Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson...
-
Upload
asher-doyle -
Category
Documents
-
view
217 -
download
0
Transcript of Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson...
![Page 1: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/1.jpg)
Web Databases for Drosophila
An introduction to web tools, databases and NCBI BLAST
Wilson Leung08/2015
![Page 2: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/2.jpg)
Agenda
• GEP annotation project overview
• Web databases for Drosophila annotation– UCSC Genome Browser– NCBI / BLAST– FlyBase– Gene Record Finder
![Page 3: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/3.jpg)
AAACAACAATCATAAATAGAGGAAGTTTTCGGAATATACGATAAGTGAAATATCGTTCTTAAAAAAGAGCAAGAACAGTTTAACCATTGAAAACAAGATTATTCCAATAGCCGTAAGAGTTCATTTAATGACAATGACGATGGCGGCAAAGTCGATGAAGGACTAGTCGGAACTGGAAATAGGAATGCGCCAAAAGCTAGTGCAGCTAAACATCAATTGAAACAAGTTTGTACATCGATGCGCGGAGGCGCTTTTCTCTCAGGATGGCTGGGGATGCCAGCACGTTAATCAGGATACCAATTGAGGAGGTGCCCCAGCTCACCTAGAGCCGGCCAATAAGGACCCATCGGGGGGGCCGCTTATGTGGAAGCCAAACATTAAACCATAGGCAACCGATTTGTGGGAATCGAATTTAAGAAACGGCGGTCAGCCACCCGCTCAACAAGTGCCAAAGCCATCTTGGGGGCATACGCCTTCATCAAATTTGGGCGGAACTTGGGGCGAGGACGATGATGGCGCCGATAGCACCAGCGTTTGGACGGGTCAGTCATTCCACATATGCACAACGTCTGGTGTTGCAGTCGGTGCCATAGCGCCTGGCCGTTGGCGCCGCTGCTGGTCCCTAATGGGGACAGGCTGTTGCTGTTGGTGTTGGAGTCGGAGTTGCCTTAAACTCGACTGGAAATAACAATGCGCCGGCAACAGGAGCCCTGCCTGCCGTGGCTCGTCCGAAATGTGGGGACATCATCCTCAGATTGCTCACAATCATCGGCCGGAATGNTAANGAATTAATCAAATTTTGGCGGACATAATGNGCAGATTCAGAACGTATTAACAAAATGGTCGGCCCCGTTGTTAGTGCAACAGGGTCAAATATCGCAAGCTCAAATATTGGCCCAAGCGGTGTTGGTTCCGTATCCGGTAATGTCGGGGCACAATGGGGAGCCACACAGGCCGCGTTGGGGCCCCAAGGTATTTCCAAGCAAATCACTGGATGGGAGGAACCACAATCAGATTCAGAATATTAACAAAATGGTCGGCCCCGTTGTTATGGATAAAAAATTTGTGTCTTCGTACGGAGATTATGTTGTTAATCAATTTTATTAAGATATTTAAATAAATATGTGTACCTTTCACGAGAAATTTGCTTACCTTTTCGACACACACACTTATACAGACAGGTAATAATTACCTTTTGAGCAATTCGATTTTCATAAAATATACCTAAATCGCATCGTC
Start codon Coding region Stop codon
Intron donor Intron acceptor UTR
![Page 4: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/4.jpg)
Annotation – adding labels to a sequence
• Genes: Novel or known genes, pseudogenes
• Regulatory Elements: Promoters, enhancers, silencers
• Non-coding RNA: tRNAs, miRNAs, siRNAs, snoRNAs
• Repeats: Transposable elements, simple repeats
• Structural: Origins of replication
• Experimental Results: – DNase I Hypersensitive sites– ChIP-chip and ChIP-Seq datasets (e.g. modENCODE)
![Page 5: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/5.jpg)
GEP Drosophila annotation projectsD. melanogasterD. simulansD. sechelliaD. yakubaD. erectaD. ficusphilaD. eugracilisD. biarmipesD. takahashiiD. elegansD. rhopaloaD. kikkawai
D. ananassaeD. bipectinata
D. pseudoobscura
D. persimilisD. willistoni
D. mojavensisD. virilisD. grimshawi
ReferencePublished
Annotation projects for Fall 2015 / Spring 2016
Species in the Four Genomes Paper
New species sequenced by modENCODE
Phylogenetic tree produced by Thom Kaufman as part of the modENCODE project
Manuscript in progress
![Page 6: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/6.jpg)
Gene annotation workflow
Visualize a genomic region with evidence tracks
GEP UCSC Genome Browser
Learn about the putative D. melanogaster ortholog
NCBI / FlyBase
Identify interesting features and putative orthologs
NCBI BLAST
Understand the gene and isoform structure
Gene Record Finder
![Page 7: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/7.jpg)
UCSC Genome Browser• Provide graphical view of genomic regions
– Sequence conservation – Gene and splice site predictions– RNA-Seq and splice junction predictions
• BLAT – BLAST-Like Alignment Tool– Map protein or nucleotide sequences against an assembly– Faster but less sensitive than BLAST
• Table Browser– Access data used to create the graphical browser
![Page 8: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/8.jpg)
UCSC Genome Browser overviewGenomic sequence
Evidence tracks
BLASTX alignments
Gene predictions
RNA-seq
Repeats
Comparative genomics
