Next Generation Sequencing Data Visualization GBrowse2 from GMOD Andreas Gisel Institute for Biomedical Technologies CNR Bari - Italy
GMOD is the Generic Model Organism Database project GMOD is a collection of interconnected applications and databases that biologists use as repositories and as tools. That connectivity is really the key here. There's no lack of tools, but many of these tools will be little used since the typical prospective user may not have the resources or expertise required to install the tool and connect it, in some way, to the data in hand. http://www.gmod.org/
The tutorial will show: how to display a reference sequence with feature in GBrowse2 how to display Next Generation Sequencing (NGS) mapping data. We will use Escherichia coli str. K-12 substr. DH10B, complete genome - NC_010473.1 (ref1.fa) and Illumina pair-end reads1.fa and reads2.fa
GBrowse2 http://localhost/cgi-bin/gb2/gbrowse/yeast_advanced/
GBrowse2 http://localhost/cgi-bin/gb2/gbrowse/yeast_advanced/
GBrowse2 /etc/gbrowse2/
GBrowse2 /var/lib/gbrowse2/databases Text
GBrowse2 Set-up the gbrowser for E.coli data We have: Reference sequence data in FASTA Annotation for the reference sequence in GFF
GBrowse2 Set-up the gbrowser for E.coli data We have: Reference sequence data in FASTA Annotation for the reference sequence in GFF We need: E. coli configuration file Add E.coli informatio to the general GBrowse.conf file Add data to the coresponding database
Annotation Data Formats EMBL annotation files GeneBank annotation files GFF files
GeneBank format LOCUS HQ336405 157790 bp DNA circular PLN 22-DEC-2010 DEFINITION Prunus persica chloroplast, complete genome. ACCESSION HQ336405 VERSION HQ336405.1 GI:309321413 KEYWORDS. SOURCE chloroplast Prunus persica (peach) ORGANISM Prunus persica Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; rosids; fabids; Rosales; Rosaceae; Maloideae; Amygdaleae; Prunus. REFERENCE 1 (bases 1 to 157790) AUTHORS Jansen,R.K., Saski,C., Lee,S.B., Hansen,A.K. and Daniell,H. TITLE Complete Plastid Genome Sequences of Three Rosids (Castanea, Prunus, Theobroma): Evidence for At Least Two Independent Transfers of rpl22 to the Nucleus JOURNAL Mol. Biol. Evol. 28 (1), 835-847 (2011) PUBMED 20935065 REFERENCE 2 (bases 1 to 157790) AUTHORS Jansen,R.K., Saski,C., Lee,S.-B., Hansen,A.K. and Daniell,H. TITLE JOURNAL Direct Submission Submitted (28-SEP-2010) Integrative Biology, University of Texas at Austin, 1 University Station C0930, Austin, TX 78712, USA
GeneBank format FEATURES Location/Qualifiers source 1..157790 /organism="prunus persica" /organelle="plastid:chloroplast" /mol_type="genomic DNA" /db_xref="taxon:3760" gene complement(join(99804..100597,71346..71459)) /gene="rps12" /trans_splicing CDS complement(join(99804..99829,100366..100597,71346..71459)) /gene="rps12" /trans_splicing /codon_start=1 /transl_table=11 /product="ribosomal protein S12" /protein_id="ado64999.1" /db_xref="gi:309321458" /translation="mptikqlirntrqpirnvtkspalggcpqrrgtctrvytitpkk PNSALRKVARVRLTSGFEITAYIPGIGHNLQEHSVVLVRGGRVKDLPGVRYHIVRGTL DAVGVKDRQQGRSKYGVKKPK"
