1. A Field Guide part 2 National Center for Biotechnology Information
February 14, 2006
UT-Health Science Center

2. GenBank Records
Header
The Flatfile Format
Feature Table
Sequence

3. A Typical GenBank Record
LOCUS NM_ bp mRNA linear INV 28-OCT-2004
DEFINITION Mus musculus REV1-like(S. cerevisiae)(Rev1l),mRNA
ACCESSION NM_019570
VERSION NM_ GI:
KEYWORDS .
A Typical GenBank Record
= Title

4. GenBank Record: Feature Table

5. GenBank Record: Feature Table, con’t.
GenPept identifier

6. GenBank Record: sequence
skip

7. Indexing for Nucleotide UID 59958365
Field Indexed Terms
[primary accession] NM_
[title] Bos taurus hemochromatosis (hfe), mRNA.
[organism] Bos taurus
[sequence length] 1168
[modification date] 2005/02/19
[properties] biomol mrna
gbdiv mam
srcdb refseq
[accn]
[orgn]
[mdat]
[prop]

8. Global Entrez Search: HFE

9. Entrez Nucleotide: HFE
137 records
[Title]
Not HFE

10. Smarter Query hfe[title] AND human[orgn] 42 records Curated HFE
splice variants
(11 total)

11. hfe[title] AND human[orgn] (con’t)
Primary data

12. Preview/Index Gateway to Advanced Searches

13. Preview/Index

14. Preview/Index: Properties, srcdb

15. Preview/Index: Properties, srcdb
…AND srcdb refseq[Properties]

16. Preview/Index: Properties, srcdb
…AND srcdb ddbj/embl/genbank[Properties]

17. Database Queries #1 hfe 137 #2 hfe[title] AND human[orgn] 42
#3 #2 AND srcdb refseq[prop]
#4 #2 AND srcdb ddbj/embl/genbank[prop]
#5 #4 AND gbdiv pri[prop]
#4 #4 AND gbdiv est[prop]
Primate division gbdiv pri[prop]
EST division gbdiv est[prop]

18. Molecule Queries #1 hfe 116 #2 hfe[title] AND human[orgn] 42
#3 #2 AND biomol mrna[prop]
#4 #2 AND biomol genomic[prop]
Genomic DNA biomol genomic[prop]
cDNA biomol mrna[prop]

19. srcdb refseq reviewed[prop] AND transcript[title] AND variant[title]
More Queries…
Fields are database-specific
Entrez Nucleotide
Reviewed RefSeqs with transcript variants:
srcdb refseq reviewed[prop] AND transcript[title] AND variant[title]

20. More Queries… Fields are database-specific Entrez Nucleotide
Reviewed RefSeqs with transcript variants:
srcdb refseq reviewed[prop] AND transcript[title] AND variant[title]
Topoisomerase genes from Archaea:
topoisomerase[gene name] AND archaea[organism]
Entrez Gene
Genes on human chromosome 2 with OMIM links
2[chromosome] AND human[organism] AND “gene omim”[filter]
Membrane proteins linked to cancer:
“integral to plasma membrane”[gene ontology] AND cancer[dis]

21. Other Entrez Databases
UniGene: rat clusters that have at least one mRNA
rat[organism] NOT 0[mrna count]
SNP: uniquely mapped microsatellites on human chr2
microsat[SNP Class] AND 1[Map Weight] AND 2[Chromosome]) AND human[orgn]
UniSTS: markers on the Genethon map of human chromosome 12
Genethon[Map Name] AND human[organism] AND 12[chromosome]
Structure: structures of bacterial kinases with resolutions below 2 Å
bacteria[organism] AND kinase AND :002.00[resolution]

22. Genome Resources Genomic Biology Genomic Biology UniGene E-PCR
Map Viewer
Trace Archive

23. Genomic Biology

24. Gen Biol: Gen Resources

25. Map Viewer – Genome Annotation Updates

26. Gen Biol: Gen Resources

27. Genome Projects: microb

28. Genome Projects: microb
13 Eukaryotic Genome Sequencing Projects Selected: Complete – 0, Assembly – 2,
In Progress - 11

