Biology 595M – Practical Biocomputing

Biol 59500-033 - Practical Biocomputing1

BioPerl

General capabilities (packages)

•Sequences

○fetching, reading, writing, reformatting, annotating, groups

○Access to remote databases

•Applications

○BLAST, Blat, FASTA, HMMer, Clustal, Alignment, many others

•Gene modeling

○Genscan, Sim4, Grail, Genemark, ESTScan, MZEF, EPCR

•XML formats

○GAME, BSML and AGAVE

•GFF

•Trees

•Genetic maps

•3D structure

•Literature

•Graphics

Biol 59500-033 - Practical Biocomputing2

BioPerl

Auxilliary packages

•possibly of less general interest

•require additional modules

•BioPerl-run – running applications

○EMBOSS

○PISE

•Bioperl-ext – extensions

•Bioperl-db and BioSQL

Biol 59500-033 - Practical Biocomputing3

BioPerl

Simple

•use Bio::Perl;

•easy access to a small part of Bioperl's functionality in an easy touse manner

use Bio::Perl;

# this script will only work if you have an internet connection on the

# computer you're using, the databases you can get sequences from

# are 'swiss', 'genbank', 'genpept', 'embl', and 'refseq'

my $seq_object = get_sequence('swiss',"ROA1_HUMAN");

write_sequence(">roa1.fasta",'fasta',$seq_object);

use Bio::Perl;

my $seq = get_sequence('swiss',"ROA1_HUMAN");

# uses the default database - nr in this case

my $blast_result = blast_sequence($seq);

write_blast(">roa1.blast",$blast_result);

Biol 59500-033 - Practical Biocomputing4

BioPerl

Bio::Perl

Bio::Perl has a number of other easy-to-use functions, including

•get_sequence - gets a sequence from standard, internet accessibledatabases

•read_sequence - reads a sequence from a file

•read_all_sequences - reads all sequences from a file

•new_sequence - makes a Bioperl sequence just from a string

•write_sequence - writes a single or an array of sequence to a file

•translate - provides a translation of a sequence

•translate_as_string - provides a translation of a sequence, returningback just the sequence as a string

•blast_sequence - BLASTs a sequence against standard databasesat NCBI

•write_blast - writes a blast report out to a file

Biol 59500-033 - Practical Biocomputing5

BioPerl

Sequence Objects

•Seq, PrimarySeq, LocatableSeq, RelSegment, LiveSeq, LargeSeq,RichSeq, SeqWithQuality, SeqI

•Common formats are interpreted automatically

•Simple formats - without features

○FASTA (Pearson), Raw, GCG

•Rich Formats - with features and annotations

○GenBank, EMBL

○Swissprot, GenPept

○XML - BSML, GAME, AGAVE, TIGRXML, CHADO

Biol 59500-033 - Practical Biocomputing6

BioPerl

Sequences, Features and Annotations

•Sequence - DNA, RNA, Amino Acid

•Sequences are feature containers

○Feature - Information with a Sequence Location

○Annotation - Information without explicit Sequence location

Parsing sequences

•Bio::SeqIO

○for automatically reading most types

○multiple drivers: genbank, embl, fasta,...

•Sequence objects

○Bio::PrimarySeq

○Bio::Seq

○Bio::Seq::RichSeq

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BioPerl

Simple examples

#!/bin/perl -w

use Bio::Seq;

$seq_obj = Bio::Seq->new( -seq => "aaaatgggggggggggccccgtt",

-alphabet => 'dna' );

#!/bin/perl -w

use Bio::Seq;

$seq_obj = Bio::Seq->new( -seq => "aaaatgggggggggggccccgtt",

-display_id => "#12345",

-desc => "example 1",

-alphabet => "dna" );

print $seq_obj->seq();

Biol 59500-033 - Practical Biocomputing8

BioPerl

Reading sequences from files & databases

#!/bin/perl -w

use Bio::SeqIO;

$seqio_obj = Bio::SeqIO->new(-file => '>sequence.fasta', -format => 'fasta' );

# if there is more than one sequence in the file

while ($seq_obj = $seqio_obj->next_seq){ # print the sequence

print $seq_obj->seq,"\n";

}

#!/bin/perl -w

use Bio::DB::GenBank;

$db_obj = Bio::DB::GenBank->new;

$seq_obj = $db_obj->get_Seq_by_id( AE006468 );

Biol 59500-033 - Practical Biocomputing9

BioPerl

Getting sequences directly from database

#!/bin/perl -w

use Bio::DB::GenBank;

