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PERL. subroutines and references. Andrew Emerson, High Performance Systems, CINECA. # Counts Gs in various bits of DNA $dna=“CGGTAATTCCTGCA”; $G_count=0; for ($pos=0; $pos <length $dna; $pos++) { $base=substr($dna,$pos,1); ++$G_count if ($base eq ‘G’); } # end for

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slide1

PERL

subroutines and references

Andrew Emerson, High Performance Systems, CINECA

slide2

# Counts Gs in various bits of DNA

$dna=“CGGTAATTCCTGCA”;

$G_count=0;

for ($pos=0; $pos <length $dna; $pos++) {

$base=substr($dna,$pos,1);

++$G_count if ($base eq ‘G’);

} # end for

. . .do something else

$new_dna = <DNA_FILE>;

$G_count=0;

for ($pos=0; $pos <length $new_dna; $pos++) {

$base=substr($new_dna,$pos,1);

++$G_count if ($base eq ‘G’);

} # end for

Consider the following code:

slide3

count_G subroutine

for ($pos=0;$pos<length $dna; $pos++) {

$base=substr($dna,$pos,1);

++$G_count if ($base eq ‘G’);

}

Inconvenient to repeat pieces of code many times if it does the same thing.

Better if we could write something like…

# Counts Gs in various bits of DNA # improved version (PSEUDO PERL)

# Main program

$dna=“CGGTAATTCCTGCA”;

count_gusing $dna;

.

. do something else

$new_dna = <DNA_FILE>;

count_g using $new_dna;

.

.

subroutines
Subroutines
  • The pieces of code used in this way are often called subroutines and are common to all programming languages (but with different names)
    • subroutines (Perl, FORTRAN)
    • functions (C,C++*,FORTRAN, Java*)
    • procedures (PASCAL)
  • Essential for procedural or structured programming.

*object-oriented programming languages

advantages of using subroutines
Advantages of using subroutines
  • Saves typing → fewer lines of code →less likely to make a mistake
  • re-usable
    • if subroutine needs to be modified, can be changed in only one place
    • other programs can use the same subroutine
    • can be tested separately
  • makes the overall structure of the program clearer
program design using subroutines conceptual flow
Program design using subroutinesConceptual flow

subroutines can use other subroutines to make more complex and flexible programs

program design using subroutines pseudo code
Program design using subroutines-pseudo code

#

# Main program

# pseudo-code

..set variables

.

call sub1

.

call sub2

.

call sub3

.

exit program

sub 1

# code for sub 1

exit subroutine

sub 2

# code for sub 1

exit subroutine

sub 4

# code sub4

exit

sub 3

# code for sub 1

call sub 4

exit subroutine

using subroutines in perl
Using subroutines in Perl

Example 1.

# Program to count Gs in DNA sequences

# (valid perl)

# Main program

$dna=“GGCCTAACCTCCGGT”;

count_G;

print “no. of G in $dna=$number_of_g\n”;

# subroutines

sub count_G {

for ($pos=0;$pos<length $dna; $pos++) {

$base=substr($dna,$pos,1);

++$number_of_g if ($base eq ‘G’);

} # end for

}

subroutines in perl
Subroutines in Perl
  • Defined using the sub command:
    • sub name {
    • ...
    • }
  • Called from the main program or another subroutine using its name:
    • name;
  • Sometimes you will see in old Perl programs (like mine)
    • &name;
  • But is optional in modern Perl.
subroutines in perl10
Subroutines in Perl

Subroutines can be placed anywhere in the program but best to group them at the end;

# main program

.

.

exit;

# subroutines defined here

sub sub1 {

...

}

sub sub2 {

...

}

sub sub3 {

...

}

exitnot strictly necessary, but makes it clear we want to leave the program here.

return to example 1 why is this bad

What does count_G need ?

Where did this come from?

Return to example 1- why is this bad?

# Program to count Gs in DNA sequences

# Main program

$dna=“GGCCTAACCTCCGGT”;

count_G;

print “no. of G in $dna=$number_of_g\n”;

exit;

# subroutines

sub count_G {

for ($pos=0;$pos<length $dna; $pos++) {

$base=substr($dna,$pos,1);

++$number_of_g if ($base eq ‘G’);

}# end for

}

perl subroutines passing parameters
Perl subroutines-passing parameters

The input/outputs to a subroutine are specified using parameters(or arguments) given when the subroutine is called:

$no_of_G = count_g($dna);

It is now clear what the subroutine expects as input and what is returned as output.

