package Boulder::Genbank; use strict; use Boulder::Stream; use Stone::GB_Sequence; use Carp; use vars qw(@ISA $VERSION); @ISA = qw(Boulder::Stream); $VERSION = 1.10; # Hard-coded defaults - must modify for your site use constant YANK => '/usr/local/bin/yank'; #use constant BATCH_URI => '/cgi-bin/Entrez/qserver.cgi/result'; #use constant BATCH_URI => '/htbin-post/Entrez/query'; use constant HOST => 'www.ncbi.nlm.nih.gov'; use constant BATCH_URI => '/IEB/ToolBox/XML/xbatch.cgi'; # Genbank entry parsing constants # (may need to adjust!) my $KEYCOL=0; my $VALUECOL=12; my $FEATURECOL=5; my $FEATUREVALCOL=21; =head1 NAME Boulder::Genbank - Fetch Genbank data records as parsed Boulder Stones =head1 SYNOPSIS use Boulder::Genbank # network access via Entrez $gb = Boulder::Genbank->newFh( qw(M57939 M28274 L36028) ); while ($data = <$gb>) { print $data->Accession; @introns = $data->features->Intron; print "There are ",scalar(@introns)," introns.\n"; $dna = $data->Sequence; print "The dna is ",length($dna)," bp long.\n"; my @features = $data->features(-type=>[ qw(Exon Source Satellite) ], -pos=>[90,310] ); foreach (@features) { print $_->Type,"\n"; print $_->Position,"\n"; print $_->Gene,"\n"; } } # another syntax $gb = new Boulder::Genbank(-accessor=>'Entrez', -fetch => [qw/M57939 M28274 L36028/]); # local access via Yank $gb = new Boulder::Genbank(-accessor=>'Yank', -fetch=>[qw/M57939 M28274 L36028/]); while (my $s = $gb->get) { # etc. } # parse a file of Genbank records $gb = new Boulder::Genbank(-accessor=>'File', -fetch => '/usr/local/db/gbpri3.seq'); while (my $s = $gb->get) { # etc. } # parse flatfile records yourself open (GB,"/usr/local/db/gbpri3.seq"); local $/ = "//\n"; while () { my $s = Boulder::Genbank->parse($_); # etc. } =head1 DESCRIPTION Boulder::Genbank provides retrieval and parsing services for NCBI Genbank-format records. It returns Genbank entries in L format, allowing easy access to the various fields and values. Boulder::Genbank is a descendent of Boulder::Stream, and provides a stream-like interface to a series of Stone objects. >> IMPORTANT NOTE << As of January 2002, NCBI has changed their Batch Entrez interface. I have modified Boulder::Genbank so as to use a "demo" interface, which fixes things, but this isn't guaranteed in the long run. I have written to NCBI, and they may fix this -- or they may not. >> IMPORTANT NOTE << Access to Genbank is provided by three different I, which together give access to remote and local Genbank databases. When you create a new Boulder::Genbank stream, you provide one of the three accessors, along with accessor-specific parameters that control what entries to fetch. The three accessors are: =over 4 =item Entrez This provides access to NetEntrez, accessing the most recent Genbank information directly from NCBI's Web site. The parameters passed to this accessor are either a series of Genbank accession numbers, or an Entrez query (see http://www.ncbi.nlm.nih.gov/Entrez/linking.html). If you provide a list of accession numbers, the stream will return a series of stones corresponding to the numbers. Otherwise, if you provided an Entrez query, the entries returned will be in the order returned by Entez. =item File This provides access to local Genbank entries by reading from a flat file (typically one of the .seq files downloadable from NCBI's Web site). The stream will return a Stone corresponding to each of the entries in the file, starting from the top of the file and working downward. The parameter in this case is the path to the local file. =item Yank This provides access to local Genbank entries using Will Fitzhugh's Yank program. Yank provides fast indexed access to a Genbank flat file using the accession number as the key. The parameter passed to the Yank accessor is a list of accession numbers. Stones will be returned in the requested order. By default the yank binary lives in /usr/local/bin/yank. To support other locations, you