29. Genome Projects: microb
13 Eukaryotic Genome Sequencing Projects Selected: Complete – 0, Assembly – 2,
In Progress - 11

30. Gen Biol: Gen Resources

31. Gen Biol: Gen Resources

32. Gen Biol: Gen Resources

33. Gen Biol: Gen Resources

34. Gen Biol: Gen Resources

35. Genome Resources UniGene Genomic Biology E-PCR Map Viewer
Trace Archive

36. Gene-oriented clusters of expressed sequences
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

37. A Cluster of ESTs
query
5’ EST hits
3’ EST hits

38. UniGene Collections

39. UniGene Collections
Species UniGene

40. UniGene Hs build 188

41. UniGene Cluster Hs.95351 Lipase, hormone-sensitive (LIPE)

42. UniGene Cluster Hs.95351

43. UniGene Cluster Hs.95351: expression

44. UniGene Cluster Hs.95351: seqs

45. Get Sequences
web page
ftp://ftp.ncbi.nih.gov/repository/UniGene/Homo_sapiens/

46. Genome Resources E-PCR Genomic Biology UniGene Map Viewer
Trace Archive

47. E-PCR
Genomic sequence here

48. Options

49. Results

50. reverse e-pcr

51. reverse e-pcr

52. reverse e-pcr

53. LY6G6D: lymphocyte antigen 6 complex, locus G6D
reverse e-pcr
Gene
STS
LY6G6D: lymphocyte antigen 6 complex, locus G6D

54. Genome Resources Map Viewer Genomic Biology UniGene E-PCR
Trace Archive

55. List View

56. Human MapViewer
adar

57. MapViewer: Human ADAR

58. 3’ UTR
5’ UTR
MV Hs ADAR

59. Maps & Options Maps & Options --Sequence maps-- --Cytogenetic maps--
Ab initio
Assembly
Repeats
BES_Clone
Clone
NCI_Clone
Contig
Component
CpG island
dbSNP haplotype
Fosmid
GenBank_DNA
Gene
Phenotype
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
Ssc_EST
Bt_UniGene
Bt_EST
Gga_UniGene
Gga_EST
Variation
= SNP

60. Component
MapViewer
Gene
UniGene
Repeats
Customize the annotation

61. Phenotype
Variation
Gene
Customize the annotation

62. Maps & Options Maps & Options
DR52/53 = alternate MHC haplotypes on chr6; hsc_tcag = alternate chr7 assembly.

63. Chimp ADAR
Human ADAR
Mouse ADAR
Customize the annotation

64. Genome Resources Trace Archive Genomic Biology UniGene E-PCR
Map Viewer
Trace Archive

65. Trace Archive Page

66. Ciona savignyi Traces

68. Trace Archive BLAST Page
Potential access to sequences NOT yet in GenBank

69. Basic Local Alignment Search Tool

70. BLAST Web Searches, 2005
200,000

71. Precomputed BLAST Services
Nucleotide or protein: Related Sequences
BLAST link: BLink
Transcript clusters: UniGene
Protein homologs: HomoloGene

72. Link to Related Sequences

73. Related Sequences
Most similar
Least similar

74. BLink (BLAST Link)

75. BLink Output
Best hits
3D structures
CDD-Search

76. Why Is BLAST So Popular? Fast Local alignments
- 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

77. Global vs Local Alignment
Seq 1
Seq 2
Global alignment
Seq 1
Seq 2
Local alignment

78. Global vs Local Alignment
Seq1: WHEREISWALTERNOW (16aa)
Seq2: HEWASHEREBUTNOWISHERE (21aa)
Global
Seq1: W--HEREISWALTERNOW 16
W HERE
Seq2: 1 HEWASHEREBUTNOWISHERE
Local
Seq1: 1 W--HERE Seq1: 1 W--HERE 5
W HERE W HERE
Seq2: 3 WASHERE Seq2: 15 WISHERE 21

79. 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)
-X X dropoff value for gapped alignment (in bits) (zero invokes default
behavior) blastn 30, megablast 20, tblastx 0, all others 15 [Integer]
default = 0
=X2 (in output)
-y X dropoff value for ungapped extensions in bits (0.0 invokes default
behavior) blastn 20, megablast 10, all others 7 [Real]
default = 0.0
=X1 (in output)
-Z X dropoff value for final gapped alignment in bits (0.0 invokes default
behavior) blastn/megablast 50, tblastx 0, all others 25 [Integer]
=X3 (in output)

