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1. אנא מלאו את סקר ההוראה הסקר ייפתח ב-06.01.13 ויהיה ניתן למלאו במשך שלושה שבועות הסקר יהיה זמין במערכת המידע האישי print “It is your chance to give us feedback\n”; use strict;

2. BioPerl

3. BioPerl • BioPerl is a collection of Perl modules for bioinformatics applications. • It is an active open source software project founded in 1996. • BioPerl provides software modules for many of the typical bioinformatic tasks. • Among these are: • Format conversions • Manipulating biological sequences • Searching for similar sequences • Creating sequence alignments • Searching for ORFs on genomic DNA • And more…

4. BioPerl BioPerl modules are called Bio::XXX You can use the BioPerl wiki: http://bio.perl.org/ with documentation and examples for how to use them – which is the best way to learn this. We recommend beginning with the "How-tos": http://www.bioperl.org/wiki/HOWTOs To a more hard-core inspection of BioPerl modules: BioPerl 1.6.1 Module Documentation

5. Object-oriented use of packages $obj0x225d14 func()anotherFunc() Many packages are meant to be used as objects. In Perl, an object is a data structure that can use subroutines that are associated with it. We will not learn object oriented programming,but we will learn how to create and use objects defined by BioPerl packages.

6. BioPerl: the SeqIO module $in0x25e211 next_seq()write_seq() BioPerl modules are named Bio::xxxx The Bio::SeqIO module deals with Sequences Input and Output: We will pass arguments to the new argument of the file name and format use Bio::SeqIO; my $in = Bio::SeqIO->new("-file"=> "<seq.gb","-format"=> "GenBank"); File argument(filename as would be in open) Format argument A list of all the sequence formats BioPerl can read is in:http://www.bioperl.org/wiki/HOWTO:SeqIO#Formats

7. BioPerl: the SeqIO module $in0x25e211 next_seq() write_seq() use Bio::SeqIO; my $in = Bio::SeqIO->new("-file" => "<seq.gb", "-format" => "GenBank"); my $seqObj = $in->next_seq(); Perform next_seq()subroutine on $in You could think of it as:SeqIO::next_seq($in) next_seq() returns the next sequence in the file as a Bio::Seq object (we will talk about them soon)

8. BioPerl: the SeqIO module use Bio::SeqIO; my $in = Bio::SeqIO->new("-file" => "<adeno12.gb", "-format" => "GenBank"); my $out = Bio::SeqIO->new("-file" => ">adeno12.out.fas", "-format" => "Fasta"); my $seqObj = $in->next_seq(); while ( defined($seqObj) ){ $out->write_seq($seqObj); $seqObj = $in->next_seq(); } write_seq()write a Bio::Seq object to $out according to its format

9. BioPerl: the Seq module use Bio::SeqIO; my $in = Bio::SeqIO->new( "-file" => "<Ecoli.prot.fasta", "-format" => "Fasta"); my $seqObj = $in->next_seq(); while (defined($seqObj)) { print "ID:".$seqObj->id()."\n"; #1st word in header print "Desc:".$seqObj->desc()."\n"; #rest of header print "Sequence:".$seqObj->seq()."\n"; #seq string print "Length:".$seqObj->length()."\n"; #seq length $seqObj = $in->next_seq() } You can read more about the Bio::Seq subroutines in: http://www.bioperl.org/wiki/HOWTO:Beginners#The_Sequence_Object

10. Print last 30aa of each sequence (no BioPerl) open (my $in, "<","seq.fasta") or die "Cannot open seq.fasta..."; my $fastaLine = <$in>; while (defined $fastaLine) { chomp $fastaLine; my $header=""; # Read first word of header if (fastaLine =~ m/^>(\S*)/) { $header = substr($fastaLine,1); $fastaLine = <$in>;} # Read seq until next header my $seq = ""; while ((defined $fastaLine) and(substr($fastaLine,0,1) ne ">" )) { chomp $fastaLine; $seq = $seq.$fastaLine; $fastaLine = <$in>; } # print last 30aa my $subseq = substr($seq,-30); print "$header\n“."$subseq\n"; }

11. Now using BioPerl use Bio::SeqIO; my $in = Bio::SeqIO->new("-file"=>"<seq.fasta","-format"=>"Fasta"); my $seqObj = $in->next_seq(); while (defined($seqObj)) { # Read first word of header my $header = $seqObj->id(); # print last 30aa my $seq = $seqObj->seq(); my $subseq = substr($seq,-30); print "$header\n"; print "$subseq\n"; $seqObj = $in->next_seq(); } Note: BioPerl warnings about:Subroutine ... redefined at ... Should not trouble you, it is a known issue – it is not your fault and won't effect your script's performances.

12. Class exercise 12a • Use Bio::SeqIO to read a FASTA file and print to an output FASTA file only sequences shorter than 3,000 bases.(use the EHD nucleotide FASTA from the webpage) • Use Bio::SeqIO to read a FASTA file, and print (to the screen) header lines that contain the words "Mus musculus". • 3. Write a script that uses Bio::SeqIO to read a GenPept file and convert it to FASTA.(use preProInsulinRecords.gp from the course’s webpage) • 4*. Same as Q1, but print to the FASTA the reverse complement of each sequence. (Do not use the reverse or tr// functions! BioPerl can do it for you - read the BioPerl documentation).