may define the environment variable YANK to contain the full path. =back It is also possible to parse a single Genbank entry from a text string stored in a scalar variable, returning a Stone object. =head2 Boulder::Genbank methods This section lists the public methods that the I class makes available. =over 4 =item new() # Network fetch via Entrez, with accession numbers $gb=new Boulder::Genbank(-accessor => 'Entrez', -fetch => [qw/M57939 M28274 L36028/]); # Same, but shorter and uses -> operator $gb = Boulder::Genbank->new qw(M57939 M28274 L36028); # Network fetch via Entrez, with a query # Network fetch via Entrez, with a query $query = 'Homo sapiens[Organism] AND EST[Keyword]'; $gb=new Boulder::Genbank(-accessor => 'Entrez', -fetch => $query); # Local fetch via Yank, with accession numbers $gb=new Boulder::Genbank(-accessor => 'Yank', -fetch => [qw/M57939 M28274 L36028/]); # Local fetch via File $gb=new Boulder::Genbank(-accessor => 'File', -fetch => '/usr/local/genbank/gbpri3.seq'); The new() method creates a new I stream on the accessor provided. The three possible accessors are B, B and B. If successful, the method returns the stream object. Otherwise it returns undef. new() takes the following arguments: -accessor Name of the accessor to use -fetch Parameters to pass to the accessor -proxy Path to an HTTP proxy, used when using the Entrez accessor over a firewall. Specify the accessor to use with the B<-accessor> argument. If not specified, it defaults to B. B<-fetch> is an accessor-specific argument. The possibilities are: For B, the B<-fetch> argument may point to a scalar, in which case it is interpreted as an Entrez query string. See http://www.ncbi.nlm.nih.gov/Entrez/linking.html for a description of the query syntax. Alternatively, B<-fetch> may point to an array reference, in which case it is interpreted as a list of accession numbers to retrieve. If B<-fetch> points to a hash, it is interpreted as extended information. See L<"Extended Entrez Parameters"> below. For B, the B<-fetch> argument must point to an array reference containing the accession numbers to retrieve. For B, the B<-fetch> argument must point to a string-valued scalar, which will be interpreted as the path to the file to read Genbank entries from. For Entrez (and Entrez only) Boulder::Genbank allows you to use a shortcut syntax in which you provde new() with a list of accession numbers: $gb = new Boulder::Genbank('M57939','M28274','L36028'); =item newFh() This works like new(), but returns a filehandle. To recover each GenBank record read from the filehandle with the <> operator: $fh = Boulder::GenBank->newFh('M57939','M28274','L36028'); while ($record = <$fh>) { print $record->asString; } =item get() The get() method is inherited from I, and simply returns the next parsed Genbank Stone, or undef if there is nothing more to fetch. It has the same semantics as the parent class, including the ability to restrict access to certain top-level tags. The object returned is a L object, which is a descendent of L. =item put() The put() method is inherited from the parent Boulder::Stream class, and will write the passed Stone to standard output in Boulder format. This means that it is currently not possible to write a Boulder::Genbank object back into Genbank flatfile form. =back =head2 Extended Entrez Parameters The Entrez accessor recognizes extended parameters that allow you the ability to customize the search. Instead of passing a query string scalar or a list of accession numbers as the B<-fetch> argument, pass a hash reference. The hashref should contain one or more of the following keys: =over =item B<-query> The Entrez query to process. =item B<-accession> The list of accession numbers to fetch, as an array ref. =item B<-db> The database to search. This is a single-letter database code selected from the following list: m MEDLINE p Protein n Nucleotide s Popset =item B<-proxy> An HTTP proxy to