Annotate-a-genome: Difference between revisions

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imported>Weigang
imported>Weigang
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|-
|-
| 115 || CA8 || B. burgdorferi (California) || Lyme Disease ||  
| 115 || CA8 || B. burgdorferi (California) || Lyme Disease ||  
* [http://www.ncbi.nlm.nih.gov/nuccore/?term=ADMY01000001:ADMY01000007%5Baccn%5D 8 unassembled contigs]  
* [http://www.ncbi.nlm.nih.gov/nuccore/?term=ADMY01000001:ADMY01000007%5Baccn%5D 7 unassembled contigs]  
||  
||  
* Accession: ADMY01000001; Assigned to: AA
* Accession: ADMY01000001; Assigned to: AA
* Accession: ADMY01000002; Assigned to: AA
* Accession: ADMY01000002; Assigned to: TAA
* Accession: ADMY01000003; Assigned to: AA
* Accession: ADMY01000003; Assigned to: KD
* Accession: ADMY01000004; Assigned to: AA
* Accession: ADMY01000004; Assigned to: JG
* Accession: ADMY01000005; Assigned to: AA
* Accession: ADMY01000005; Assigned to: KPG
* Accession: ADMY01000006; Assigned to: AA
* Accession: ADMY01000006; Assigned to: GG
* Accession: ADMY01000007; Assigned to: AA
* Accession: ADMY01000007; Assigned to: TDH
* Accession: ADMY01000008; Assigned to: AA
|-
|-
| 304 || BgVir || B. garinii (Russia) || Lyme Disease ||
| 304 || BgVir || B. garinii (Russia) || Lyme Disease ||
Line 36: Line 35:
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003201.1 cp26]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003201.1 cp26]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003202.1 lp54]  
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003202.1 lp54]  
|| Example
||  
* Accession: CP003151; Assigned to: LH
* Accession: CP003201; Assigned to: SK
* Accession: CP003202; Assigned to: BK
|-
|-
| 305 ||  NMJW1 || B. garinii (China) || Lyme Disease ||  
| 305 ||  NMJW1 || B. garinii (China) || Lyme Disease ||  
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003866.1 main chromosome]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003866.1 main chromosome]
|| Example
|| Accession: CP003866.1; Assigned to: AL
|-
|-
| 402 || HLJ01 || B. afzelii (China) || Lyme Disease ||  
| 402 || HLJ01 || B. afzelii (China) || Lyme Disease ||  
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003882.1 main chromosome]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003882.1 main chromosome]
|| Example
|| Accession: CP003882; Assigned to: RL
|-
|-
| 1003|| Ly || B. duttonii (Tanzania) || Relapsing Fever ||
| 1003|| Ly || B. duttonii (Tanzania) || Relapsing Fever ||
Line 50: Line 52:
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000980.1 lp23 (homolog of cp26 in LD genomes)]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000980.1 lp23 (homolog of cp26 in LD genomes)]
* Many other plasmids (not to include)
* Many other plasmids (not to include)
|| Example
||  
* Accession: CP000976; Assigned to: HL
* Accession: CP000980; Assigned to: NM
|-
|-
| 1001 || A1 ||B. recurrentis (Ethiopia) || Relapsing Fever ||  
| 1001 || A1 ||B. recurrentis (Ethiopia) || Relapsing Fever ||  
Line 56: Line 60:
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000994.1 lp124 (homologous to lp54 in LD genomes)]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000994.1 lp124 (homologous to lp54 in LD genomes)]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000995.1 lp23]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000995.1 lp23]
|| Example
||  
* Accession: CP000993; Assigned to: JP
* Accession: CP000994; Assigned to: DP
* Accession: CP000995; Assigned to: GAR
|-
|-
| 1100 || DAH || B. hermsii (Washington State) || Relapsing Fever ||  
| 1100 || DAH || B. hermsii (Washington State) || Relapsing Fever ||  
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000048.1 main chromosome]  
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000048.1 main chromosome]  
|| Example
|| Accession: CP000048; Assigned to: KR
|-
|-
| 1200 || 91E135 || B. turicatae (Texas) || Relapsing Fever ||
| 1200 || 91E135 || B. turicatae (Texas) || Relapsing Fever ||
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000049.1 main chromosome]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP000049.1 main chromosome]
|| Example
|| Accession: CP000049; Assigned to: MDR
|-
|-
| 1002 || Achema || B. crocidurae (Mauritania) || Relapsing Fever ||
| 1002 || Achema || B. crocidurae (Mauritania) || Relapsing Fever ||
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003426.1 main chromosome]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP003426.1 main chromosome]
* Many unassembled plasmids (not to include)  
* Many unassembled plasmids (not to include)  
|| Example
|| Accession: CP003426; Assigned to: VS
|-
|-
|1400 || HR1 || B. parkeri (??) || Relapsing Fever ||  
|1400 || HR1 || B. parkeri (??) || Relapsing Fever ||  
* [http://www.ncbi.nlm.nih.gov/nuccore/CP007022.1 main chromosome]
* [http://www.ncbi.nlm.nih.gov/nuccore/CP007022.1 main chromosome]
|| Example
|| Accession: CP0007022; Assigned to: AV
|-
|-
| 1300 || LB-2001 || B. miyamotoi (Northeast US) || Relapsing Fever ||  
| 1300 || LB-2001 || B. miyamotoi (Northeast US) || Relapsing Fever ||  
* [http://www.ncbi.nlm.nih.gov/nuccore/CP006647.1 main chromosome]  
* [http://www.ncbi.nlm.nih.gov/nuccore/CP006647.1 main chromosome]  
|| Example
|| Accession: CP006647; Assigned to: LLW
|}
|}



