1. Advanced Perl For Bioinformatics Part 1 2/23/06 1-4pm Module structure Module path Module export Object oriented programming Part 2 2/24/06 1-4pm Bioperl modules Sequence access Sequence manipulation Parsing BLAST records

2. Module and main program package Hello1; sub greet { return "Hello, World!"; } 1; Hello1.pm test1.pl #!/usr/bin/perl use Hello1; print Hello1::greet();

3. Why use module? Reusable by different programs. Keep your code well organized.

4. Module structure package Hello1; sub greet { return "Hello, World!\n"; } 1; Declare a package; file must be saved as Hello.pm Contents of the package: functions, and variables. Return a true value at end

5. Path to module Default path to look for module: @INC perl -e “print @INC” If your module is placed under one of the path in @INC, you can refer to your module use relative path. E.g. If @INC contains /usr/my/lib, and (1)your Mod.pm is /usr/my/lib/Mod.pm, you can refer to your module by “use Mod.pm”. (2)Your Mod.pm is /usr/my/lib/Mymod/Seq/Mod.pm, then you say: use Mymod::Seq::Mod If your module is not placed under any of @INC, e.g. /some/dir/Mod.pm, then: use lib “/some/dir”; --- this adds the path to the beginning of @INC use Mod;

6. Variable scope in module my $var --- accessible only in module our $var --- accessible from outside $var ---same as “our $var” use strict; --- This forces all variables to be qualified with ‘my’ or ‘our’. package Hello2; use strict; our $var1 = 1; my $var2 = 3; my $str = "Hello World!\n"; sub greet { return $str; } 1; Hello2.pm #!/usr/bin/perl use Hello2; print "var1= $Hello2::var1\n"; print "var2= $Hello2::var2\n"; pring Hello2::greet(); test2.pl

7. Export Export functions and variables, so that they can be accessed without qualifier package Hello3; use strict; require Exporter; our @ISA=“Exporter”; our @EXPORT_OK = qw(greet); our $var1 = 1; my $var2 = 3; my $str = "Hello World!\n"; sub greet { return $str; } 1; Hello3.pm #!/usr/bin/perl use Hello3 qw(greet); print "var1= $Hello3::var1\n"; print "var2= $Hello3::var2\n"; print greet(); test3.pl

8. package Hello3; use strict; use Exporter; our @ISA=“Exporter”; our @EXPORT_OK = qw(greet); our $var1 = 1; my $var2 = 3; my $str = "Hello World!\n"; sub greet { return $str; } 1; Hello3.pm Need functionality in Exporter.pm to do exporting. This programs inherits functions Exporter module, rather than creates its own. Exporter this sub routine upon request by other program

9. #!/usr/bin/perl use Hello3 qw(greet); print "var1= $Hello3::var1\n"; print "var2= $Hello3::var2\n"; print greet(); test3.pl Request “greet”

10. package Hello4; use strict; use Exporter; our @ISA=“Exporter”; our @EXPORT_OK = qw(greet); our @EXPORT = qw(greet2); our $var1 = 1; my $var2 = 3; my $str = "Hello World!"; sub greet { return $str; } sub greet2 { return “Hi.\n”; } 1; Hello4.pm Export this automatically

11. #!/usr/bin/perl use Hello4 qw(greet); use Hello4; print "var1= $Hello4::var1\n"; print "var2= $Hello4::var2\n"; print greet(); print greet2(); test4.pl Request “greet” This automatically imports whatever in @EXPORT.

12. Exercise 1 Create a module which has functions to calculate the area and boundary of a rectangle. The width and length are to be supplied in your main program and passed into your module. Practice the @EXPORT, and @EXPORT_OK.

13. Object Orientied Programming A package (or module) is a class. A reference to a hash becomes an object of this class. The object contains member variables which are stored in the hash. The object also contains member functions.

14. Hello5.pm package Hello; use strict; sub new { my $class = shift; my $ref = {}; bless ( $ref, $class ); return $ref; } sub greet { my ($ref, $str)= @_; return $str; } sub greet2 { return "Hi\n"; } 1; #!/usr/local/bin/perl use Hello5; $h = new Hello5; print $h->greet("Good morning\n"); print $h->greet2; test5.pl

15. Rectangle.pm package Rectangle; sub new { my ($class, $width, $length)=@_; my $hashref = {W=>$width, L=>$length }; bless ( $hashref, $class); return $hashref; } sub getArea { my $self = shift; return $self->{W} * $self->{L}; } sub getBoundary { my $self=shift; return 2*($self->{W}+$self->{L}); } 1; #!/usr/bin/perl use Rectangle; my $w = 3; my $l = 4; my $rect = new Rectangle($w,$l); my $area = $rect->getArea(); print "Area = $area\n"; my $b = $rect->getBoundary(); Print “Boundary=$b\n”; recttest.pl

16. Exercise 2 Create a class called “Cube”. It should have methods to calculate volume based on the cube’s width, length and height.

17. More Pratices on Class Sequence.pm: clean, wrap, reverse complement, shuffle, GC content, translate Main program: seq.pl

18. Bioperl A collection of perl modules for bioinformatics Facilitates sequence retrieval, manipulation, and parsing results of programs like blast, clustalw. http://bioperl.org for download and documentation.http://bioperl.org Individual.pm file has info on how to use modules. Usually installed: /usr/local/lib/perl5/site_perl/5.8.0/Bio

19. Some Bioperl modules Bio::Perl, Bio::DB -- access seq databases. Examples: seqret.pl Bio::Seq -- sequence and its annotation. E.g. seqio.pl Bio::SeqIO – read sequence from file, and write to file. E.g. seqio.pl Bio::Tools:SeqStats -- molecular weight, etc. E.g. seqmw.pl Bio::SearchIO -- parse blast results.

20. Accessing Remote Databases use Bio::Perl; $seqobj = get_sequence(‘swiss’, “ROA1_HUMAN”); write_sequence(“roa1.fasta”, ‘fasta’, $seqobj); Databases can be: swiss, genbank, genpept, refseq, etc.

21. Bio::Seq Contain sequence and annotation Methods: display_id, desc, seq, revcom, translate, etc. The revcom and translate methods create new Bio::Seq object. One way to create a Bio::Seq object: $seq = Bio::Seq->new(-seq => 'actgtggcgtcaact', -desc => 'Sample Bio::Seq object', -display_id => 'something', -accession_number => 'accnum', -alphabet => 'dna' ); An other way: read the sequence from file via Bio::SeqIO object.

22. Parsing blast results Module: Bio::SearchIO my $in = new Bio::SearchIO(-format => 'blast', -file => 'report.bls'); while( my $result = $in->next_result ) { while( my $hit = $result->next_hit ) { while( my $hsp = $hit->next_hsp ) { if( $hsp->length('total') > 100 ) { if ( $hsp->percent_identity >= 75 ) { print "Hit= ", $hit->name, ", Length=", $hsp->length('total'), ", Percent_id=", $hsp->percent_identity, "\n"; } Example: blastparse.pl