use. For example: -proxy => http://www.firewall.com:9000 If you think you need this, get the correct URL from your system administrator. =back As an example, here's how to search for ESTs from Oryza sativa that have been entered or modified since 1999. my $gb = new Boulder::Genbank( -accessor=>Entrez, -query=>'Oryza sativa[Organism] AND EST[Keyword] AND 1999[MDAT]', -db => 'n' }); =head1 METHODS DEFINED BY THE GENBANK STONE OBJECT Each record returned from the Boulder::Genbank stream defines a set of methods that correspond to features and other fields in the Genbank flat file record. L gives the full details, but they are listed for reference here: =head2 $length = $entry->length Get the length of the sequence. =head2 $start = $entry->start Get the start position of the sequence, currently always "1". =head2 $end = $entry->end Get the end position of the sequence, currently always the same as the length. =head2 @feature_list = $entry->features(-pos=>[50,450],-type=>['CDS','Exon']) features() will search the entry feature list for those features that meet certain criteria. The criteria are specified using the B<-pos> and/or B<-type> argument names, as shown below. =over 4 =item -pos Provide a position or range of positions which the feature must B. A single position is specified in this way: -pos => 1500; # feature must overlap postion 1500 or a range of positions in this way: -pos => [1000,1500]; # 1000 to 1500 inclusive If no criteria are provided, then features() returns all the features, and is equivalent to calling the Features() accessor. =item -type, -types Filter the list of features by type or a set of types. Matches are case-insensitive, so "exon", "Exon" and "EXON" are all equivalent. You may call with a single type as in: -type => 'Exon' or with a list of types, as in -types => ['Exon','CDS'] The names "-type" and "-types" can be used interchangeably. =head2 $seqObj = $entry->bioSeq; Returns a L object from the Bioperl project. Dies with an error message unless the Bio::Seq module is installed. =back =head1 OUTPUT TAGS The tags returned by the parsing operation are taken from the NCBI ASN.1 schema. For consistency, they are normalized so that the initial letter is capitalized, and all subsequent letters are lowercase. This section contains an abbreviated list of the most useful/common tags. See "The NCBI Data Model", by James Ostell and Jonathan Kans in "Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins" (Eds. A. Baxevanis and F. Ouellette), pp 121-144 for the full listing. =head2 Top-Level Tags These are tags that appear at the top level of the parsed Genbank entry. =over 4 =item Accession The accession number of this entry. Because of the vagaries of the Genbank data model, an entry may have multiple accession numbers (e.g. after a merging operation). Accession may therefore be a multi-valued tag. Example: my $accessionNo = $s->Accession; =item Authors The list of authors, as they appear on the AUTHORS line of the Genbank record. No attempt is made to parse them into individual authors. =item Basecount The nucleotide basecount for the entry. It is presented as a Boulder Stone with keys "a", "c", "t" and "g". Example: my $A = $s->Basecount->A; my $C = $s->Basecount->C; my $G = $s->Basecount->G; my $T = $s->Basecount->T; print "GC content is ",($G+$C)/($A+$C+$G+$T),"\n"; =item Blob The entire flatfile record as an unparsed chunk of text (a "blob"). This is a handy way of reassembling the record for human inspection. =item Comment The COMMENT line from the Genbank record. =item Definition The DEFINITION line from the Genbank record, unmodified. =item Features The FEATURES table. This is a complex stone object with multiple subtags. See the L<"The Features Tag"> for details. =item Journal The JOURNAL line from the Genbank record, unmodified. =item Keywords The KEYWORDS line from the Genbank record, unmodified. No attempt is made to parse the keywords into separate