistics-GaussHelmert-0.05 - 18 Apr 2002 - Stephan Heuel

9. Statistics::LTU
An implementation of Linear Threshold Units
Statistics-LTU-2.8 - 27 Feb 1997 - Tom Fawcett

10. Statistics::Lite
Small stats stuff.
Statistics-Lite-1.02 - 15 Apr 2002 - Brian Lalonde 

11.  Statistics::MaxEntropy
Statistics-MaxEntropy-0.9 - 26 Nov 1998 - Hugo WL ter Doest

12. Statistics::OLS
perform ordinary least squares and associated statistics, v 0.07.
Statistics-OLS-0.07 - 13 Oct 2000 - Sanford Morton

13. Statistics::ROC
receiver-operator-characteristic (ROC) curves with nonparametric confidence bounds
Statistics-ROC-0.01 - 22 Jul 1998 - Hans A. Kestler

14. Statistics::Regression
weighted linear regression package (line+plane fitting)
StatisticsRegression - 26 May 2001 - ivo welch

15. Statistics::SparseVector
Perl5 extension for manipulating sparse bitvectors
Statistics-MaxEntropy-0.9 - 26 Nov 1998 - Hugo WL ter Doest

16. Statistics::Descriptive::Discrete
Compute descriptive statistics for discrete data sets.
Statistics-Descriptive-Discrete-0.07 - 13 Jun 2002 - Rhet Turnbull

17. Bio::Tree::Statistics
Calculate certain statistics for a Tree
bioperl-1.0.2 - 16 Jul 2002 - Ewan Birney

18. Device::ISDN::OCLM::Statistics
OCLM statistics superclass
Device-ISDN-OCLM-0.40 - 02 Jan 2000 - Merlin Hughes

19. Device::ISDN::OCLM::CurrentStatistics
OCLM current call statistics
Device-ISDN-OCLM-0.40 - 02 Jan 2000 - Merlin Hughes

20. Device::ISDN::OCLM::ISDNStatistics
OCLM ISDN statistics
Device-ISDN-OCLM-0.40 - 02 Jan 2000 - Merlin Hughes 

21.  Device::ISDN::OCLM::Last10Statistics
OCLM Last10 call statistics
Device-ISDN-OCLM-0.40 - 02 Jan 2000 - Merlin Hughes

22. Device::ISDN::OCLM::LastStatistics
OCLM last call statistics
Device-ISDN-OCLM-0.40 - 02 Jan 2000 - Merlin Hughes

23. Device::ISDN::OCLM::ManualStatistics
OCLM manual call statistics
Device-ISDN-OCLM-0.40 - 02 Jan 2000 - Merlin Hughes

24. Device::ISDN::OCLM::SPStatistics
OCLM service provider statistics
Device-ISDN-OCLM-0.40 - 02 Jan 2000 - Merlin Hughes

25. Device::ISDN::OCLM::SystemStatistics
OCLM system statistics
Device-ISDN-OCLM-0.40 - 02 Jan 2000 - Merlin Hughes

Letâs check out the Statistics::ChiSquare module.

First, click on the link to Statistics::ChiSquare; youâll see a summary of the module, complete with a description, overview, discussion of the method, examples of use, and information about the author.

One of the modules looks interesting; letâs download and install it. How big is the source code? If you click on the source link, youâll find that the module is really just one short subroutine with the documentation defined right in the module. Hereâs the subroutine definition part of the module:

package Statistics::ChiSquare;

# ChiSquare.pm
#
# Jon Orwant, orwant@media.mit.edu
#
# 31 Oct 95, revised Mon Oct 18 12:16:47 1999, and again November 2001
# to fix an off-by-one error
#
# Copyright 1995, 1999, 2001 Jon Orwant.  All rights reserved.
# This program is free software; you can redistribute it and/or
# modify it under the same terms as Perl itself.
# 
# Version 0.3.  Module list status is "Rdpf"

use strict;
use vars qw($VERSION @ISA @EXPORT);

require Exporter;
require AutoLoader;

