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use strict;

13.2

BioPerl

12.3

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…

BioPerl

12.4 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

12.5

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.

Object-oriented use of packages

$obj0x225d14

func()anotherFunc()

12.6

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");

BioPerl: the SeqIO module

File argument(filename as

would be in open)

A list of all the sequence formats BioPerl can read is in:

http://www.bioperl.org/wiki/HOWTO:SeqIO#Formats

Format argument $in

0x25e211

next_seq()write_seq()

12.7

use Bio::SeqIO;

my $in = Bio::SeqIO->new("-file" => "<seq.gb",


my $seqObj = $in->next_seq();


$in0x25e211

next_seq() write_seq()

next_seq() returns the next sequence in the file as

a Bio::Seq object (we will talk about them soon)

Perform next_seq()subroutine on $in You could think of it as:SeqIO::next_seq($in)

12.8

use Bio::SeqIO;

my $in = Bio::SeqIO->new("-file" => "<adeno12.gb",


my $out = Bio::SeqIO->new("-file" => ">adeno12.out.fas",

"-format" => "Fasta");


while ( defined($seqObj) ){

$out->write_seq($seqObj);

$seqObj = $in->next_seq();

}


write_seq()write a Bio::Seq object to $out

according to its format

12.9

use Bio::SeqIO;

my $in = Bio::SeqIO->new( "-file" => "<Ecoli.prot.fasta",

"-format" => "Fasta");


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

BioPerl: the Seq module

12.10 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 30aamy $subseq = substr($seq,-30);

print "$header\n“."$subseq\n";

}

12.11 Now using BioPerl use Bio::SeqIO;

my $in = Bio::SeqIO->new("-file"=>"<seq.fasta","-format"=>"Fasta");


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.

13.13Class exercise 12a

1. 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)

2. 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).

12.14

The Bio::DB::Genbank module allows us to download

a 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

BioPerl: downloading files from the web

12.15

BLAST

Congrats, you just sequenced yourself some DNA.

And you want to see if it exists in any other organism#$?!?

12.16

BLAST

BLAST helps you find similarity between your

sequence and other sequences

BLAST - Basic Local Alignment and Search Tool

12.17

BLAST



BLAST - Basic Local Alignment and Search Tool

12.18

BLAST



12.19

BLAST

Database

query hit

high scoring pair (HSP)

12.21

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):

BioPerl: reading BLAST output

Text

Download

An alternative is to use BLASTALL on your computer

12.22

Query= gi|52840257|ref|YP_094056.1| chromosomal replication initiatorprotein DnaA [Legionella pneumophila subsp. pneumophila str.Philadelphia 1] (452 letters)

Database: Coxiella.faa 1818 sequences; 516,956 total letters

Searching..................................................done

Score ESequences 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-14gi|29654861|ref|NP_820553.1| Holliday junction DNA helicase B [C... 32 0.033gi|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

BioPerl: reading BLAST outputQuery

Results info

12.23

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 +RISbjct: 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

12.24

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)

Bio::SearchIO : reading BLAST output

12.25

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


13.26Class exercise 12b

1. Uses Bio::SearchIO to parse the BLAST results: (LegCox.blastp provided in the course’s website)

a) 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.

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