values. Example: my $keywords = $s->Keywords =item Locus The LOCUS line from the Genbank record. It is not further parsed. =item Medline, Nid References to other database accession numbers. =item Organism The taxonomic name of the organism from which this entry was derived. This line is taken from the Genbank entry unmodified. See the NCBI data model documentation for an explanation of their taxonomic syntax. =item Reference The REFERENCE line from the Genbank entry. There are often multiple Reference lines. Example: my @references = $s->Reference; =item Sequence The DNA or RNA sequence of the entry. This is presented as a single lower-case string, with all base numbers and formatting characters removed. =item Source The entry's SOURCE field; often giving clues on how the sequencing was performed. =item Title The TITLE field from the paper describing this entry, if any. =back =head2 The Features Tag The Features tag points to a Stone record that contains multiple subtags. Each subtag is the name of a feature which points, in turn, to a Stone that describes the feature's location and other attributes. The full list of feature is beyond this document, but the following are the features that are most often seen: Cds a CDS Intron an intron Exon an exon Gene a gene Mrna an mRNA Polya_site a putative polyadenylation signal Repeat_unit a repetitive region Source More information about the organism and cell type the sequence was derived from Satellite a microsatellite (dinucleotide repeat) Each feature will contain one or more of the following subtags: =over 4 =item DB_xref A cross-reference to another database in the form DB_NAME:accession_number. See the NCBI Web site for a description of these cross references. =item Evidence The evidence for this feature, either "experimental" or "predicted". =item Gene If the feature involves a gene, this will be the gene's name (or one of its names). This subtag is often seen in "Gene" and Cds features. Example: foreach ($s->Features->Cds) { my $gene = $_->Gene; my $position = $_->Position; Print "Gene $gene ($position)\n"; } =item Map If the feature is mapped, this provides a map position, usually as a cytogenetic band. =item Note A grab-back for various text notes. =item Number When multiple features of this type occur, this field is used to number them. Ordinarily this field is not needed because Boulder::Genbank preserves the order of features. =item Organism If the feature is Source, this provides the source organism. =item Position The position of this feature, usually expresed as a range (1970..1975). =item Product The protein product of the feature, if applicable, as a text string. =item Translation The protein translation of the feature, if applicable. =back =head1 SEE ALSO L, L =head1 AUTHOR Lincoln Stein . Copyright (c) 1997-2000 Lincoln D. Stein This library is free software; you can redistribute it and/or modify it under the same terms as Perl itself. See DISCLAIMER.txt for disclaimers of warranty. =head1 EXAMPLE GENBANK OBJECT The following is an excerpt from a moderately complex Genbank Stone. The Sequence line and several other long lines have been truncated for readability. Authors=Spritz,R.A., Strunk,K., Surowy,C.S.O., Hoch,S., Barton,D.E. and Francke,U. Authors=Spritz,R.A., Strunk,K., Surowy,C.S. and Mohrenweiser,H.W. Locus=HUMRNP7011 2155 bp DNA PRI 03-JUL-1991 Accession=M57939 Accession=J04772 Accession=M57733 Keywords=ribonucleoprotein antigen. Sequence=aagcttttccaggcagtgcgagatagaggagcgcttgagaaggcaggttttgcagcagacggcagtgacagcccag... Definition=Human small nuclear ribonucleoprotein (U1-70K) gene, exon 10 and 11. Journal=Nucleic Acids Res. 15, 10373-10391 (1987) Journal=Genomics 8, 371-379 (1990) Nid=g337441 Medline=88096573 Medline=91065657 Features={ Polya_site={ Evidence=experimental Position=1989 Gene=U1-70K } Polya_site={ Position=1990 Gene=U1-70K } Polya_site={ Evidence=experimental Position=1992 Gene=U1-70K } Polya_site={ Evidence=experimental Position=1998 Gene=U1-70K } Source={ Organism=Homo sapiens Db_xref=taxon:9606 Position=1..2155 Map=19q13.3 } Cds={ Codon_start=1 Product=ribonucleoprotein antigen Db_xref=PID:g337445 Position=join(M57929:329..475,M57930:183..245,M57930:358..412, ... Gene=U1-70K Translation=MTQFLPPNLLALFAPRDPIPYLPPLEKLPHEKHHNQPYCGIAPYIREFEDPRDAPPPTR... } Cds={ Codon_start=1 Product=ribonucleoprotein antigen Db_xref=PID:g337444 Evidence=experimental Position=join(M57929:329..475,M57930:183..245,M57930:358..412, ... Gene=U1-70K Translation=MTQFLPPNLLALFAPRDPIPYLPPLEKLPHEKHHNQPYCGIAPYIREFEDPR... } Polya_signal={ Position=1970..1975 Note=putative Gene=U1-70K } Intron={ Evidence=experimental Position=1100..1208 Gene=U1-70K } Intron={ Number=10 Evidence=experimental Position=1100..1181 Gene=U1-70K } Intron={ Number=9 Evidence=experimental Position=order(M57937:702..921,1..1011) Note=2.1 kb gap Gene=U1-70K } Intron={ Position=order(M57935:272..406,M57936:1..284,M57937:1..599, <1..>1208) Gene=U1-70K } Intron={ Evidence=experimental Position=order(M57935:284..406,M57936:1..284,M57937:1..599, <1..>1208) Note=first gap-0.14 kb, second gap-0.62 kb Gene=U1-70K } Intron={ Number=8 Evidence=experimental Position=order(M57935:272..406,M57936:1..284,M57937:1..599, <1..>1181) Note=first gap-0.14 kb, second gap-0.62 kb Gene=U1-70K } Exon={ Number=10 Evidence=experimental Position=1012..1099 Gene=U1-70K } Exon={ Number=11 Evidence=experimental Position=1182..(1989.1998) Gene=U1-70K } Exon={ Evidence=experimental Position=1209..(1989.1998) Gene=U1-70K } Mrna={ Product=ribonucleoprotein antigen Position=join(M57928:358..668,M57929:319..475,M57930:183..245, ... Gene=U1-70K } Mrna={ Product=ribonucleoprotein antigen Citation=[2] Evidence=experimental Position=join(M57928:358..668,M57929:319..475,M57930:183..245, ... Gene=U1-70K } Gene={ Position=join(M57928:207..719,M57929:1..562,M57930:1..577, ... Gene=U1-70K } } Reference=1 (sites) Reference=2 (bases 1 to 2155) = =cut # new() takes named parameters: # -accessor=> Reference to an object class that will return a series of # Genbank records. Predefined objects include 'Yank', 'Entrez' and 'File'. # (defaults to 'Entrez'). # -fetch=> Parameters to pass to the subroutine. Can be a list of accession numbers # or an entrez query. # -out=> Output filehandle. Defaults to STDOUT. # # If you don't use named parameters, then will assume method 'yank' on # a list of accession numbers. # e.g. # $gb = new Boulder::Genbank(-accessor=>'Yank',-fetch=>[qw/M57939 M28274 L36028/]); sub new { my($package,@parameters) = @_; # superclass constructor my($self) = $package->SUPER::new(); # figure out whether parameters are named. Look for # an initial '-' my %parameters; if ($parameters[0]=~/^-/) { %parameters = @parameters; $self->{accessor} = $parameters{'-accessor'} || 'Entrez'; $self->{OUT} = $parameters{'-out'} || \*STDOUT; $self->{format} = $parameters{'-format'}; } else { $self->{accessor}='Entrez'; $parameters{-fetch} = \@parameters; } $self->{format} ||= 'stone'; $parameters{-format} = $self->{format}; $self->{accessor} = new {$self->{accessor}}(\%parameters); return bless $self,$package; } sub read_record { my($self,@tags) = @_; my($s); my $query = $self->{query}; if (wantarray) { my(@result); while (!$self->{EOF}) { $s = $self->read_one_record(@tags); next unless $s; next if $query && !(&$query); push(@result,$s); } return @result; } # we get here if in a scalar context while (!$self->{EOF}) { $s = $self->read_one_record(@tags); next unless $s; return $s unless $query; return $s if &$query; } return undef; } sub parse { my $self = shift; my $record = shift; return unless $record; my $tags = shift; my %ok; %ok = map {$_ => 1} @$tags if ref($tags) eq 'ARRAY'; my($s,@lines,$line,$accumulated,$key,$keyword,$value,$feature,@features); $s = Stone::GB_Sequence->new; @lines = split("\n",$record); foreach $line (@lines) { if ($line=~/^ACCESSION\s+(.