@ISA = qw(Exporter AutoLoader);
# Items to export into callers namespace by default. Note: do not export
# names by default without a very good reason. Use EXPORT_OK instead.
# Do not simply export all your public functions/methods/constants.
@EXPORT = qw(chisquare);

$VERSION = '0.3';

my @chilevels = (100, 99, 95, 90, 70, 50, 30, 10, 5, 1);
my %chitable = (  );

# assume the expected probability distribution is uniform
sub chisquare {
    my @data = @_;
    @data = @{$data[0]} if @data =  = 1 and ref($data[0]);
    my $degrees_of_freedom = scalar(@data) - 1;
    my ($chisquare, $num_samples, $expected, $i) = (0, 0, 0, 0);
    if (! exists($chitable{$degrees_of_freedom})) {
        return "I can't handle ", scalar(@data), 
        " choices without a better table.";
    }
    foreach (@data) { $num_samples += $_ }
    $expected = $num_samples / scalar(@data);
    return "There's no data!" unless $expected;
    foreach (@data) {
        $chisquare += (($_ - $expected) ** 2) / $expected;
    }
    foreach (@{$chitable{$degrees_of_freedom}}) {
        if ($chisquare < $_) {
            return
             "There's a <$chilevels[$i+1]% and <$chilevels[$i]% chance that this data 
                    is random.";
        }
        $i++;
    }
    return "There's a <$chilevels[$#chilevels]% chance that this data is random.";
}
$chitable{1} = [0.00016, 0.0039, 0.016, 0.15, 0.46, 1.07, 2.71, 3.84, 6.64];
$chitable{2} = [0.020,   0.10,   0.21,  0.71, 1.39, 2.41, 4.60, 5.99, 9.21];
$chitable{3} = [0.12,    0.35,   0.58,  1.42, 2.37, 3.67, 6.25, 7.82, 11.34];
$chitable{4} = [0.30,    0.71,   1.06,  2.20, 3.36, 4.88, 7.78, 9.49, 13.28];
$chitable{5} = [0.55,    1.14,   1.61,  3.00, 4.35, 6.06, 9.24, 11.07, 15.09];
$chitable{6} = [0.87,    1.64,   2.20,  3.83, 5.35, 7.23, 10.65, 12.59, 16.81];
$chitable{7} = [1.24,    2.17,   2.83,  4.67, 6.35, 8.38, 12.02, 14.07, 18.48];
$chitable{8} = [1.65,    2.73,   3.49,  5.53, 7.34, 9.52, 13.36, 15.51, 20.09];
$chitable{9} = [2.09,    3.33,   4.17, 6.39, 8.34, 10.66, 14.68, 16.92, 21.67];
$chitable{10} = [2.56,   3.94,   4.86, 7.27, 9.34, 11.78, 15.99, 18.31, 23.21];
$chitable{11} = [3.05,   4.58,  5.58, 8.15, 10.34, 12.90, 17.28, 19.68, 24.73];
$chitable{12} = [3.57,   5.23, 6.30, 9.03, 11.34, 14.01, 18.55, 21.03, 26.22];
$chitable{13} = [4.11,   5.89, 7.04, 9.93, 12.34, 15.12, 19.81, 22.36, 27.69];
$chitable{14} = [4.66,   6.57, 7.79, 10.82, 13.34, 16.22, 21.06, 23.69, 29.14];
$chitable{15} = [5.23,   7.26, 8.55, 11.72, 14.34, 17.32, 22.31, 25.00, 30.58];
$chitable{16} = [5.81,   7.96, 9.31, 12.62, 15.34, 18.42, 23.54, 26.30, 32.00];
$chitable{17} = [6.41,  8.67, 10.09, 13.53, 16.34, 19.51, 24.77, 27.59, 33.41];
$chitable{18} = [7.00,  9.39, 10.87, 14.44, 17.34, 20.60, 25.99, 28.87, 34.81];
$chitable{19} = [7.63, 10.12, 11.65, 15.35, 18.34, 21.69, 27.20, 30.14, 36.19];
$chitable{20} = [8.26, 10.85, 12.44, 16.27, 19.34, 22.78, 28.41, 31.41, 37.57];

1;

Some of this code will look familiar; some may not. Check out the use of package, use strict, and require Exporter; theyâre parts of Perl youâve just seen.

