A Field Guide part 2

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A Field Guide part 2
National Center for Biotechnology Information
UT-Health Science Center
February 14, 2006
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GenBank Records
The Flatfile Format
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A Typical GenBank Record
LOCUS NM_019570 4279 bp mRNA linear INV
28-OCT-2004 DEFINITION Mus musculus REV1-like(S.
cerevisiae)(Rev1l),mRNA ACCESSION
NM_019570 VERSION NM_019570.3 GI50811869
KEYWORDS .
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GenBank Record Feature Table
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GenBank Record Feature Table, cont.
GenPept identifier
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GenBank Record sequence
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Indexing for Nucleotide UID 59958365
Field Indexed Terms primary
accession NM_001012399 title Bos taurus
hemochromatosis (hfe), mRNA. organism Bos
taurus sequence length 1168 modification
date 2005/02/19 properties biomol
mrna gbdiv mam srcdb refseq
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Global Entrez Search HFE
HFE
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Entrez Nucleotide HFE
137 records
Not HFE
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Smarter Query
hfetitle
AND humanorgn
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hfetitle AND humanorgn (cont)
Primary data
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Preview/IndexGateway to Advanced Searches
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Preview/Index
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Preview/Index Properties, srcdb
Properties
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Preview/Index Properties, srcdb
AND srcdb refseqProperties
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Preview/Index Properties, srcdb
AND srcdb ddbj/embl/genbankProperties
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Database Queries

• 1 hfe 137
• 2 hfetitle AND humanorgn 42
• 3 2 AND srcdb refseqprop 11
• 4 2 AND srcdb ddbj/embl/genbankprop
  31

5 4 AND gbdiv priprop 29 4
4 AND gbdiv estprop 2
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Molecule Queries

• 1 hfe 116
• 2 hfetitle AND humanorgn
  42
• 3 2 AND biomol mrnaprop 29
• 4 2 AND biomol genomicprop
  13

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More Queries
Fields are database-specific
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More Queries
Fields are database-specific
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Other Entrez Databases
UniGene rat clusters that have at least one
mRNA ratorganism NOT 0mrna count
SNP uniquely mapped microsatellites on human
chr2 microsatSNP Class AND 1Map Weight AND
2Chromosome) AND humanorgn
UniSTS markers on the Genethon map of human
chromosome 12 GenethonMap Name AND
humanorganism AND 12chromosome
Structure structures of bacterial kinases with
resolutions below 2 Å bacteriaorganism AND
kinase AND 000.00002.00resolution
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Genome Resources
Genomic Biology
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Genomic Biology
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Gen Biol Gen Resources
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Map Viewer Genome Annotation Updates
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Gen Biol Gen Resources
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Genome Projects microb
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Genome Projects microb
13 Eukaryotic Genome Sequencing Projects
Selected Complete 0, Assembly 2, In Progress
- 11
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Genome Projects microb
13 Eukaryotic Genome Sequencing Projects
Selected Complete 0, Assembly 2, In Progress
- 11
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Gen Biol Gen Resources
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Genome Resources
Genomic Biology
UniGene
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UniGene
Gene-oriented clusters of expressed sequences

• Automatic clustering using MegaBlast
• Each cluster represents a unique gene
• Informed by genome hits
• Information on tissue types and map locations
• Useful for gene discovery and selection of
  mapping reagents

