Web Databases for Drosophila
An introduction to web tools, databases
and NCBI BLAST
Wilson Leung
01/2016
Agenda
• GEP annotation project overview
• Web databases for Drosophila annotation
– UCSC Genome Browser
– NCBI / BLAST
– FlyBase
– Gene Record Finder
AAACAACAATCATAAATAGAGGAAGTTTTCGGAATATACGATAAGTGAAATATCGTTCT
TAAAAAAGAGCAAGAACAGTTTAACCATTGAAAACAAGATTATTCCAATAGCCGTAAGA
GTTCATTTAATGACAATGACGATGGCGGCAAAGTCGATGAAGGACTAGTCGGAACTGGA
AATAGGAATGCGCCAAAAGCTAGTGCAGCTAAACATCAATTGAAACAAGTTTGTACATC
GATGCGCGGAGGCGCTTTTCTCTCAGGATGGCTGGGGATGCCAGCACGTTAATCAGGAT
ACCAATTGAGGAGGTGCCCCAGCTCACCTAGAGCCGGCCAATAAGGACCCATCGGGGGG
GCCGCTTATGTGGAAGCCAAACATTAAACCATAGGCAACCGATTTGTGGGAATCGAATT
TAAGAAACGGCGGTCAGCCACCCGCTCAACAAGTGCCAAAGCCATCTTGGGGGCATACG
CCTTCATCAAATTTGGGCGGAACTTGGGGCGAGGACGATGATGGCGCCGATAGCACCAG
CGTTTGGACGGGTCAGTCATTCCACATATGCACAACGTCTGGTGTTGCAGTCGGTGCCA
TAGCGCCTGGCCGTTGGCGCCGCTGCTGGTCCCTAATGGGGACAGGCTGTTGCTGTTGG
TGTTGGAGTCGGAGTTGCCTTAAACTCGACTGGAAATAACAATGCGCCGGCAACAGGAG
CCCTGCCTGCCGTGGCTCGTCCGAAATGTGGGGACATCATCCTCAGATTGCTCACAATC
ATCGGCCGGAATGNTAANGAATTAATCAAATTTTGGCGGACATAATGNGCAGATTCAGA
ACGTATTAACAAAATGGTCGGCCCCGTTGTTAGTGCAACAGGGTCAAATATCGCAAGCT
CAAATATTGGCCCAAGCGGTGTTGGTTCCGTATCCGGTAATGTCGGGGCACAATGGGGA
GCCACACAGGCCGCGTTGGGGCCCCAAGGTATTTCCAAGCAAATCACTGGATGGGAGGA
ACCACAATCAGATTCAGAATATTAACAAAATGGTCGGCCCCGTTGTTATGGATAAAAAA
TTTGTGTCTTCGTACGGAGATTATGTTGTTAATCAATTTTATTAAGATATTTAAATAAA
TATGTGTACCTTTCACGAGAAATTTGCTTACCTTTTCGACACACACACTTATACAGACA
GGTAATAATTACCTTTTGAGCAATTCGATTTTCATAAAATATACCTAAATCGCATCGTC
Start codon
Coding region
Stop codon
Intron donor
Intron acceptor
UTR
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)
GEP Drosophila annotation projects
D. melanogaster
D. simulans
D. sechellia
D. yakuba
D. erecta
D. ficusphila
D. eugracilis
D. biarmipes
D. takahashii
D. elegans
D. rhopaloa
D. kikkawai
D. bipectinata
D. ananassae
D. pseudoobscura
D. persimilis
D. willistoni
D. mojavensis
D. virilis
D. grimshawi
Reference
Published
Species in the Four
Genomes Paper
Annotation projects for
Fall 2015 / Spring 2016
Manuscript in progress
New species sequenced
by modENCODE
Phylogenetic tree produced by Thom Kaufman as part of the modENCODE project
Gene annotation workflow
Visualize a genomic region
with evidence tracks
GEP UCSC Genome Browser
Identify interesting features
and putative orthologs
NCBI BLAST
Learn about the putative
D. melanogaster ortholog
NCBI / FlyBase
Understand the gene and
isoform structure
Gene Record Finder
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
UCSC Genome Browser overview
Genomic sequence
Gene
predictions
RNA-seq
Repeats
Comparative
genomics
Evidence tracks
BLASTX
alignments
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
Two different versions of the
UCSC Genome Browser
Official UCSC
Version
http://genome.ucsc.edu
Published data, lots of
species, whole genomes,
used for “Chimp Chunks”
GEP Version
http://gander.wustl.edu
GEP data, parts of
genomes, used for
annotation of Drosophila
species
Additional training resources
• Training section on the UCSC web site
– http://genome.ucsc.edu/training/index.html
– User guides
– Mailing lists
• OpenHelix tutorials and training materials
– http://www.openhelix.com/ucsc
– Pre-recorded tutorial
– Reference cards
UCSC GENOME BROWSER DEMO
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
Common types of BLAST programs
• 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:
Program
BLASTN
BLASTP
Query
Nucleotide
Protein
Database (Subject)
Nucleotide
Protein
BLASTX
TBLASTN
TBLASTX
Nucleotide → Protein
Protein
Nucleotide → Protein
Protein
Nucleotide → Protein
Nucleotide → Protein
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
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
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
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/
NCBI BLAST DEMO
National Center for Biotechnology Information (NCBI)
http://www.ncbi.nlm.nih.gov
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)
FlyBase - Database for the
Drosophila research community
http://flybase.org/
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 20
Drosophila species
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
FLYBASE DEMO
Gene Record Finder
http://gander.wustl.edu/~wilson/dmelgenerecord/index.html
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
GENE RECORD FINDER DEMO
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
Questions?
http://www.flickr.com/photos/jac_opo/240254763/sizes/l/
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
Ensembl Metazoa:
Databases for 12 Drosophila species
http://metazoa.ensembl.org/index.html
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