Youâll also see references to version, Autoloader, use vars, and an initialization of a multidimensional array chitable, which will be covered later. For now, you may want to take a quick read-through of the code and get some personal satisfaction at how much of it makes sense.

Indeed, one of the really nice things about most modules is that you donât really have to read the code very often. Usually you can just install the module, read enough of the documentation to see how to call it from your program, and youâre off and running. Letâs take that approach now.

Installing Modules Using CPAN.pm

Our next task is to install the module using CPAN.pm. This section contains a log from when I installed Statistics::ChiSquare on my Linux computer using CPAN.pm.

In fact, to make things easy, hereâs the section of the CPAN FAQ that addresses installing modules:

How do I install Perl modules?

Installing a new module can be as simple as typing

perl -MCPAN -e 'install Chocolate::Belgian'.

The CPAN.pm documentation has more complete instructions on how to use
this convenient tool.  If you are uncomfortable with having something
take that much control over your software installation, or it otherwise
doesn't work for you, the perlmodinstall documentation covers
module installation for UNIX, Windows and Macintosh in more familiar terms.

Finally, if you're using ActivePerl on Windows, the PPM (Perl Package Manager)
has much of the same functionality as CPAN.pm.

The following is my install log. Notice that all I have to do is type a couple of lines, and everything else that follows is automatic!

[tisdall@coltrane tisdall]$ perl -MCPAN -e 'install Statistics::ChiSquare'
CPAN: Storable loaded ok
mkdir /root/.cpan: Permission denied at /usr/local/lib/perl5/5.6.1/CPAN.pm line 2218
[tisdall@coltrane tisdall]$ su
Password: 
[root@coltrane tisdall]# perl -MCPAN -e 'install Statistics::ChiSquare'
CPAN: Storable loaded ok
Going to read /root/.cpan/Metadata
  Database was generated on Wed, 20 Mar 2002 00:39:29 GMT
CPAN: LWP::UserAgent loaded ok
Fetching with LWP:
  ftp://cpan.cse.msu.edu/authors/01mailrc.txt.gz
Going to read /root/.cpan/sources/authors/01mailrc.txt.gz
CPAN: Compress::Zlib loaded ok
Fetching with LWP:
  ftp://cpan.cse.msu.edu/modules/02packages.details.txt.gz
Going to read /root/.cpan/sources/modules/02packages.details.txt.gz
  Database was generated on Mon, 26 Aug 2002 00:22:07 GMT

  There's a new CPAN.pm version (v1.62) available!
  [Current version is v1.59_54]
  You might want to try
    install Bundle::CPAN
    reload cpan
  without quitting the current session. It should be a seamless upgrade
  while we are running...

Fetching with LWP:
  ftp://cpan.cse.msu.edu/modules/03modlist.data.gz
Going to read /root/.cpan/sources/modules/03modlist.data.gz
Going to write /root/.cpan/Metadata
Running install for module Statistics::ChiSquare
Running make for J/JO/JONO/Statistics-ChiSquare-0.3.tar.gz
Fetching with LWP:
  ftp://cpan.cse.msu.edu/authors/id/J/JO/JONO/Statistics-ChiSquare-0.3.tar.gz
CPAN: MD5 loaded ok
Fetching with LWP:
  ftp://cpan.cse.msu.edu/authors/id/J/JO/JONO/CHECKSUMS
Checksum for /root/.cpan/sources/authors/id/J/JO/JONO/Statistics-ChiSquare-0.3.
     tar.gz ok
Scanning cache /root/.cpan/build for sizes
Deleting from cache: /root/.cpan/build/IO-stringy-2.108 (21.4>20.0 MB)
Deleting from cache: /root/.cpan/build/XML-Node-0.11 (20.8>20.0 MB)
Deleting from cache: /root/.cpan/build/bioperl-0.7.2 (20.7>20.0 MB)
Statistics/ChiSquare-0.3/
Statistics/ChiSquare-0.3/ChiSquare.pm
Statistics/ChiSquare-0.3/Makefile.PL
Statistics/ChiSquare-0.3/test.pl
Statistics/ChiSquare-0.3/Changes
Statistics/ChiSquare-0.3/MANIFEST
Package seems to come without Makefile.PL.
  (The test -f "/root/.cpan/build/Statistics/Makefile.PL" returned false.)
  Writing one on our own (setting NAME to StatisticsChiSquare)