![Page 9: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/9.jpg)
Control how evidence tracks are displayed on the Genome Browser
• Most evidence tracks have five display modes:– Hide: track is hidden– Dense: all features (including overlapping features) are
displayed on a single line– Squish: overlapping features are drawn on separate lines,
features are half the height compared to full mode– Pack: overlapping features are drawn on separate lines,
features are the same height as full mode– Full: Each feature is displayed on its own line
• Some annotation tracks (e.g. RepeatMasker) only have a subset of these display modes
![Page 10: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/10.jpg)
Two different versions of the UCSC Genome Browser
http://genome.ucsc.edu
http://gander.wustl.edu
Official UCSC Version
GEP Version
Published data, lots of species, whole genomes, used for “Chimp Chunks”
GEP data, parts of genomes, used for annotation of Drosophila species
![Page 11: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/11.jpg)
Additional training resources
• Training section on the UCSC web site – http://genome.ucsc.edu/training.html– User guides– Mailing lists
• OpenHelix tutorials and training materials– http://www.openhelix.com/ucsc – Pre-recorded tutorial– Reference cards
![Page 12: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/12.jpg)
UCSC GENOME BROWSER DEMO
![Page 13: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/13.jpg)
Use BLAST to detect sequence similarity
• BLAST = Basic Local Alignment Search Tool
• Why is BLAST popular?– Provide statistical significance for each match– Good balance of sensitivity and speed
• Find local regions of similarity irrespective of where they are in the sequence
![Page 14: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/14.jpg)
Common types of BLAST programs
Program Query Database (Subject)BLASTN Nucleotide NucleotideBLASTP Protein ProteinBLASTX Nucleotide -> Protein Protein
TBLASTN Protein Nucleotide -> ProteinTBLASTX Nucleotide -> Protein Nucleotide -> Protein
• Except for BLASTN, all alignments are based on comparisons of protein sequences
• Decide which BLAST program to use based on the type of query and subject sequences:
![Page 15: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/15.jpg)
Common BLAST programs use cases• BLASTN: Search for similar nucleotide sequences
– Map contigs to genome, mRNAs/ESTs to genome
• BLASTP: Search for proteins similar to predicted genes
• BLASTX: Map protein / exons against genomic sequence
• TBLASTN: Map protein against genomic assemblies
• TBLASTX: Identify genes in unannotated sequences
• See the BLAST Homepage and Selected Search Pages document for details:• ftp://ftp.ncbi.nlm.nih.gov/pub/factsheets/HowTo_BLASTGuide.pdf
![Page 16: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/16.jpg)
NCBI BLAST nucleotide databases
• GenBank Non-Redundant Nucleotide Database (nr/nt)– Most comprehensive but some entries are low quality– Exclude sequences from whole genome assembly, ESTs
• RefSeq RNA Database– mRNA and non-coding RNA entries from the NCBI Reference
Sequence Project– Include real and computationally predicted sequences
• Expressed Sequence Tag (EST) Database– ESTs are short single reads of cDNA clones– High error rate but useful for identifying transcribed loci
![Page 17: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/17.jpg)
NCBI BLAST protein databases• GenBank Non-Redundant Protein Database (nr)
– Most comprehensive but some entries are low quality– Include sequences from both RefSeq and UniProtKB
• RefSeq Protein Database– Sequences from the NCBI Reference Sequence Project– Higher quality than the nr database– Include real and computationally predicted sequences
• UniProtKB / Swiss-Prot Protein Database– Manually curated proteins from literature– Real proteins with known functions– Much smaller database than either RefSeq or nr
![Page 18: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/18.jpg)
Where can I run BLAST?