80. Protein Words GTQITVEDLFYNIATRRKALKN GTQ TQI QIT ITV TVE VED EDL DLF
Query:
Word size = 3 (default)
Word size can only be 2 or 3
GTQ
TQI
QIT
ITV
TVE
VED
EDL
DLF
...
Make a lookup
table of words
Neighborhood Words
VTV, LTV, VSV, etc.
VTV 12
LTV 11
VSV 8
Neighborhood score threshold

81. Highest score – current score
BLASTP Summary
Query: IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILEV…
example query words
HFL 18
HFV 15
HFS 14
HWL 13
NFL 13
DFL 12
HWV 10
etc …
YLS 15
YLT 12
YVS 12
YIT 10
Neighborhood words
Neighborhood score threshold
T (-f) =11
YLS HFL
Sbjct 287 LEETYAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEI 333
Query 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESI 47
+E YA YL K F+ L +SP+ +DVNVHP+K V I
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

82. BLASTP Summary High-scoring pair (HSP) Final HSP example query words
Query: IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILEV…
example query words
HFL 18
HFV 15
HFS 14
HWL 13
NFL 13
DFL 12
HWV 10
etc …
YLS 15
YLT 12
YVS 12
YIT 10
Neighborhood words
Neighborhood score threshold
T (-f) =11
YLS HFL
Sbjct 287 LEETYAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEI 333
Query 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESI 47
+E YA YL K F+ L +SP+ +DVNVHP+K V I
High-scoring pair (HSP)
Gapped extension with trace back
Query 1 IETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESI-LEV… 50
+E YA YL K F+YLSL +SP+ +DVNVHP+K VHFL+++ I + +
Sbjct 287 LEETYAKYLHKGASYFVYLSLNMSPEQLDVNVHPSKRIVHFLYDQEIATSI… 337
Final HSP

83. Scoring Systems - Nucleotides
Identity matrix
A G C T
A +1 –3 –3 -3
G –3 +1 –3 -3
C –3 –
T –3 –3 –3 +1
[ -r 1 -q -3 ]
The default values for M and N, respectively 5 and -4, having a ratio of 1.25, correspond to about 47 nucleic acid PAMs, or about 58 amino acid PAMs; an M:N ratio of 1 corresponds to 30 nucleic acid PAMs or 38 amino acid PAMs. At higher than about 40 nucleic acid PAMs, or 50 amino acid PAMs, better sensitivity at detecting similarities between coding regions is expected by performing comparisons at the amino acid level (States et al., 1991).
CAGGTAGCAAGCTTGCATGTCA
|| |||||||||||| ||||| raw score = 19-9 = 10
CACGTAGCAAGCTTG-GTGTCA

84. 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

85. BLOSUM62 Negative for less likely substitutionsD C Q E G H I L K M F P S T W Y V X
A R N D C Q E G H I L K M F P S T W Y V X D F
Negative for less likely substitutions Y F
Positive for more likely substitutions

86. Position-Specific Score Matrix
Serine/Threonine protein kinases
catalytic loop 1 7 4
PSSM scores 5
DAF-1
abnormal DAuer Formation DAF-1, cell-surface receptor daf-1 [Caenorhabditis elegans] gi| |ref|NP_

87. 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
436 E
437 S
438 N
439 K
440 P
441 A
442 M
443 A
444 H
445 R
446 D
447 I
448 K
449 S
450 K
451 N
452 I
453 M
454 V
455 K
456 N
457 D
458 L
catalytic loop
cell-surface receptor daf-1 [Caenorhabditis elegans] ; gi| |ref|NP_ ; -j3 vs nr
>blastpgp -i NP_ d nr -j 3 -Q NP_ pssm