Other examples:

$day_in_yr = calc_day($day,$month);

$num_sequences = read_sequence_file(@database);

input parameters
Input parameters

All the parameters in a Perl subroutine (including arrays) end up in a single array called @_

Therefore in the code:

#

$pos = find_motif($motif,$protein);

.

sub find_motif {

$a = $_[0];

$b = $_[1];

...

}

$a takes the value of $motif

$b takes the value of $protein

subroutine output return values
Subroutine output (return values)

A subroutine does not have to explicitly return something to the main program:

print_title;

sub print_title{

print “Sequence Manipulation program\n”;

print “-----------------------------\n”;

print “Written by: A.Nother \n”;

print “Version 1.1: \n”

}

but often it does, even if only to signal the procedure went well or gave an error.

subroutine return values
Subroutine return values

By default the subroutine returns the last thing evaluated but you can use the return statement to make this explicit:

input

sub count_G {

$dna=@_[0];

for ($pos=0;$pos<length $dna; $pos++) {

$base=substr($dna,$pos,1);

++$number_of_g if ($base eq ‘G’);

} # end for

return $number_of_g;

}

output

returnalso exits the subroutine

return values
Return Values

You can return from more than 1 point in the sub – can be useful for signalling errors:

if ($dna eq “”) {

return “No DNA was given”; #exit with error message

} else {

..

} # end if

return $number_of_G;

} # end sub

In which case the calling program or sub should check the return value before continuing. However, in general best to return from only 1 point, otherwise difficult to follow the logic.

return values17
Return values

Can also return multiple scalars, arrays, etc. but just as for the input everything ends up in a single array or list:

@DNA = read_file($filename);

.

.

($nG,$nC,$nT,$nA) = count_bases($dna);

sub count_bases {

...

return ($num_G,$num_C,$num_T,$num_A);

} # end sub

note ( and ) for the list

counting bases attempt 2

All the variables inside are also visible outside the sub, not only params !

Counting bases – Attempt 2

# Program to count Gs in DNA sequences

# using input/output parameters

# Main program

$dna=“GGCCTAACCTCCGGT”;

$num_g = count_G($dna);

print “no. of G in $dna=$num_g\n”;

exit;

# subroutines

sub count_G {

$dna=$_[0];

for ($pos=0;$pos<length $dna; $pos++) {

$base=substr($dna,$pos,1);

++$number_of_g if ($base eq ‘G’);

} # end for

return $number_of_g; # return value of sub

}

better but ...

the need for variable scoping
The need for variable scoping

A subroutine written this way is in danger of overwriting a variable used elsewhere in the program. Remember that a subroutine should work like a black box, apart from well-defined inputs/outputs it should not affect the rest of the program.

input

Apart from input/output all vars needed by the sub should appear and disappear within the sub.

Allows us also to use the same names for vars outside and inside the sub without conflict.

sub

output

variable scoping in perl
Variable scoping in Perl
  • By default, all variables defined outside a sub are visible within it and vice-versa – all variables are global.
  • Therefore the sub can change variables used outside the sub.
  • Solution ?
    • Restrict the scope of the variables by making them local to the subroutine → eliminate the risk of altering a variable present outside the sub. Also makes it clear what the subroutine needs to function.

.

variable scoping in perl21

Attenzione

Must use () if multiple vars per line

my $seq1, $seq2;

This means

my $seq1;

$seq2;

Which is valid Perl so the compiler won’t give an error.

Variable scoping in Perl

In Perl, variables are made local to a subroutine (or a block) using the my keyword. For example,

my variable1; # simple declaration

my $dna=“GGTTCACCACCTG”; # with initialization

my ($seq1,$seq2,$seq3); # more than 1

subroutines with local variables
Subroutines with local variables

# Program to count Gs in DNA sequences – final version

# Main program

$dna=“GGCCTAACCTCCGGT”;

$num_g = count_G($dna);

print “no. of G in $dna=$num_g\n”;

exit;

# subroutines

sub count_G {

my $dna=$_[0];

my ($pos,$base);

my $number_of_g=0;

for ($pos=0;$pos<length $dna; $pos++) {

$base=substr($dna,$pos,1);

++$number_of_g if ($base eq ‘G’);

} # end for

return $number_of_g; # return value

}

Remember that my makes a copy of the variable.