# also Bio::DB::GenBank, Bio::DB::GenPept, Bio::DB::SwissProt, Bio::DB::RefSeq and Bio::DB::EMBL

#keyword query

$query_obj = Bio::DB::Query::GenBank->new(

-query =>'gbdiv est[prop] AND Trypanosoma brucei [organism]',

-db => 'nucleotide' );

$gb = new Bio::DB::GenBank;

# this returns a Seq object :

$seq1 = $gb->get_Seq_by_id('MUSIGHBA1');

# this also returns a Seq object :

$seq2 = $gb->get_Seq_by_acc('AF303112');

# this returns a SeqIO object, which can be used to get a Seq object :

$seqio = $gb->get_Stream_by_id(["J00522","AF303112","2981014"]);

$seq3 = $seqio->next_seq;

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BioPerl

Getting more sequence information

•Some methods

−accession_number()get the accession number

−display_id()get identifier string

−description() or desc()get description string

−seq()get the sequence as a string

−length()get the sequence length

−subseq($start, $end)get a subsequence (char string)

−translate()translate to protein (seq obj)

−revcom()reverse complement (seq obj)

−species()Returns an Bio::Species object

#!/usr/bin/env perl

use strict;

use Bio::SeqIO;

use Bio::DB::GenBank;

my $genBank = new Bio::DB::GenBank;

my $seq = $genBank->get_Seq_by_acc('AF060485'); # get a record by accession

my $dna = $seq->seq; # get the sequence as a string

my $id = $seq->display_id; # identifier

my $acc = $seq->accession; # accession number

my $desc = $seq->desc; # get the description

print "ID: $id\naccession: $acc\nDescription: $desc\n$dna\n";

Biol 59500-033 - Practical Biocomputing11

BioPerl

SequenceObjects

LOCUS ECORHO 1880 bp DNA linear BCT 26-APR-1993

DEFINITION E.coli rho gene coding for transcription termination factor.

ACCESSION J01673 J01674

VERSION J01673.1 GI:147605

KEYWORDS attenuator; leader peptide; rho gene; transcription terminator.

SOURCE Escherichia coli

ORGANISM Escherichia coli

Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales;

Enterobacteriaceae; Escherichia.

REFERENCE 1 (bases 1 to 1880)

AUTHORS Brown,S., Albrechtsen,B., Pedersen,S. and Klemm,P.

TITLE Localization and regulation of the structural gene for

transcription-termination factor rho of Escherichia coli

JOURNAL J. Mol. Biol. 162 (2), 283-298 (1982)

MEDLINE 83138788

PUBMED 6219230

REFERENCE 2 (bases 1 to 1880) AUTHORS Pinkham,J.L. and Platt,T.

TITLE The nucleotide sequence of the rho gene of E. coli K-12

COMMENT Original source text: Escherichia coli (strain K-12) DNA.

A clean copy of the sequence for [2] was kindly provided by

J.L.Pinkham and T.Platt.

FEATURES Location/Qualifiers

source 1..1880

/organism="Escherichia coli"

/mol_type="genomic DNA"

/strain="K-12"

/db_xref="taxon:562"

mRNA 212..>1880

/product="rho mRNA"

gene 468..1727

/gene="rho"

CDS 468..1727

/gene="rho"

/note="transcription termination factor"

/codon_start=1

/translation="MNLTELKNTPVSELITLGENMGLENLARMRKQDIIFAILKQHAK

. . .

IDAMEFLINKLAMTKTNDDFFEMMKRS"

ORIGIN 15 bp upstream from HhaI site.

1 aaccctagca ctgcgccgaa atatggcatc cgtggtatcc cgactctgct gctgttcaaa

61 aacggtgaag tggcggcaac caaagtgggt gcactgtcta aaggtcagtt gaaagagttc

...deleted...

1801 tgggcatgtt aggaaaattc ctggaatttg ctggcatgtt atgcaatttg catatcaaat

1861 ggttaatttt tgcacaggac

Biol 59500-033 - Practical Biocomputing12

BioPerl

Bio::Seq object methods

•add_SeqFeature($feature) - attach feature(s)

•get_SeqFeatures() - get all the attached features.

•species() - a Bio::Species object

•annotation() - Bio::Annotation::Collection

Features

•Bio::SeqFeatureI - interface

•Bio::SeqFeature::Generic - basic implementation

•SeqFeature::Similarity - some score info

•SeqFeature::FeaturePair - pair of features

Biol 59500-033 - Practical Biocomputing13

BioPerl

Sequence Features

•Bio::SeqFeatureI - interface - GFF derived

○start(), end(), strand() for location information

○location() - Bio::LocationI object (to represent complex locations)

○score,frame,primary_tag, source_tag - feature information

○spliced_seq() - for attached sequence, get the sequence spliced.