Revision as of 02:49, 9 March 2014

Project Goals

A Borrelia Phylogeny
  • Annotate and add newly sequenced Borrelia genomes to BorreliaBase
  • Build an informatics pipeline for gene prediction, ortholog calls, databasing, and synteny analysis

Download genome sequences from GenBank

Genome_id Strain Species Group Genome Sequences Notes
100 B31 B. burgdorferi (reference genome) Lyme Disease Reference. Already downloaded as "ref.pep"
114 CA382 B. burgdorferi (California) Lyme Disease Accession: CP005925; Assigned to: HA
115 CA8 B. burgdorferi (California) Lyme Disease
  • Accession: ADMY01000001; Assigned to: AA
  • Accession: ADMY01000002; Assigned to: TAA
  • Accession: ADMY01000003; Assigned to: KD
  • Accession: ADMY01000004; Assigned to: JG
  • Accession: ADMY01000005; Assigned to: KPG
  • Accession: ADMY01000006; Assigned to: GG
  • Accession: ADMY01000007; Assigned to: TDH
304 BgVir B. garinii (Russia) Lyme Disease
  • Accession: CP003151; Assigned to: LH
  • Accession: CP003201; Assigned to: SK
  • Accession: CP003202; Assigned to: BK
305 NMJW1 B. garinii (China) Lyme Disease Accession: CP003866.1; Assigned to: AL
402 HLJ01 B. afzelii (China) Lyme Disease Accession: CP003882; Assigned to: RL
1003 Ly B. duttonii (Tanzania) Relapsing Fever
  • Accession: CP000976; Assigned to: HL
  • Accession: CP000980; Assigned to: NM
1001 A1 B. recurrentis (Ethiopia) Relapsing Fever
  • Accession: CP000993; Assigned to: JP
  • Accession: CP000994; Assigned to: DP
  • Accession: CP000995; Assigned to: GAR
1100 DAH B. hermsii (Washington State) Relapsing Fever Accession: CP000048; Assigned to: KR
1200 91E135 B. turicatae (Texas) Relapsing Fever Accession: CP000049; Assigned to: MDR
1002 Achema B. crocidurae (Mauritania) Relapsing Fever Accession: CP003426; Assigned to: VS
1400 HR1 B. parkeri (??) Relapsing Fever Accession: CP0007022; Assigned to: AV
1300 LB-2001 B. miyamotoi (Northeast US) Relapsing Fever Accession: CP006647; Assigned to: LLW