GeneBank format ORIGIN 1 tgggcgaacg acgggaattg aacccgcgca tggtggattc acaatccact gccttgatcc 61 acttggctac atccgcccct tatactatta caaatattta caccatttat cattacttgt 121 aagataaaat acaacataaa ataaactgaa acttttaata ttttaattaa attttgtagt 181 aaattaacta aaaaaaaata tagaacaaaa caatatagta aagttaagta gtaaataaaa 241 aaaatactaa atagtaaagg agcaataaca aacctcttga tataacaaga aatttattat 301 tgctccttta ctttcaagaa ctcctatata ctaagaccaa agtcttatcc atttatagat 361 ggaacttcaa cagcagctag atctagaggg aaattatggg cattacgttc atgcataact 421 tccataccaa ggttagcgcg gttaataata tcagcccaag tattaattac acgaccctga 481 ctatcaacta cagattgatt gaaattaaaa ccatttaagt tgaaagccat agtgctgata 541 cctaaagcgg taaaccagat acctactaca ggccaagcag ctaggaagaa atgtaaagaa 601 cgagaattgt tgaaactagc atattggaag atcaatcggc caaaataacc atgagcggct 661 acgatattat aggtttcttc ctcttgaccg aatctgtaac cttcattagc agattcattt 721 tctgtggttt ccctgatcaa actagaggtt accaaggacc catgcatagc actgaatagg 781 gagccgccga atacaccagc tacgcctaac atgtgaaatg ggtgcataag gatgttgtgc 841 tcggcttgga atacaatcat gaagttgaaa gtaccggaga ttcctagggg cataccgtca 901 gaaaagcttc cttgaccaat tggatatatc aagaaaacag cagtagcagc tgcaacagga 961 gctgaatatg caacagcaat ccaagggcgc atacccagac ggaaactaag ttcccactca 1021 cgacccatgt agcaagctac accaagtaag aagtgtagaa caattagttc ataaggacca 1081 ccgttgtata accattcatc aacggaagcc gcttcccata tcgggtaaaa gtgcaaacct 1141 atagctgcag aggtaggaat aatggcacca gaaataatat tgtttccata aagtaaagat 1201 ccagaaacag gttcacgaat accatcaata tctactggag gtgcagcaat gaaagcaata
GFF format ##gff-version 3 # sequence-region HQ336405 1 157790 # conversion-by bp_genbank2gff3.pl # organism Prunus persica # date 22-DEC-2010 # Note Prunus persica chloroplast, complete genome. # working on region:hq336405, Prunus persica, 22-DEC-2010, Prunus persica chloroplast, complete genome. # Possible gene unflattening error withhq336405: consult STDERR HQ336405!GenBank! region! 1! 157790!.! +!.! ID=HQ336405;Dbxref=taxon:3760;Note=Prunus persica chloroplast complete genome;date=22-dec-2010;mol_type=genomic DNA;organelle=plastid:chloroplast;organism=Prunus persica HQ336405!GenBank! CDS!99804! 99829!.! -!.! ID=rps12;Dbxref=GI: 309321458;codon_start=1;gene=rps12;product=ribosomal protein S12;protein_id=ADO64999.1;trans_splicing=_no_value;transl_table=11;translation=length.123 HQ336405!GenBank! CDS!100366! 100597!.! -!.! ID=rps12;Dbxref=GI: 309321458;codon_start=1;gene=rps12;product=ribosomal protein S12;protein_id=ADO64999.1;trans_splicing=_no_value;transl_table=11;translation=length.123 HQ336405!GenBank! CDS!71346! 71459!.! -!.! ID=rps12;Dbxref=GI: 309321458;codon_start=1;gene=rps12;product=ribosomal protein S12;protein_id=ADO64999.1;trans_splicing=_no_value;transl_table=11;translation=length.123 HQ336405!GenBank! gene!99804! 100597!.! -!. ID=rps12;gene=rps12;trans_splicing=_no_value HQ336405!GenBank! gene!71346! 71459!.! -!. ID=rps12;gene=rps12;trans_splicing=_no_value HQ336405!GenBank! gene!3! 77!.! -!.! ID=trnH-GUG;gene=trnH-GUG HQ336405!GenBank! trna!3! 77!.! -!.! ID=trnH-GUG.r01;Parent=trnH GUG;Note=anticodon:GUG;gene=trnH-GUG;product=tRNA-His
GBrowse2 Create and configure the tracks visualizing the date of E.coli [GENERAL] description = Escherichia coli str. K-12 substr. DH10B database = annotations initial landmark = NC_010473.1:1..10000 # bring in the special Submitter plugin for the rubber-band select menu plugins = FastaDumper RestrictionAnnotator SequenceDumper TrackDumper Submitter S autocomplete = 1 default tracks = Genes ORFs trnas CDS Transp Centro:overview GC:region # examples to show in the introduction examples = NC_010473.1:3170000..3180000 # "automatic" classes to try when an unqualified identifier is given automatic classes = Symbol Gene Clone