•Bio::SeqFeature::Generic

○add_tag_value($tag,$value) - add a tag/value pair

○get_tag_value($tag) - get all the values for this tag

○has_tag($tag) - test if a tag exists

○get_all_tags() - get all the tags

Biol 59500-033 - Practical Biocomputing14

BioPerl

Sequence Annotations

•Each Bio::Seq has a Bio::Annotation::Collection via$seq->annotation()

•Annotations are stored with keys like ‘comment’ and ‘reference’

•Annotation::Comment

○comment field

•Annotation::Reference

○author,journal,title, etc

•Annotation::DBLink

○database,primary_id,optional_id,comment

•Annotation::SimpleValue

@com=$annotation-> get_Annotations(’comment’)

$annotation-> add_Annotation(’comment’,$an)

Biol 59500-033 - Practical Biocomputing15

BioPerl

Sequences, Features, and Annotations

Bio::LocationI

has-a

Bio::SeqFeature::Generic

Bio::Annotation::Comment

has-a

Annotations

Bio::Seq

Bio::Annotation::Collection

Features

Biol 59500-033 - Practical Biocomputing16

BioPerl

Writing sequences

•write in a different format than read = reformatting

use Bio::SeqIO;

#convert swissprot to fasta formatmy $in = Bio::SeqIO->new(-format => ‘swiss’, -file => ‘file.sp’);

my $out = Bio::SeqIO->new(-format => ‘fasta’, -file => ‘>file.fa’);`

while( my $seq = $in->next_seq ) { $out->write_seq($seq);}

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BioPerl

Remote Blast

•Retrieve sequence, setup and submit

use Bio::DB::GenBank;

use Bio::Tools::Run::RemoteBlast;

# retrieve sequence from genbank

my $db_obj = Bio::DB::GenBank->new;

my $seq_obj = $db_obj->get_Seq_by_acc( '1942116' );

my $seq = $seq_obj->seq;

print "seq:$seq\n";

#remote BLAST setup and query submission

my $v = 1;# turn on verbose output

my $remote_blast = Bio::Tools::Run::RemoteBlast->new(

'-prog' => 'blastp',

'-data' => 'swissprot',

'-expect' => '1e-10' );

my $r = $remote_blast->submit_blast( $seq_obj );

print STDERR "waiting…" if( $v > 0 );

Biol 59500-033 - Practical Biocomputing18

BioPerl

Remote Blast

•Retrieve sequence, setup and submit

--------------------- WARNING ---------------------

MSG: Unrecognized DBSOURCE data: pdb: molecule 2NLL, chain 65, release Aug 27, 2007; deposition: Nov 20, 1996;

class: TranscriptionDNA;

source: Mol_id: 1; Organism_scientific: Homo Sapiens;

Organism_common: Human; Genus: Homo; Species: Sapiens;

Expression_system: Escherichia Coli; Expression_system_common:

Bacteria; Expression_system_genus: Escherichia;

Expression_system_species: Coli; Mol_id: 2; Organism_scientific:

Homo Sapiens; Organism_common: Human; Genus: Homo; Species:

Sapiens; Expression_system: Escherichia Coli;

Expression_system_common: Bacteria; Expression_system_genus:

Escherichia; Expression_system_species: Coli; Mol_id: 3; Synthetic:

Yes; Mol_id: 4; Synthetic: Yes;

Exp. method: X-Ray Diffraction.

---------------------------------------------------

seq:CAICGDRSSGKHYGVYSCEGCKGFFKRTVRKDLTYTCRDNKDCLIDKRQRNRCQYCRYQKCLAMGM

Biol 59500-033 - Practical Biocomputing19

BioPerl

Remote Blast

•Results

•list of searchrids are storedin theremoteblastobject

#while (my @rids = $remote_blast->each_rid ) {

foreach my $rid ( @rids ) {

# Try to retrieve a search, $rc is not a reference until the search is done

# when the serch is complete, $rc is a Bio::SearchIO object

my $rc = $remote_blast->retrieve_blast($rid);

if( !ref($rc) ) {

# if the search is not done, wait 5 sec and try again

# it would be a good idea to put a maximum limit here so the script

# doesn't run forever in the event of an error

if ( $rc < 0 ) {

$remote_blast->remove_rid($rid);