  CPAN.pm: Going to build J/JO/JONO/Statistics-ChiSquare-0.3.tar.gz

Checking if your kit is complete...
Looks good
Writing Makefile for Statistics::ChiSquare
Writing Makefile for StatisticsChiSquare
make[1]: Entering directory `/root/.cpan/build/Statistics/ChiSquare-0.3'
cp ChiSquare.pm ../blib/lib/Statistics/ChiSquare.pm
AutoSplitting ../blib/lib/Statistics/ChiSquare.pm (../blib/lib/auto/
     Statistics/ChiSquare)
Manifying ../blib/man3/Statistics::ChiSquare.3
make[1]: Leaving directory `/root/.cpan/build/Statistics/ChiSquare-0.3'
  /usr/bin/make  -- OK
Running make test
make[1]: Entering directory `/root/.cpan/build/Statistics/ChiSquare-0.3'
make[1]: Leaving directory `/root/.cpan/build/Statistics/ChiSquare-0.3'
make[1]: Entering directory `/root/.cpan/build/Statistics/ChiSquare-0.3'
PERL_DL_NONLAZY=1 /usr/bin/perl -I../blib/arch -I../blib/lib -I/usr/local/lib/
     perl5/5.6.1/i686-linux -I/usr/local/lib/perl5/5.6.1 test.pl
1..2
ok 1
ok 2
make[1]: Leaving directory `/root/.cpan/build/Statistics/ChiSquare-0.3'
  /usr/bin/make test -- OK
Running make install
make[1]: Entering directory `/root/.cpan/build/Statistics/ChiSquare-0.3'
make[1]: Leaving directory `/root/.cpan/build/Statistics/ChiSquare-0.3'
Installing /usr/local/lib/perl5/site_perl/5.6.1/Statistics/ChiSquare.pm
Installing /usr/local/lib/perl5/site_perl/5.6.1/auto/Statistics/ChiSquare/
     autosplit.ix
Installing /usr/local/man/man3/Statistics::ChiSquare.3
Writing /usr/local/lib/perl5/site_perl/5.6.1/i686-linux/auto/
    StatisticsChiSquare/.packlist
Appending installation info to /usr/local/lib/perl5/5.6.1/i686-linux/perllocal.pod
  /usr/bin/make install UNINST=1 -- OK
[root@coltrane tisdall]#

This may seem like a confusing amount of output, but, again, all you have to do is type a couple of lines, and the installation follows automatically.

You may get something like the following message when you try to install a CPAN module:

[tisdall@coltrane tisdall]$ perl -MCPAN -e 'install Statistics::ChiSquare'
CPAN: Storable loaded ok
mkdir /root/.cpan: Permission denied at /usr/local/lib/perl5/5.6.1/CPAN.pm line 2218

As you can see, it didnât work, and it produced an error message. On Unix machines, itâs often necessary to become root to install things.^[2] In that case, use the Unix su command and try the CPAN command again:

[tisdall@coltrane tisdall]$ su
Password: 
[root@coltrane tisdall]# perl -MCPAN -e 'install Statistics::ChiSquare'

Great, it worked. If you look over the rather verbose output, youâll see that it finds the module, installs it, tests it, and logs the installation.