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A Cluster of ESTs
query
5 EST hits
3 EST hits
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UniGene Collections
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UniGene Collections
Species UniGene
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UniGene Hs build 188
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UniGene Cluster Hs.95351Lipase,
hormone-sensitive (LIPE)
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UniGene Cluster Hs.95351
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UniGene Cluster Hs.95351 expression
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UniGene Cluster Hs.95351 seqs
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Get Sequences
web page
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Genome Resources
Genomic Biology
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E-PCR
Genomic sequence here
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Options
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Results
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reverse e-pcr
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reverse e-pcr
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reverse e-pcr
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reverse e-pcr
Gene
STS
LY6G6D lymphocyte antigen 6 complex, locus G6D
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Genome Resources
Genomic Biology
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List View
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Human MapViewer
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MapViewer Human ADAR
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MV Hs ADAR
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Maps Options
Maps Options
--Sequence maps-- Ab initio Assembly Repeats BES_C
lone Clone NCI_Clone Contig Component CpG
island dbSNP haplotype Fosmid GenBank_DNA Gene Phe
notype SAGE_Tag STS TCAG_RNA Transcript
(RNA) Hs_UniGene Hs_EST
--Cytogenetic maps-- Ideogram FISH
Clone Gene_Cytogenetic Mitelman
Breakpoint Morbid/Disease --Genetic
Maps-- deCODE Genethon Marshfield --RH
maps-- GeneMap99-G3 GeneMap99-GB4 NCBI
RH Standford-G3 TNG Whitehead-RH Whitehead-YAC
Mm_UniGene Mm_EST Rn_UniGene Rn_EST Ssc_UniGene Ss
c_EST Bt_UniGene Bt_EST Gga_UniGene Gga_EST Variat
ion
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MapViewer
Component
Gene
UniGene
Repeats
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Phenotype
Variation
Gene
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Maps Options
Maps Options
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Chimp ADAR
Human ADAR
Mouse ADAR
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Genome Resources
Genomic Biology
Trace Archive
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Trace Archive Page
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Ciona savignyi Traces
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(No Transcript)
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Trace Archive BLAST Page
Potential access to sequences NOT yet in GenBank
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Basic Local Alignment Search Tool
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BLAST Web Searches, 2005
200,000
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• Precomputed BLAST Services

• Nucleotide or protein Related Sequences
• BLAST link BLink

• Transcript clusters UniGene
• Protein homologs HomoloGene

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Link to Related Sequences
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Related Sequences
Most similar
Least similar
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BLink (BLAST Link)
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BLink Output
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Why Is BLAST So Popular?

• Fast
• - heuristic approach based on Smith Waterman
• Local alignments
• Statistical significance
• - Expect value
• Versatile
• - blastn, blastp, blastx, tblastn, tblastx,
  rps-blast, psi-blast
• - www, standalone, and network clients

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Global vs Local Alignment
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Global vs Local Alignment
Seq1 WHEREISWALTERNOW (16aa) Seq2
HEWASHEREBUTNOWISHERE (21aa)
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How BLAST Works

• Make lookup table of words for query
• Scan database for hits
• Extend alignment both directions
• Ungapped extensions of hits (initial HSPs)
• Gapped extensions (no traceback)
• Gapped extensions (traceback - alignment details)

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Protein Words
GTQ TQI QIT ITV TVE VED
EDL DLF ...
Make a lookup table of words
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BLASTP Summary
Drop-off score Highest score current
score -X X dropoff value for gapped alignment
(in bits) blastn 30, megablast 20, tblastx 0, all
others 15
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BLASTP Summary
High-scoring pair (HSP)
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Scoring Systems - Nucleotides
Identity matrix
A G C T A 1 3 3 -3 G 3 1 3 -3 C 3 3
1 -3 T 3 3 3 1
-r 1 -q -3
CAGGTAGCAAGCTTGCATGTCA
raw score 19-9 10 CACGTAGCAAGCTTG-GTGTCA
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Scoring Systems - Proteins

• Position Independent Matrices
• PAM Matrices (Percent Accepted Mutation)
• Derived from observation small dataset of
  alignments
• Implicit model of evolution
• All calculated from PAM1
• PAM250 widely used
• BLOSUM Matrices (BLOck SUbstitution Matrices)
• Derived from observation large dataset of
  highly conserved blocks
• Each matrix derived separately from blocks with
  a defined percent identity cutoff
• BLOSUM62 - default matrix for BLAST
• Position Specific Score Matrices (PSSMs)
• PSI- and RPS-BLAST