}

print STDERR "." if ( $v > 0 );

sleep 5;

} else {

# search result successfully retrieved

my $result = $rc->next_result(); # see Bio::Search::Result

#save the output

my $filename = $result->query_name()."\.out";

$remote_blast->save_output($filename);

$remote_blast->remove_rid($rid);

print "\nQuery Name: ", $result->query_name(), "\n";

while ( my $hit = $result->next_hit ) {a # see Bio::Search::Hit::HitI

next unless ( $v > 0);

print "\thit name is ", $hit->name, "\n";

while( my $hsp = $hit->next_hsp ) {

print "\t\tscore is ", $hsp->score, "\n";

}

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BioPerl

Remote Blast

waiting....

Query Name: 2NLL_A

hit name is sp|P28700.1|RXRA_MOUSE score is 275

hit name is sp|P19793.1|RXRA_HUMAN score is 275

hit name is sp|Q05343.1|RXRA_RAT score is 275

hit name is sp|Q90415.1|RXRAB_DANRE score is 273

hit name is sp|A2T929.2|RXRAA_DANRE score is 272

hit name is sp|Q7SYN5.1|RXRBA_DANRE score is 270

hit name is sp|Q90416.2|RXRGA_DANRE score is 268

hit name is sp|P51128.1|RXRA_XENLA score is 268

hit name is sp|P28701.1|RXRG_CHICK score is 268

hit name is sp|Q90417.1|RXRBB_DANRE score is 266

hit name is sp|Q0GFF6.2|RXRG_PIG score is 264

hit name is sp|Q0VC20.1|RXRG_BOVIN score is 264

hit name is sp|Q5BJR8.1|RXRG_RAT score is 264

hit name is sp|Q5REL6.1|RXRG_PONAB score is 264

hit name is sp|P51129.1|RXRG_XENLA score is 264

hit name is sp|P48443.1|RXRG_HUMAN score is 264

hit name is sp|P28705.2|RXRG_MOUSE score is 264

hit name is sp|Q6DHP9.1|RXRGB_DANRE score is 261

hit name is sp|Q5TJF7.1|RXRB_CANFA score is 258

…

hit name is sp|Q505F1.2|NR2C1_MOUSE score is 200

hit name is sp|Q9TTR7.1|COT2_BOVIN score is 200

hit name is sp|Q90733.1|COT2_CHICK score is 200

hit name is sp|P16375.1|7UP1_DROME score is 200

hit name is sp|P16376.3|7UP2_DROME

hit name is sp|P24468.1|COT2_HUMAN

hit name is sp|O09018.1|COT2_RAT

hit name is sp|A0JNE3.1|NR2C1_BOVIN

hit name is sp|Q6PH18.1|N2F1B_DANRE

hit name is sp|Q9N4B8.4|NHR41_CAEEL

hit name is sp|O45666.2|NHR49_CAEEL

hit name is sp|P49866.2|HNF4_DROME

hit name is sp|P79926.1|HNF4B_XENLA

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BioPerl

Database Search

•BLAST - 3 Components

○Result: Bio::Search::Result::ResultI

○Hit: Bio::Search::Hit::HitI

○HSP: Bio::Search::HSP::HSPI

Biol 59500-033 - Practical Biocomputing22

BioPerl

Blast

use Bio::Perl;

my $seq = get_sequence('swiss',"ROA1_HUMAN");

# uses the default database - nr in this case

my $blast_result = blast_sequence($seq);

write_blast(">roa1.blast",$blast_result);}

$report_obj = new Bio::SearchIO(-format => 'blast',

-file => 'report.bls');

while( $result = $report_obj->next_result ) {

while( $hit = $result->next_hit ) {

while( $hsp = $hit->next_hsp ) {

if ( $hsp->percent_identity > 75 ) {

print "Hit\t", $hit->name, "\n", "Length\t", $hsp->length('total'),

"\n", "Percent_id\t", $hsp->percent_identity, "\n";