• NCBI BLAST web service– http://blast.ncbi.nlm.nih.gov/Blast.cgi
• EBI BLAST web service– http://www.ebi.ac.uk/Tools/sss/
• FlyBase BLAST (Drosophila and other insects)– http://flybase.org/blast/
![Page 19: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/19.jpg)
NCBI BLAST DEMO
![Page 20: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/20.jpg)
National Center for Biotechnology Information (NCBI)
http://www.ncbi.nlm.nih.gov
![Page 21: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/21.jpg)
Key features of NCBI• Strengths
– Most comprehensive among publicly available databases– PubMed for literature searches– Comprehensive BLAST web service
• Weaknesses– Web site is large and complex– Quality of GenBank records may vary
• Use cases– Perform BLAST searches against Refseq, nr/nt databases– Compare one sequence against another (bl2seq)
![Page 23: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/23.jpg)
Key Features of FlyBase• Lots of ancillary data for each gene in Drosophila• Curation of literature for each gene• Reference Drosophila annotations for all the
other databases (including NCBI)• Fast release cycle (6-8 releases per year)
• Use cases– Species-specific BLAST searches– Genome browser (GBrowse) and access datasets for
the 20 sequenced Drosophila species
![Page 24: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/24.jpg)
Web databases and tools
• Many genome databases available– Be aware of different annotation releases– Use FlyBase as the canonical reference
• Web databases are being updated constantly– Update GEP materials before semester starts– Discrepancies in exercise screenshots– Minor changes in search results– Let us know about errors or revisions
![Page 25: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/25.jpg)
FLYBASE DEMO
![Page 26: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/26.jpg)
Gene Record Finder
http://gander.wustl.edu/~wilson/dmelgenerecord/index.html
![Page 27: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/27.jpg)
Key features of the Gene Record Finder• List of unique coding and non-coding exons for
each gene in D. melanogaster• CDS and exon usage maps for each isoform• Optimized for exon-by-exon annotation strategy• Slower update release cycle than FlyBase
– Database is updated every semester
• Use cases:– Get amino acid sequences and nucleotide
sequences of each exon for BLAST 2 Sequences (bl2seq) searches
![Page 28: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/28.jpg)
GENE RECORD FINDER DEMO
![Page 29: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/29.jpg)
Summary• GEP annotation project seeks to generate high quality
manually curated gene models for multiple Drosophila species
• Use BLAST to characterize a genomic sequence
• Use web databases to gather information on a gene– UCSC Genome Browser– NCBI– FlyBase– Gene Record Finder
![Page 30: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/30.jpg)
Questions?
http://www.flickr.com/photos/jac_opo/240254763/sizes/l/
![Page 31: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/31.jpg)
![Page 32: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/32.jpg)
Brief overview of the BLAST algorithm• BLAST is based on the Smith-Waterman algorithm
but use the following heuristics:– Build word list with the query and subject sequences
• Word size = 3 for protein, 11 for nucleotide
– Generate a list of high-scoring words• Determine by scoring system or scoring matrix
– Scan database for exact matches to high-scoring words– Extend matches to generate High-scoring Segment Pairs– Merge multiple HSP’s into a longer alignment– Calculate E-value for the alignments– Report alignments below E-value threshold
![Page 33: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/33.jpg)
Ensembl Metazoa: Databases for 12 Drosophila species
http://metazoa.ensembl.org
![Page 34: Web Databases for Drosophila An introduction to web tools, databases and NCBI BLAST Wilson Leung08/2015.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f455503460f94c679a7/html5/thumbnails/34.jpg)
Key features of Ensembl Metazoa• Lots of ancillary data for each gene• Data for 12 Drosophila species available• Detailed information on each gene available at the
transcript, peptide, and exon level• Not always up-to-date
– Annotations are from FlyBase Release 6.02
• Use cases– Get amino acid sequences and nucleotide sequences
of each exon for bl2seq searches– Perform species-specific BLAST searches