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BLOSUM62
A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3
-3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2
5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0
0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2
-3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1
-2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2
-1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0
6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4
7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1
4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1
1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1
-4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2
-1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3
3 1 -2 1 -1 -2 -2 0 -3 -1 4 X 0 -1 -1 -1 -2
-1 -1 -1 -1 -1 -1 -1 -1 -1 -2 0 0 -2 -1 -1 -1
A R N D C Q E G H I L K M F P S
T W Y V X
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Position-Specific Score Matrix
Serine/Threonine protein kinases catalytic loop
DAF-1
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Position-Specific Score Matrix
A R N D C Q E G H I L K M
F P S T W Y V 435 K -1 0 0 -1 -2 3
0 3 0 -2 -2 1 -1 -1 -1 -1 -1 -1 -1 -2 436 E
0 1 0 2 -1 0 2 -1 0 -1 -1 0 0 0 -1 0
0 -1 -1 -1 437 S 0 0 -1 0 1 1 0 1 1
0 -1 0 0 0 2 0 -1 -1 0 -1 438 N -1 0
-1 -1 1 0 -1 3 3 -1 -1 1 -1 0 0 -1 -1 1
1 -1 439 K -2 1 1 -1 -2 0 -1 -2 -2 -1 -2
5 1 -2 -2 -1 -1 -2 -2 -1 440 P -2 -2 -2 -2
-3 -2 -2 -2 -2 -1 -2 -1 0 -3 7 -1 -2 -3 -1 -1
441 A 3 -2 1 -2 0 -1 0 1 -2 -2 -2 0 -1 -2
3 1 0 -3 -3 0 442 M -3 -4 -4 -4 -3 -4 -4
-5 -4 7 0 -4 1 0 -4 -4 -2 -4 -1 2 443 A
4 -4 -4 -4 0 -4 -4 -3 -4 4 -1 -4 -2 -3 -4 -1
-2 -4 -3 4 444 H -4 -2 -1 -3 -5 -2 -2 -4 10
-6 -5 -3 -4 -3 -2 -3 -4 -5 0 -5 445 R -4 8
-3 -4 0 -1 -2 -3 -2 -5 -4 0 -3 -2 -4 -3 -3 0
-4 -5 446 D -4 -4 -1 8 -6 -2 0 -3 -3 -5 -6
-3 -5 -6 -4 -2 -3 -7 -5 -5 447 I -4 -5 -6 -6
-3 -4 -5 -6 -5 3 5 -5 1 1 -5 -5 -3 -4 -3 1
448 K 0 0 1 -3 -5 -1 -1 -3 -3 -5 -5 7 -4 -5
-3 -1 -2 -5 -4 -4 449 S 0 -3 -2 -3 0 -2
-2 -3 -3 -4 -4 -2 -4 -5 2 6 2 -5 -4 -4 450 K
0 3 0 1 -5 0 0 -4 -1 -4 -3 4 -3 -2 2 1
-1 -5 -4 -4 451 N -4 -3 8 -1 -5 -2 -2 -3 -1
-6 -6 -2 -4 -5 -4 -1 -2 -6 -4 -5 452 I -3 -5
-5 -6 0 -5 -5 -6 -5 6 2 -5 2 -2 -5 -4 -3 -5
-3 3 453 M -4 -4 -6 -6 -3 -4 -5 -6 -5 0 6
-5 1 0 -5 -4 -3 -4 -3 0 454 V -3 -3 -5 -6
-3 -4 -5 -6 -5 3 3 -4 2 -2 -5 -4 -3 -5 -3 5
455 K -2 1 1 4 -5 0 -1 -2 1 -4 -2 4 -3
-2 -3 0 -1 -5 -2 -3 456 N 1 1 3 0 -4 -1
1 0 -3 -4 -4 3 -2 -5 -2 2 -2 -5 -4 -4 457 D
-3 -2 5 5 -1 -1 1 -1 0 -5 -4 0 -2 -5 -1 0
-2 -6 -4 -5 458 L -3 -1 0 -3 0 -3 -2 3 -4
-2 3 0 1 1 -2 -2 -3 5 -1 -3
catalytic loop
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Local Alignment Statistics
Expect Value E number of database hits you
expect to find by chance, S
More info The Statistics of Sequence
Similarity Scores
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An Alignment BLAST Cannot Make
1 GAATATATGAAGACCAAGATTGCAGTCCTGCTGGCCTGAACC
ACGCTATTCTTGCTGTTG
1
GAGTGTACGATGAGCCCGAGTGTAGCAGTGAAGATCTGGACCACGGTGTA
CTCGTTGTCG 61 GTTACGGAACCGAGAATGGTAAAGACTACTGGA
TCATTAAGAACTCCTGGGGAGCCAGTT
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GCTATGGTGTTAAGGGTGGGAAGAAGTACTGGCTCGTCAAGAACAGCTGG
GCTGAATCCT 121 GGGGTGAACAAGGTTATTTCAGGCTTGCTCGTG
GTAAAAAC
121 GGGGAGACCAAGGCTACATCCTTATGTCCCGTGACAAC
AAC
Reason no contiguous exact match of 7 bp.
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An Alignment BLAST Can Make
Solution compare protein sequences BLASTX
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Other BLAST Algorithms