}

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BioPerl

BLAST – Processed result

Query is: BOSS_DROME Bride of sevenless protein precursor. 896 aa

Matrix was BLOSUM62

Hit is F35H10.10

HSP Len is 315 E-value is 4.9e-11 Bit score 182

Query loc: 511 813 Sbject loc: 1006 1298

HSP Len is 28 E-value is 1.4e-09 Bit score 39

Query loc: 508 535 Sbject loc: 427 454

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BioPerl

BLAST – Using the search::Hit object

use Bio::SearchIO;use strict;

my $parser = new Bio::SearchIO(-format => ‘blast’, -file => ‘file.bls’);

while( my $result = $parser->next_result ){ while( my $hit = $result->next_hit ) { print “hit name=”,$hit->name, “ desc=”, $hit->description, “\n len=”, $hit->length, “ acc=”, $hit->accession, ”\n”; print “raw score “, $hit->raw_score, “ bits “, $hit->bits, “ significance/evalue=“, $hit->evalue, “\n”; }}

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BioPerl

Search::Hit methods

•start(), end()

○get overall alignment start and end for all HSPs

•strand()

○get best overall alignment strand

•matches()

○get total number of matches across entire set of HSPs

○can specify only exact ‘id’ or conservative ‘cons’

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BioPerl

Using Search::HSP

use Bio::SearchIO;use strict;

my $parser = new Bio::SearchIO(-format => ‘blast’, -file => ‘file.bls’);

while( my $result = $parser->next_result ){ while( my $hit = $result->next_hit ) { while( my $hsp = $hit->next_hsp ) { print “hsp evalue=“, $hsp->evalue, “ score=“ $hsp->score, “\n”; print “total length=“, $hsp->hsp_length, “ qlen=”, $hsp->query->length, “ hlen=”,$hsp->hit->length, “\n”; print “qstart=”,$hsp->query->start, “ qend=”,$hsp->query->end, “ qstrand=“, $hsp->query->strand, “\n”; print “hstart=”,$hsp->hit->start, “ hend=”,$hsp->hit->end, “ hstrand=“, $hsp->hit->strand, “\n”; print “percent identical “, $hsp->percent_identity, “ frac conserved “, $hsp->frac_conserved(), “\n”; print “num query gaps “, $hsp->gaps(’query’), “\n”; print “hit str =”, $hsp->hit_string, “\n”; print “query str =”, $hsp->query_string, “\n”; print “homolog str=”, $hsp->homology_string, “\n”; }}

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BioPerl

Search::HSP methods

•rank()

○order in the alignment

○ by score, size

•matches

•seq_inds

○residue positions that are

−conserved, identical, mismatches, gaps

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BioPerl

SearchIO object correspond to many results

•BLAST (WU-BLAST, NCBI, XML, PSIBLAST, BL2SEQ, MEGABLAST,TABULAR (-m8/m9))

•FASTA (m9 and m0)

•HMMER (hmmpfam, hmmsearch)

•UCSC formats (WABA, AXT, PSL)

•Gene based alignments

○Exonerate, SIM4, {Gene,Genome}wise

•Can write searches in alternative formats

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BioPerl

Sequence Alignment

•Bio::AlignIO to read alignment files

•Produces Bio::SimpleAlign objects

○Phylip

○Clustal

•Interface and objects designed for round-tripping and somefunctional work

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BioPerl

Graphics

use Bio::Graphics;

use Bio::SeqIO;

use Bio::SeqFeature::Generic;

my $file = shift

or die "provide a sequence file as the argument";

my $io = Bio::SeqIO->new(-file=>$file)

or die "couldn't create Bio::SeqIO";

my $seq = $io->next_seq

or die "couldn't find a sequence in thefile";

my @features = $seq->all_SeqFeatures;

# sort features by their primary tags

my %sorted_features;

for my $f (@features) {

my $tag = $f->primary_tag;

push @{$sorted_features{$tag}},$f;

}

my $panel = Bio::Graphics::Panel->new(

-length => $seq->length,

-key_style => 'between',

-width => 800,

-pad_left => 10,

-pad_right => 10);

$panel->add_track(arrow =>

Bio::SeqFeature::Generic->new(-start => 1,

-end => $seq->length),

-bump => 0,

-double=>1,

-tick => 2);

$panel->add_track(generic =>

Bio::SeqFeature::Generic->new(-start => 1,

-end => $seq->length,

-bgcolor => 'blue',

-label => 1,);

# general case

my @colors = qw(cyan orange blue purple green

chartreuse magenta yellow aqua);

my $idx = 0;

for my $tag (sort keys %sorted_features) {

my $features = $sorted_features{$tag};

$panel->add_track($features,

-glyph => 'generic',

-bgcolor => $colors[$idx++ %@colors],

-fgcolor => 'black',

-font2color => 'red',

-key => "${tag}s",

-bump => +1,

-height => 8,

-label => 1,

-description => 1,

);

}

print $panel->png;

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BioPerl

Graphics