13. BioPerl: downloading files from the web The Bio::DB::Genbank module allows us to downloada specific record from the NCBI website: use Bio::DB::GenBank;my $gb = Bio::DB::GenBank->new;my $seqObj = $gb->get_Seq_by_acc("J00522"); print $seqObj->seq(); see more options in: http://www.bioperl.org/wiki/HOWTO:Beginners#Retrieving_a_sequence_from_a_database http://doc.bioperl.org/releases/bioperl-1.4/Bio/DB/GenBank.html

14. BLAST Congrats, you just sequenced yourself some DNA. #$?!? And you want to see if it exists in any other organism

15. BLAST BLAST - Basic Local Alignment and Search Tool BLAST helps you find similarity between your sequence and other sequences

16. BLAST BLAST - Basic Local Alignment and Search Tool BLAST helps you find similarity between your sequence and other sequences

17. BLAST BLAST helps you find similarity between your sequence and other sequences

18.  high scoring pair (HSP) BLAST Database hit query

19. BioPerl: reading BLAST output First we need to have the BLAST results in a text file BioPerl can read.Here is one way to achieve this (using NCBI BLAST): Download Text An alternative is to use BLASTALL on your computer

20. BioPerl: reading BLAST output Query Query= gi|52840257|ref|YP_094056.1| chromosomal replication initiator protein DnaA [Legionella pneumophila subsp. pneumophila str. Philadelphia 1] (452 letters) Database: Coxiella.faa 1818 sequences; 516,956 total letters Searching..................................................done Score E Sequences producing significant alignments: (bits) Value gi|29653365|ref|NP_819057.1| chromosomal replication initiator p... 633 0.0 gi|29655022|ref|NP_820714.1| DnaA-related protein [Coxiella burn... 72 4e-14 gi|29654861|ref|NP_820553.1| Holliday junction DNA helicase B [C... 32 0.033 gi|29654871|ref|NP_820563.1| ATPase, AFG1 family [Coxiella burne... 27 1.4 gi|29654481|ref|NP_820173.1| hypothetical protein CBU_1178 [Coxi... 25 3.1 gi|29654004|ref|NP_819696.1| succinyl-diaminopimelate desuccinyl... 25 3.1 Results info

21. BioPerl: reading BLAST output gi|215919162|ref|NP_820316.2| threonyl-tRNA synthetase [Coxiella... 25 5.3 gi|29655364|ref|NP_821056.1| transcription termination factor rh... 24 9.0 gi|215919324|ref|NP_821004.2| adenosylhomocysteinase [Coxiella b... 24 9.0 gi|29653813|ref|NP_819505.1| putative phosphoribosyl transferase... 24 9.0 >gi|29653365|ref|NP_819057.1| chromosomal replication initiator protein [Coxiella burnetii RSA 493] Length = 451 Score = 633 bits (1632), Expect = 0.0 Identities = 316/452 (69%), Positives = 371/452 (82%), Gaps = 5/452 (1%) Query: 1 MSTTAWQKCLGLLQDEFSAQQFNTWLRPLQAYMDEQR-LILLAPNRFVVDWVRKHFFSRI 59 + T+ W KCLG L+DE QQ+NTW+RPL A +Q L+LLAPNRFV+DW+ + F +RI Sbjct: 3 LPTSLWDKCLGYLRDEIPPQQYNTWIRPLHAIESKQNGLLLLAPNRFVLDWINERFLNRI 62 Query: 60 EELIKQFSGDDIKAISIEVGSKPVEAVDTPAETIVTSSSTAPLKSAPKKAVDYKSSHLNK 119 EL+ + S D I +++GS+ E + + AP + + +++N Sbjct: 63 TELLDELS-DTPPQIRLQIGSRSTEMPTKNSHEPSHRKAAAPPAGT---TISHTQANINS 118 Query: 120 KFVFDSFVEGNSNQLARAASMQVAERPGDAYNPLFIYGGVGLGKTHLMHAIGNSILKNNP 179 F FDSFVEG SNQLARAA+ QVAE PG AYNPLFIYGGVGLGKTHLMHA+GN+IL+ + Sbjct: 119 NFTFDSFVEGKSNQLARAAATQVAENPGQAYNPLFIYGGVGLGKTHLMHAVGNAILRKDS 178 Result header  high scoring pair (HSP) data HSP Alignment Note: There could be more than one HSP for each result, in case of homology in different parts of the protein

22. Bio::SearchIO : reading BLAST output The Bio::SearchIO module can read and parse BLAST output: use Bio::SearchIO; my $blast_report = Bio::SearchIO->new("-file" => "<LegCox.blastp", "-format" => "blast" ); my ($resultObj, $hitObj, $hspObj); while( defined($resultObj = $blast_report->next_result()) ){ print "Checking query ".$resultObj->query_name()."\n"; while( defined($hitObj = $resultObj->next_hit()) ) { print "Checking hit ". $hitObj->name()."\n"; $hspObj = $hitObj->next_hsp(); print "Best score: ".$hspObj->score()."\n"; } } (See the BLAST output example in the course’s website)

23. BioPerl: reading BLAST output You can send parameters to the subroutines of the objects: # Get length of HSP (including gaps) $hspObj->length("total"); # Get length of hit part of alignment (without gaps) $hspObj->length("hit"); # Get length of query part of alignment (without gaps) $hspObj->length("query"); More about what you can do with query, hit and hsp see in: http://www.bioperl.org/wiki/HOWTO:SearchIO#Table_of_Methods

24. Class exercise 12b • Uses Bio::SearchIO to parse the BLAST results:(LegCox.blastp provided in the course’s website) • For each query print out its name and the name of its first hit. • b*) Print the percent identity of each HSP of the first hit of each query. • c*) Print the e-value of each HSP of the first hit of each query.