Pretty easy, huh?

Itâs usually this easy, but not always. Occasionally, errors result, and the module may not be installed. In that case, the error messages may be enough to explain the problem; for instance, the module may depend on another module you have to install first. Another problem is that some modules havenât been tested on, or even designed to work on, all operating systems; if you try to install a Windows-specific module on Linux, it is likely to complain. In extreme cases, the module documentation usually provides the authorâs email address.

Using the Newly Installed CPAN Module

Now comes the payoff. Letâs look again at the documentation for the module and see if we can use it from our own Perl code.

Now that the module is installed, you can see the documentation by typing:

perldoc Statistics::ChiSquare

You can also simply go back to the web documentation found at http://search.cpan.org. Either way, youâll find the following example using this ChiSquare module:

NAME
       "Statistics::ChiSquare" - How random is your data?

SYNOPSIS
        use Statistics::Chisquare;
        print chisquare(@array_of_numbers);
        Statistics::ChiSquare is available at a CPAN site near
        you.

DESCRIPTION
        Suppose you flip a coin 100 times, and it turns up heads
        70 times.  Is the coin fair?
        Suppose you roll a die 100 times, and it shows 30 sixes.
        Is the die loaded?
        In statistics, the chi-square test calculates "how random"
        a series of numbers is.  But it doesn't simply say "yes"
        or "no".  Instead, it gives you a confidence interval,
        which sets upper and lower bounds on the likelihood that
        the variation in your data is due to chance.  See the
        examples below.
...

The documentation continues with more discussion and some concrete examples that use the module and interpret the results.

Very often, the SYNOPSIS part of the documentation is all you need to look at. It shows you specific examples of how to call the code in the module. In this case, because itâs a very simple module, there is just one subroutine that can be used. As you see from the documentation excerpt, you just need to pass the chisquare subroutine an array of numbers and print out the return value to use the code. Letâs try it. Weâll take as our input an array of numbers that corresponds to the stops of the Broadway-7th Avenue local subway train on the west side of Manhattan, from 14th Street up to 137th Street in Harlem. (Weâll assume you didnât run fast enough and missed the A train.) Letâs see how random these stops really are:

use strict;
use warnings;

use Statistics::ChiSquare;

my(@subwaystops) = (14, 18, 23, 28, 34, 42, 50, 59, 66, 72, 79, 86, 96, 103, 110, 
116, 125, 137);

print chisquare(@subwaystops);

This produces the output:

There's a <1% chance that this data is random.

(Knowing firsthand the feelings of long-suffering New York City Subway riders, I predict that this result might provoke some spirited discussion. Nevertheless, we seem to have working code.)

Problems with CPAN Modules

Actually, the sharp-eyed reader may have noticed a problem in our mad dash uptown. In the first line of the SYNOPSIS section, thereâs the following:

use Statistics::Chisquare;

The name of the module is spelled Chisquare, whereas in all other places in the documentation the module is spelled ChiSquare with a capital S. In Perl, the case of a letter, uppercase or lowercase, is important, and this looks suspiciously like a typographical error in the documentation. If you try use Statistics::Chisquare, youâll discover that the module canât be found, whereas if you try use Statistics::ChiSquare, the module is there. This is a minor bug, but some modules have poor documentation, and it can be a time-consuming problem, especially if you are forced to wade into the module code or try various tests, to figure out how the module works.

Apart from bugs, Iâve also mentioned the problem that some modules are not tested, or designed, for all operating systems. In addition, many modules require other modules to be present. Itâs possible to configure CPAN to automatically install all the required modules a requested module uses, as described in the CPAN documentation, but you may need to intervene personally. Itâs useful to remember that if you have a program that uses a certain module running on one computer, and you move the program to another computer, you may have to install the required modules on the new computer as well.

Saving the worst for last, itâs also important to remember that contributing to CPAN is open to one and all, and not all the code there is well-written or well-tested. The heavily used modules are, but counterexamples can be found. So, donât bet the farm on your code just because it uses a CPAN module; you should still carefully read the documentation for the module and test your program.