• Megablast
• Discontiguous Megablast
• PSI-BLAST
• PHI-BLAST

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Megablast NCBIs Genome Annotator

• Long alignments of similar DNA sequences
• Greedy algorithm
• Concatenation of query sequences
• Faster than blastn less sensitive

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MegaBLAST Word Size
Trade-off sensitivity vs speed
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Discontiguous Megablast

• Uses discontiguous word matches
• Better for cross-species comparisons

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Templates for Discontiguous Words
W 11, t 16, coding 1101101101101101 W 11,
t 16, non-coding 1110010110110111 W 12, t
16, coding 1111101101101101 W 12, t 16,
non-coding 1110110110110111 W 11, t 18,
coding 101101100101101101 W 11, t 18,
non-coding 111010010110010111 W 12, t 18,
coding 101101101101101101 W 12, t 18,
non-coding 111010110010110111 W 11, t 21,
coding 100101100101100101101 W 11, t 21,
non-coding 111010010100010010111 W 12, t
21, coding 100101101101100101101 W 12, t
21, non-coding 111010010110010010111
W word size matches in template t template
length
Reference Ma, B, Tromp, J, Li, M. PatternHunter
faster and more sensitive homology search.
Bioinformatics March, 2002 18(3)440-5
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(No Transcript)
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Discontiguous (Cross-species) MegaBLAST
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Discontiguous Word Options
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Disco. Megablast Example . . .
Query NM_078651 Drosophila melanogaster
CG18582-PA (mbt) mRNA, (3244 bp) /note mushroom
bodies tiny synonyms Pak2, STE20, dPAK2
Database nr (nt), Mammaliaorgn

• MegaBLAST No significant similarity found.

• Discontiguous megaBLAST numerous hits . . .

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Ex Discontiguous MegaBLAST
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Ex BLASTN
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PSI-BLAST
Position-specific Iterated BLAST

• Example Confirming relationships of purine
• nucleotide metabolism proteins

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PSI-BLAST
E value cutoff for PSSM
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RESULTS Initial BLASTP
Same results as protein-protein BLAST different
format
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Results of First PSSM Search
Other purine nucleotide metabolizing enzymes not
found by ordinary BLAST
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Tenth PSSM Search Convergence
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PHI-BLAST
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Whats New?
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BLAST Databases

• Nucleotide
• refseq_rna NM_, XM_
• refseq_genomic NC_, NG_
• env_nt
• environmental samplefilter, e.g., 16S rRNA
• Protein
• refseq NP_, XP_
• env_nr

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New Formatter
Select lower case
Select red
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BLAST Output Alignments Filter
low complexity sequence filtered
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BLAST Output CDS Feature
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Advanced Options
Limit to Organism
allfilter NOT ma
Example Entrez Queries allFilter NOT
mammaliaOrganism ray finned fishesOrganism s
rcdb refseqProperties Nucleotide
only biomol mrnaProperties biomol
genomicProperties OtherAdvanced e
10000 expect value -v 2000 descriptions -b
2000 alignments
-e 10000 -v 2000
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Genome BLAST
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Genome BLAST via Map Viewer
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Example Human Genome BLAST
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Human Genome BLAST Results
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Human Genome BLAST MapViewer
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Example Mapping Oligos Onto a Genome
?
gtforward CCATGGCGACCCTGGAAAAGC gtreverse CAGCAGCGG
CTGTGCCTGCGG
?
?
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Map Oligos Onto Genome
gtCCATGGCGACCCTGGAAAAGCNNNNNNNNNNCAGCAGCGGCTGTGCCTG
CGG
-W 7 e 1000
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Genome BLAST Results
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Primer Alignments
reverse primer
forward primer
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MapViewer
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MapViewer
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Sequence View (sv)
forward
reverse
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Service Addresses

• BLAST blast-help_at_ncbi.nlm.nih.gov
• General Help info_at_ncbi.nlm.nih.gov
• Wayne Matten matten_at_ncbi.nlm.nih.gov