GBrowse2 Create and configure the tracks visualizing the date of E.coli ################################# # database definitions ################################# [scaffolds:database] db_adaptor = Bio::DB::SeqFeature::Store db_args = -adaptor memory -dir /var/lib/gbrowse2/databases/ecoli_seq search options = default +autocomplete [annotations:database] db_adaptor = Bio::DB::SeqFeature::Store db_args = -adaptor memory -dir /var/lib/gbrowse2/databases/ecoli_annotations search options = default +autocomplete
GBrowse2 Create and configure the tracks visualizing the date of E.coli # Default glyph settings [TRACK DEFAULTS] glyph = generic database = annotations height = 8 bgcolor = cyan fgcolor = black label density = 25 bump density = 100 show summary = 99999 # go into summary mode when zoomed out to 100k # default pop-up balloon balloon hover = <b>$name</b> is a $type spanning $ref from $start to $end. Click for more details. [CDS] feature = gene glyph = cds description = 0 height = 26 sixframe = 1 label = sub {shift->name. " reading frame"} key = CDS balloon click width = 500 balloon hover width = 350 balloon hover = <b>$name</b> is a $type spanning $ref from $start to $end. Click to search Google for $name. balloon click = http://www.google.com/search?q=$name citation = This track shows CDS reading frames.
GBrowse2 Insert E.coli in the general GBrowse config file ############################################################################## # # DATASOURCE DEFINITIONS # One stanza for each configured data source # ############################################################################## [yeast] description = Yeast chromosomes 1+2 (basic) path = yeast_simple.conf [yeast_advanced] description = Yeast chromosomes 1+2 (advanced) path = yeast_chr1+2.conf [ecoli] description = Escherichia coli str. K-12 substr. DH10B path = ecoli.conf
GBrowse2 Add data to databases ################################# # database definitions ################################# [scaffolds:database] db_adaptor = Bio::DB::SeqFeature::Store db_args = -adaptor memory -dir /var/lib/gbrowse2/databases/ecoli_seq search options = default +autocomplete [annotations:database] db_adaptor = Bio::DB::SeqFeature::Store db_args = -adaptor memory -dir /var/lib/gbrowse2/databases/ecoli_annotations search options = default +autocomplete Create: /var/lib/gbrowse2/databases/ecoli_seq /var/lib/gbrowse2/databases/ecoli_annotations
GBrowse2 Add data to databases Move to: /var/lib/gbrowse2/databases/ecoli_seq - ref1.fa - chromosomes.gff3 /var/lib/gbrowse2/databases/ecoli_annotations - NC_010473.gff
GBrowse2 http://localhost/cgi-bin/gb2/gbrowse/ecoli/
Create mapping data Map with bowtie: index: bowtie-build -f ref1.fa ref1 map: bowtie -n 1 -l 30 -I 0 -X 400 --un unmapped -p 2 -S ref/ ref1-1 illumina/reads1.fq -2 illumina/reads2.fq > pair.sam SAM to BAM: index: samtools faidx ref1.fa SAM to BAM: samtools import ref1.fa.fai pair.sam pair.bam sort BAM: samtools sort pair.bam pair_sorted index BAM: samtools index pair_sorted.bam
Modify the ecoli.conf Add database: [ecolisam:database] db_adaptor = Bio::DB::Sam db_args = -fasta /var/lib/gbrowse2/databases/ecolisam/ref1.fa -bam /var/lib/gbrowse2/databases/ecolisam/pair_sorted.bam search options = none Add tracks: [CoverageXyplot] feature = coverage glyph = wiggle_xyplot database = ecolisam height = 50 fgcolor = black bicolor_pivot = 20 pos_color = blue neg_color = red key = Coverage (xyplot) category = Reads label = 0 # Labels on wiggle tracks are redundant.
Create mapping database create directory ecolisam in /var/lib/gbrowse2/databases copy pair_sorted.bam and pair_sorted.bam.bai to ecolisam and set the right privileges to ecolisam so that the browser can access them
E.coli GBrowse