The CPAN FAQ explains in detail the way to be a good citizen when it comes to testing and reporting bugs that you discover in CPAN code.

Exercises

Exercise 1.1

What are the problems that might arise when dividing program code into separate module files?

Exercise 1.2

What are the differences between libraries, modules, packages, and namespaces?

Exercise 1.3

Write a module that finds modules on your computer.

Exercise 1.4

Where do the standard Perl distribution modules live on your computer?

Exercise 1.5

Research how Perl manages its namespaces.

Exercise 1.6

When might it be necessary to export names from a module? When might it be useful? When might it be convenient? When might it be a very bad idea?

Exercise 1.7

The program testGeneticcode contains the following loop:

# Translate each three-base codon to an amino acid, and append to a protein 
for(my $i=0; $i < (length($dna) - 2) ; $i += 3) {
        $protein .= Geneticcode::codon2aa( substr($dna,$i,3) );
}

Hereâs another way to accomplish that loop:

# Translate each three-base codon to an amino acid, and append to a protein 
my $i=0;
while (my $codon = substr($dna, $i += 3, 3) ) {
        $protein .= Geneticcode::codon2aa( $codon );
}

Compare the two methods. Which is easier to understand? Which is easier to maintain? Which is faster? Why?

Exercise 1.8

The subroutine codon2aa causes the entire program to halt when it encounters a âbadâ codon in the data. Often (usually) it is best for a subroutine to return some indication that it encountered a problem and let the calling program decide how to handle it. It makes the subroutine more generally useful if it isnât always halting the program (although that is what you want to do sometimes).

Rewrite codon2aa and the calling program testGeneticcode so that the subroutine returns some errorâperhaps the value undefâand the calling program checks for that error and performs some action.

Exercise 1.9

Write a separate module for each of the following: reading a file, extracting FASTA sequence data, and printing sequence data to the screen.

Exercise 1.10

Download, install, and use a module from CPAN.

^[1]Perl libraries were traditionally put in files ending with .pl, which stands for perl library; the term library is also used to refer to a collection of Perl modules. The common denominator is that a library is a collection of reusable subroutines.

^[2]You may need to contact your system administrator about getting root permission. The CPAN documentation discusses how to do a non-root installation. If youâre not on a Unix or Linux machine and are using ActiveStateâs Perl on a Windows machine, for instance, you need to consult that documentation.

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Mastering Perl for Bioinformatics by James Tisdall

ChapterÂ 1.Â Modular Programming with Perl

What Is a Module?

Why Perl Modules?

Subroutines and Software Engineering

Modules and Libraries

Namespaces

Namespaces Compared with Scoping: my and use strict

Packages

Defining Modules

Storing Modules

Writing Your First Perl Module

An Example: Geneticcode.pm

Expanding Geneticcode.pm

Using Modules

Exporting Names

CPAN Modules

Whatâs Available at CPAN?

Searching CPAN

Installing Modules Using CPAN.pm

Using the Newly Installed CPAN Module

Problems with CPAN Modules

Exercises

Don’t leave empty-handed

It’s yours, free.

Check it out now on O’Reilly

ChapterÂ 1.Â Modular Programming with Perl

What Is a Module?

Why Perl Modules?

Subroutines and Software Engineering

Modules and Libraries

Namespaces

Namespaces Compared with Scoping: my and use strict

Packages

Defining Modules

Storing Modules

Writing Your First Perl Module

An Example: Geneticcode.pm

Expanding Geneticcode.pm

Using Modules

Exporting Names

CPAN Modules

Whatâs Available at CPAN?

Searching CPAN

Installing Modules Using CPAN.pm

Using the Newly Installed CPAN Module

Problems with CPAN Modules

Exercises

Don’t leave empty-handed

It’s yours, free.

Check it out now on O’Reilly

Whatâs Available at CPAN?