1. subroutines and references
PERL
subroutines and references
Andrew Emerson, High Performance Systems, CINECA

2. Consider the following code:
# 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
$new_dna = <DNA_FILE>;
for ($pos=0; $pos <length $new_dna; $pos++) {
$base=substr($new_dna,$pos,1);

3. Better if we could write something like…
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_g using $dna; .
. do something else
$new_dna = <DNA_FILE>;
count_g using $new_dna;
count_G subroutine
for ($pos=0;$pos<length $dna; $pos++) {
$base=substr($dna,$pos,1);
++$G_count if ($base eq ‘G’); }

4. 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

5. 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

6. Program design using subroutines Conceptual flow
subroutines can use other subroutines to make more complex and flexible programs

7. 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

8. 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 }

9. 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.

10. 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 {
exit not strictly necessary, but makes it clear we want to leave the program here.

11. 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 }
What does count_G need ?
Where did this come from?

12. 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 =

13. Input parameters
All the parameters in a Perl subroutine (including arrays) end up in a single array
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

14. 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.

15. 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 {
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
return also exits the subroutine

16. 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.

17. 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

18. 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 ...
All the variables inside are also visible outside the sub, not only params !

19. 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

20. 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.
scope = l’ambito ? Remember, In addition modifying $_[0], $_[1], ... within the sub will change the original variables .

21. 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
Attenzione
my $seq1, $seq2;
This means
my $seq1;
$seq2;
Which is valid Perl so the compiler won’t give an error.
Must use () if multiple vars per line
how do you say initialise = inizializzare, settare

22. 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

23. Other examples of subroutine use
sub find_motif {
my $motif=shift; # shifts array
my $protein=shift; # (avoids $_[0], etc)
... }
sub count_C_and_G { my
return ($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)

24. Question: What if we want to pass two arrays ?
Remember that everything arrives in the sub in a single array. Likewise for return values: =
...
sub parse_genbank {
return }
In the first example what will be in the special ?
Solution: Use references

25. 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

26. 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

27. References
In computing, references are often addresses of objects (scalars,arrays,..) in memory:
@genbank
$dna
address of $dna scalar
address array

28. 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..

29. References in Perl
Simplest way to create a reference in Perl is with \
$scalar_ref = \$sequence; # reference to a scalar
$dna_ref = # 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 # for arrays just
%codon_lookup = %$hash_ref; # similary for hashes

30. Passing two arrays into a sub using references
# compare two databases, each held as an array #
$results = # supply refs
...
sub compare_dbase {
my ($db1_ref,$db2_ref) # params are refs to arrays
@db1 # dereference
@db2 # dereference
# now use
return $results; }
Similarly we can return 2 or more arrays by the same method

31. 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);
print \n \n”;
sub add_seqs {
my ($seq1,$seq2) }
If you don’t want this behaviour then create local copies of the arrays as in previous example.

32. 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 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.