Variables declared like this is called lexical scoping in the Perl man pages

other examples of subroutine use
Other examples of subroutine use

sub find_motif {

my $motif=shift; # shifts the @_ array

my $protein=shift; # (avoids $_[0], etc)

...

}

sub count_C_and_G {

my @fasta_lines=@_;

...

return ($num_C,$num_G);# returns a list

}

sub reverse_seq {

use strict;#the strict command enforces use of my

$seq=$_[0];# so this line will give an error (even # if defined in main program)

...

}

question what if we want to pass two arrays
Question: What if we want to pass two arrays ?

$seq=compare_seqs(@seqs1,@seqs2);

Remember that everything arrives in the sub in a single array. Likewise for return values:

(@annotations,@dna) = parse_genbank(@dna);

...

sub parse_genbank {

...

return (@annotations,@dna);

}

In the first example what will be in the special array @_ ?

Solution: Use references

subroutines call by value
subroutines – call by value

Consider

$i=2;

$j =add_100($i);

print “i=$i\n”;

sub simple_sub {

my $i=$_[0];

$i=$i+100;

return $i;

}

$i unaffected by the subroutine

This is called Call by Value because a copy is made of the parameter passed and whatever happens to this copy in the subroutine doesn’t affect the variable in the main program

what are references
What are references?

References can be considered to be an identifier or some other description of the objects rather than the objects themselves.

reference

To buy

Bananas

Beer

Fruit

Pasta

Frozen pizza

more beer

or

copy

references
References

In computing, references are often addresses of objects (scalars,arrays,..) in memory:

@genbank

$dna

100 200 300

address of $dna scalar

address of @genbank array

references28
References
  • References (sometimes called pointers in other languages) can be more convenient because in Perl they are scalars→ often much smaller than the object they refer to (e.g. an array or hash).
  • Array references can be passed around and copied very efficiently, often also using less memory.
  • Being scalars, they can be used to make complicated data structures such as arrays of arrays, arrays of hashes and so on..
references in perl
References in Perl

Simplest way to create a reference in Perl is with \

$scalar_ref = \$sequence; # reference to a scalar

$dna_ref = \@DNA_list; # reference to an array

$hash_ref = \%genetic_code; # reference to a hash

To get back the original object the reference needs to be dereferenced;

$scalar = $$scalar_ref; # for scalars just add $

@new_dna = @$dna_ref; # for arrays just add @

%codon_lookup = %$hash_ref; # similary for hashes

passing two arrays into a sub using references
Passing two arrays into a sub using references

# compare two databases, each held as an array

#

$results = compare_dbase(\@dbase1,\@dbase2); # supply refs

...

sub compare_dbase {

my ($db1_ref,$db2_ref) = @_;#params are refs to arrays

@db1 = @$db1_ref;# dereference

@db2 = @$db2_ref;# dereference

... # now use @db1,@db2

return $results;

}

Similarly we can return 2 or more arrays by the same method

references final words
References – final words

Caution: Calling by reference can change the original variables;

OUTPUT

dna1=G G T C T G A A A A A

dna2=A A A A A

@dna1=(G,G,T,C,T,G);

@dna2=(A,A,A,A,A);

add_seqs(\@dna1,\@dna2);

print “dna1=@dna1 \n dna2=@dna2 \n”;

sub add_seqs {

my ($seq1,$seq2) =@_;

push(@$seq1,$@seq2);

}

If you don’t want this behaviour then create local copies of the arrays as in previous example.

subroutines summary
subroutines-summary
  • subroutines defined with sub represent the main tool for structuring programs in Perl.
  • variables used only by the subroutine should be declared with my, to prevent conflict with external variables (lexical scoping)
  • parameters passed in to the sub end up in the single array @_; similarly for any return values
  • array references need to be used to pass two or more arrays in (call by reference) or out of a sub.