Protocol

Dependencies

Fetch genome sequences

  • Commands:
./bioseq -z 'b31_accession' -o 'genbank' > b31.gb # Reference genome for ortholog identification. Choose main, cp26, or lp54
./bioseq -z 'gb_accession' -o 'genbank' > new.gb
./gb2fas -n b31.gb > b31.nuc # Extract CDS
./gb2fas -n new.gb > new.nuc
./bioseq -t b31.nuc > b31.pep # Translate (and remove those with internal stop codons)
./bioseq -t new.nuc > new.pep
  • Perl code for "gb2fas.pl":
#!/usr/bin/env perl
# Extract sequences from a GenBank file
# Input: a GenBank file
# Output: -n: CDS sequences in FASTA; -t: CDS information in Tab-delimited
use strict;
use Bio::SeqIO;
use Getopt::Std;
use Data::Dumper;
use 5.10.0;

my %opts;
getopts('tn',\%opts);

die "$0 [-nt] <genbank_file>\n" unless @ARGV == 1;
my $gb_file = shift @ARGV;
my $in = Bio::SeqIO->new(-file=>$gb_file, -format=>'genbank');
my $cds_ct=0;
while (my $seqobj = $in->next_seq() ) {
    my @features = $seqobj->get_SeqFeatures(); # just top level
    foreach my $feat ( @features ) {
	next unless $feat->primary_tag eq "CDS";
	$cds_ct++;
	&to_db($feat, $cds_ct) if $opts{t};
	&to_nt($feat, $seqobj) if $opts{n};
    }
}

exit;

sub to_nt {
    my $ft = shift;
    my $seq = shift;
    say ">", $ft->get_tag_values("locus_tag");
    my $subseq = $seq->trunc($ft->start, $ft->end);
    if ($ft->strand > 0) {
	say $subseq->seq();
    } else {
	say $subseq->revcom()->seq();
    }
}

sub to_db {
    my $ft = shift;
    my $ct = shift;
    my $orf_id = sprintf "ORF%04d", $ct;
    my $gid = 401; # this is bad and needs improvement: nothing should be hard-coded
    my $con_id = 111114823; # the same problem
    my $locus =  sprintf "%s", $ft->get_tag_values('locus_tag');
    my $prod = sprintf "%s", $ft->get_tag_values('product');
    $prod =~ tr/\'/_/;
    my $strand = ($ft->strand > 0) ? 't' : 'f';
    say join "\t", ($gid, $con_id, $orf_id, 'f', $ft->start, $ft->end, $strand, $locus, $prod);
}

Predict orthologs with reciprocal BLAST

  • Commands:
makeblastdb -in b31.pep -parse_seqids # Prepare the reference DB
makeblastdb -in new.pep -parse_seqids # Prepare the new genome DB
blastp -query new.pep -db b31.pep -outfmt 6 -evalue 1e-3 -out forward_blast.out # Forward BLAST
blastp -query b31.pep -db new.pep -outfmt 6 -evalue 1e-3 -out reverse_blast.out # Reverse BLAST
./check-reciprocal.pl forward_blast.out reverse_blast.out > new.orthlogs 2> new.not-orthologs # Identify orthologs
  • Code for "check-reciprocal.pl":
#!/usr/bin/env perl
use strict;
use warnings;
use Data::Dumper;

die "$0 <forward_blast_output> <reverse_blast_output>\n" unless @ARGV == 2;
my ($fwd, $rev) = @ARGV;
my (@fwd_top_hits, @rev_top_hits);
open FWD, "<" . $fwd;
my (%fwd_top_hits, %rev_top_hits, @query);
my $query_ct=0;
while (<FWD>) {
    chomp;
    my @data = split;
    next if $fwd_top_hits{$data[0]};
    $fwd_top_hits{$data[0]} = $data[1];
    push @query, $data[0];
    $query_ct++;
}
close FWD;
warn "Total query having hits:" . $query_ct . "\n";

open REV, "<" . $rev;
while (<REV>) {
    chomp;
    my @data = split;
    next if $rev_top_hits{$data[0]};
    $rev_top_hits{$data[0]} = $data[1];
}
close REV;

foreach my $q (@query) { # e.g., BafPKo_0002
    my $top = $fwd_top_hits{$q}; # e.g. BB_0002
    if ( $q eq $rev_top_hits{$top}) {
	print "Found reciprocol top hits:\t", $q, "\t", $top, "\n";
    } else {
	warn "Not reciprocol top hits:\t", $q, "\t", $top, "\t", $rev_top_hits{$top}, "\n";
    }
}

exit;

Verify with synteny broswer

./gb2fas -t new.gb > new-to-orf-table.txt
# load into database with SQL
# Visualize synteny