+)/) { foreach ($1=~/(\S+)/g) { $self->_addToStone('Accession',$_,$s,\%ok); } next; } # special case for the VERSION if ($line=~/^VERSION/) { my @vernid = split(/\s+/,$line); my ($junk,$tmp)=split(/:/,$vernid[2]); $vernid[2]='g'.$tmp; $self->_addToStone('Version',$vernid[1],$s,\%ok); $self->_addToStone('Nid',$vernid[2],$s,\%ok); next; } # special case for the features table if ($line=~/^FEATURES/..$line=~/^ORIGIN/) { undef $keyword; if ($line=~/^FEATURES/) { undef @features; next; } if ($line =~ /^(BASE COUNT|ORIGIN)/) { push(@features,$feature) if $feature; $self->_addFeaturesToStone(\@features,_trim($'),$s,\%ok) if @features; undef @features; undef $feature; next if $line =~ /^BASE COUNT/; # special case for the sequence itself if ($line=~/^ORIGIN/) { # next line added by Luca Toldo $self->_addToStone('Blob',$record,$s,\%ok); $self->_addToStone($key,$accumulated,$s,\%ok) if $key; last; } } my($featurelabel) = _trim(substr($line,$FEATURECOL,$FEATUREVALCOL-$FEATURECOL)); my($featurevalue) = _trim(substr($line,$FEATUREVALCOL)); if ($featurelabel) { push(@features,$feature) if $feature; $feature = {'label'=>$featurelabel,'value'=>$featurevalue}; } else { $feature->{'value'} .= $featurevalue; } next; } $keyword = _trim(substr($line,0,$VALUECOL-1)); $value = _trim(substr($line,$VALUECOL)); if ($keyword && $key) { $self->_addToStone($key,_trim($accumulated),$s,\%ok); $accumulated = $value; next; } $accumulated .= " $value"; } continue { $key = $keyword if $keyword; } my($sequence) = $record=~/\nORIGIN[^\n]*\n(.+)\\?\\?/s; # $sequence=~s/[\s0-9-]+//g; # remove white space and numbers # $sequence =~ s/^\s*\d+\s//mg; # remove leading numbers and whitespace # $sequence =~ s/(\S{1,10}) /$1/g; # remove spacer $sequence =~ s/^.{9,10}//mg; # remove leading numbers and whitespace $sequence =~ s/(.{10}) /$1/g; # remove spacers $sequence =~ s/\n//g; $self->_addToStone('Sequence',$sequence,$s,\%ok); return $s; } sub read_one_record { my($self,@tags) = @_; my(%ok); my $accessor = $self->{'accessor'}; my $record = $accessor->fetch_next(); unless ($record) { $self->{EOF}++; return undef; } return $record unless $self->{format} eq 'stone'; return $self->parse($record,\@tags); } sub _trim { my($v) = @_; $v=~s/^\s+//; $v=~s/\s+$//; return $v; } sub _canonicalize { my $h = shift; substr($h,0)=~tr/a-z/A-Z/; substr($h,1,length($h)-1)=~tr/A-Z/a-z/; $h; } sub _addToStone { my($self,$label,$value,$stone,$ok) = @_; return unless !%{$ok} || $ok->{$label}; $stone->insert(_canonicalize($label),$value); } sub _addFeaturesToStone { my($self,$features,$basecount,$stone,$ok) = @_; # first add the basecount if (!%{$ok} || $ok->{'BASECOUNT'}) { my(%counts) = $basecount=~/(\d+)\s+([gatcGATC])/g; %counts = map { uc $_ } reverse %counts; $stone->insert('Basecount',new Stone(%counts)); } if (!%{$ok} || $ok->{'FEATURES'}) { # now add the features my($f) = new Stone; foreach (@$features) { my($p) = new Stone; my($q) = $_->{'value'}; my($label) = _canonicalize($_->{'label'}); my($position) = $q=~m!^([^/\s]+)!; my @qualifiers = $q=~m@/(\w+)=(.+?)(?=\"|/\w+=|$)@g; # slower but ?better my %qualifiers; while (my($key,$value) = splice(@qualifiers,0,2)) { $value =~ s/^\s*\"//; # trim off extra space and quotes $value =~ s/\"\s*$//; $value =~ s/^\s+//; # trim off extra space and quotes $value =~ s/\s+$//; $value =~ s/\s+//g if uc($key) eq 'TRANSLATION'; # get rid of spaces in protein translation $p->insert(_canonicalize($key)=>$value); } $p->insert('Position'=>$position); $f->insert($label=>$p); undef $p; } $stone->insert('Features',$f); } } # ---------------------------------------------------------------------------------------- # -------------------------- DEFINITION OF ACCESSOR OBJECTS ------------------------------ package GenbankAccessor; use Carp; sub new { my($class,@parameters) = @_; croak "GenbankAccessor::new: Abstract class\n"; } sub fetch_next { my($self) = @_; croak "GenbankAccessor::fetch_next: Abstract class\n"; } sub DESTROY { } package Yank; use strict; use Carp; use vars '@ISA'; @ISA=qw(GenbankAccessor); my $YANK = $ENV{YANK} || Boulder::Genbank::YANK(); sub new { my($package,$param) = @_; croak "Yank::new(): need at least one Genbank acccession number" unless $param; croak "Yank::new(): yank executable not found" unless -x $YANK; $param->{-fetch} ||= $param->{-param}; # for backward compatibility $param->{-fetch} || croak "Provide list of accession numbers to yank"; my @accession = @{$param->{-fetch}}; my $tmpfile = "/usr/tmp/yank$$"; open (TMP,">$tmpfile") || croak "Yank::new(): couldn't open tmpfile $tmpfile for write: $!"; print TMP join("\n",@accession),"\n"; close TMP; open(YANK,"$YANK -b < $tmpfile |") || croak "Yank::new(): couldn't open pipe from yank: $!"; return bless {'tmpfile'=>$tmpfile,'fh'=>\*YANK},$package; } sub fetch_next { my($self) = @_; return undef unless $self->{'fh'}; local($/) = "//\n"; my($line); my($fh) = $self->{'fh'}; chomp($line = <$fh>); return $line; } sub DESTROY { my($self) = shift; close $self->{'fh'} if $self->{'fh'}; unlink $self->{'tmpfile'} if $self->{'tmpfile'} } package File; use vars '@ISA'; use Symbol; use Carp; @ISA=qw(GenbankAccessor); sub new { my($package,$param) = @_; my $path = $param->{-fetch} || $param->{-path} || $param->{-param}; my $fh; if (!$path) { $fh = \*ARGV; } elsif (ref $path eq 'GLOB') { $fh = $path; } else { $fh = Symbol::gensym; open ($fh,$path) or croak "File::new(): couldn't open $path: $!"; } return bless {'fh'=>$fh},$package; } sub fetch_next { my $self = shift; return undef unless $self->{'fh'}; local($/)="//\n"; my $line; my $fh = $self->{'fh'}; $line = <$fh>; chomp $line if $line; return $line; } package Entrez; use Carp; use vars '@ISA'; use IO::Socket; use CGI 'escape'; # used by Entrez accessor, may need to change in the future use constant ENTREZ_HOST => 'www.ncbi.nlm.nih.gov'; use constant QUERY_URI => '/entrez/utils/pmqty.fcgi'; use constant BATCH_URI => '/entrez/utils/pmfetch.fcgi'; use constant XBATCH_URI => '/IEB/ToolBox/XML/xbatch.cgi'; use constant PROTO => 'HTTP/1.0'; use constant CRLF => "\r\n"; use constant MAX_ENTRIES => 10_000; @ISA=qw(GenbankAccessor); sub new { my($package,$param) = @_; croak "Entrez::new(): usage [list of accession numbers] or {args => values}" unless $param; my $self = bless {},ref($package) || $package; $self->{query} = $param->{-query}; $self->{accession} = $param->{-fetch} || $param->{-param}; $self->{db} = $param->{-db} || 'n'; $self->{format} = $param->{-format} || 'stone'; $self->{proxy} = $param->{-proxy}; $self->{limit} = $param->{-limit}; if ($self->{query}) { $self->{accession} = $self->get_accessions($self->{query}); } elsif ($self->{accession}) { my @accessions = ref $self->{accession} ? @{$self->{accession}} : ($self->{accession}); $self->{accession} = \@accessions; } else { croak "Must provide a 'query' or 'accession' argument" unless $self->{query} || $self->{accession} ; } $self; } sub fetch_next { my $self = shift; # if any additional records are left, then return them if ($self->{'records'} && @{$self->{'records'}}) { my $data = shift @{$self->{'records'}}; if ($data=~/\S/) { $self->_cleanup(\$data); return $data; } else { $self->{'records'} = []; } } my $format = $self->{format}; local ($/) = $format eq 'fasta' ? "\n>" : "//\n"; # if we have a socket open, then read a record if ($self->{'socket'}) { if (my $data = $self->_getline) { $self->_cleanup(\$data); return $data; } elsif (!$self->{accession} || @{$self->{accession}} == 0) { # nothing more to do return; } } die "Must provide either a list of accession numbers or an Entrez query" unless $self->{accession} || $self->{query}; return unless $self->get_entries; my $data = $self->_getline; $self->_cleanup(\$data); return $data; } sub _cleanup { my ($self,$d) = @_; $$d =~ s/\A\s+//; $$d=~s!//\n$!!; return unless $self->{format} eq 'fasta'; chomp $$d; substr($$d,0,0)='>' unless $$d =~/^>/; } sub get_accessions { my $self = shift; my $query = shift; my $sock = $self->_build_connection(ENTREZ_HOST) or return; # bug here: assume that the server will give us everything when we ask for 1 billion entries my $request = $self->_build_post(ENTREZ_HOST, QUERY_URI, undef, sprintf("db=%s&dispmax=%d&report=gen&mode=text&tool=boulder&term=%s",$self->db,$self->limit, escape($query))); print $sock $request; my $status = $self->_read_header($sock); return unless $status == 200; local $/ = ' '; my $line = $sock->getline; chomp $line; warn "*** ENTREZ: $line ***" unless $line =~ /^\d+$/; my @accessions = $line; while (defined ($line = $sock->getline)) { chomp $line; push @accessions,$line; } return \@accessions; } sub db { my $self = shift; my $db = $self->{db} || 'n'; my $translated = { m => 'medline', p => 'protein', n => 'nucleotide', s => 'popset' }->{$db}; $translated || $db; } # BUG: one billion = infinity sub limit { shift->{limit} || 1_000_000_000; } sub _build_connection { my $self = shift; my $host = shift; my ($hostent,$peer,$peerport); if (my $proxy = $self->{proxy}) { $proxy =~ m!^http://([^/]+)/?! or return; $hostent = $1; } else { $hostent = $host; } ($peer,$peerport) = split(':',$hostent); $peerport ||= 'http(80)'; my $sock = IO::Socket::INET->new( PeerAddr => $peer, PeerPort => $peerport, Proto => 'tcp' ); $sock; } sub _build_post { my $self = shift; my ($host,$uri,$type,$param) = @_; my $path = $self->{proxy} ? "http://$host$uri" : $uri; $type ||= 'application/x-www-form-urlencoded'; my $length = length($param); my $request = join (CRLF, "POST $path ".PROTO, "User-agent: Mozilla/5.0 [en] (PalmOS)", "Content-Type: $type", "Content-Length: $length", CRLF ); $request.$param; } sub _read_header { my $self = shift; my $sock = shift; local $/ = CRLF.CRLF; my $header = $sock->getline; return 500 unless $header; return 500 unless $header =~ /^HTTP\/[\d.]+ (\d+)/; $1; } sub get_entries { my $self = shift; my $format = $self->{format}; my $sock = $self->_build_connection(ENTREZ_HOST) or return; # create the multipart form... my $db = $self->{'db'}; my $boundary = '-' x 30 . int rand(10E14); my $name = 'Content-Disposition: form-data; name='; my %canned = ('db' => $db, 'FORMAT' => $format eq 'fasta' ? 1 : 0, 'REQUEST_TYPE' => $self->{accession} ? 'LIST_OF_GIS' : 'ADVANCED_QUERY', 'ORGNAME' => '', 'LIST_ORG' => '(None)', 'QUERY' => "$self->{query}\r\n", 'SAVETO' => 'YES', 'NOHEADER' => 'YES', ); my @records = map {qq(Content-Disposition: form-data; name="$_"\r\n\r\n$canned{$_}\r\n)} keys %canned; if (my $a = $self->{accession}) { my @accessions = splice(@$a,0,MAX_ENTRIES); return unless @accessions; my $accessions = join "\n",@accessions; push @records, qq{Content-Disposition: form-data; name="UID"; filename="accession.txt"\r\nContent-type: text/plain\r\n\r\n$accessions\r\n}; } my $content = "$boundary\r\n" . join("$boundary\r\n",@records) . "$boundary--\r\n"; my $request = $self->_build_post(ENTREZ_HOST, XBATCH_URI, "multipart/form-data; boundar=$boundary", $content); print $sock $request; local($/) = CRLF . CRLF; my $header = $sock->getline; return unless $header; return unless $header =~ /^HTTP\/[\d.]+ 200/; $/ = "\n"; my $line = $sock->getline; # this handles the case of Batch Entrez complaining that we're trying to # get too many sequences at once! if ($line =~ /exceed limit/) { my @accessions; while ($_ = $sock->getline) { chomp; push @accessions,$_; } delete $self->{query}; $self->{accession} = \@accessions; return $self->get_entries; # horrible recursion here! } else { while ($line =~ /(\*\*\*\* |WARNING: |ERROR: )(.+)/) { warn "**** GENBANK $1: $2\n"; $line = $sock->getline; } } $self->{bufferedline} = $line; if ($format eq 'fasta') { return unless $line =~ /^>/; } else { return unless $line =~ /LOCUS/; } $self->{socket} = $sock; return 1; } sub _getline { my $l = $_[0]->{socket}->getline; if ($_[0]->{bufferedline}) { $l = "$_[0]->{bufferedline}$l"; delete $_[0]->{bufferedline}; } return $l; } 1; __END__