88. Local Alignment Statistics
Expect Value
E = number of database hits you expect to find by chance, ≥ S
(applies to ungapped alignments)
E = Kmne-S or E = mn2-S’
K = scale for search space
 = scale for scoring system
S’ = bitscore = (S - lnK)/ln2
Lambda -- the expected increase in reliability of an alignment associated with a unit increase in alignment score.
E (range 0 to infinity) can be extrapolated to an expectation over the entire database search, by converting the pairwise expectation to a probability (range 0-1) and multiplying the result by the ratio of the entire database size to the length of the database sequence: E_database = (1 - exp(-E)) D / d where D is the size of the database; d is the length of the matching database sequence; and the quantity (1 - exp(-E)) is the probability, P, corresponding to the expectation E for the pairwise comparison.
As E approaches 0, E and P approach equality. Due to inaccuracy in the statistical methods as applied in the BLAST programs, whenever E and P are less than about 0.05, the two values can be practically treated as equal.
Effective length: computed according to: Length_eff = MAX( Length_real - Lambda S / H , 1) where H is the relative entropy of the target and background residue frequencies (Karlin and Altschul, 1990). H : the information expected to be obtained from each pair of aligned residues in a real alignment that distinguishes the alignment from a random one.
More info: The Statistics of Sequence Similarity Scores

89. An Alignment BLAST Cannot Make
1 GAATATATGAAGACCAAGATTGCAGTCCTGCTGGCCTGAACCACGCTATTCTTGCTGTTG
|| | || || || | || || || || | ||| |||||| | | || | ||| |
1 GAGTGTACGATGAGCCCGAGTGTAGCAGTGAAGATCTGGACCACGGTGTACTCGTTGTCG
61 GTTACGGAACCGAGAATGGTAAAGACTACTGGATCATTAAGAACTCCTGGGGAGCCAGTT
| || || || ||| || | |||||| || | |||||| ||||| | |
61 GCTATGGTGTTAAGGGTGGGAAGAAGTACTGGCTCGTCAAGAACAGCTGGGCTGAATCCT
121 GGGGTGAACAAGGTTATTTCAGGCTTGCTCGTGGTAAAAAC
|||| || ||||| || || | | |||| || |||
121 GGGGAGACCAAGGCTACATCCTTATGTCCCGTGACAACAAC
Reason:
no contiguous exact match of 7 bp.

90. An Alignment BLAST Can Make
Score = 290 bits (741), Expect = 7e-77 Identities = 147/331 (44%), Positives = 206/331 (61%), Gaps = 8/331 (2%) Frame = +3
Solution: compare protein sequences; BLASTX
BLAST 2 Sequences (blastx) output:

91. Other BLAST Algorithms
Megablast
Discontiguous Megablast
PSI-BLAST
PHI-BLAST

92. Megablast: NCBI’s Genome Annotator
Long alignments of similar DNA sequences
Greedy algorithm
Concatenation of query sequences
Faster than blastn; less sensitive

93. MegaBLAST & Word Size Trade-off: sensitivity vs speed blastn megablast2 3
blastp 8 28
megablast 7 11
blastn
minimum
default
WORD SIZE
Consider for each search

94. Discontiguous Megablast
Uses discontiguous word matches
Better for cross-species comparisons

95. Templates for Discontiguous Words
W = 11, t = 16, coding:
W = 11, t = 16, non-coding:
W = 12, t = 16, coding:
W = 12, t = 16, non-coding:
W = 11, t = 18, coding:
W = 11, t = 18, non-coding:
W = 12, t = 18, coding:
W = 12, t = 18, non-coding:
W = 11, t = 21, coding:
W = 11, t = 21, non-coding:
W = 12, t = 21, coding:
W = 12, t = 21, non-coding:
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

97. Discontiguous (Cross-species) MegaBLAST

98. Discontiguous Word Options
Non-affine gap scores: gap open penalty = 0; gap extension penalty = match/2 - mismatch (mismatch < 0). Non-affine gapping parameters tend to yield alignments with more gaps, but the gap lengths are shorter. The latter formula is applied automatically if -G and -E command line parameters
are both set to 0.

99. 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), Mammalia[orgn]
MegaBLAST = “No significant similarity found.”
Discontiguous megaBLAST = numerous hits . . .

100. Ex: Discontiguous MegaBLAST

101. Ex: BLASTN

102. PSI-BLAST Position-specific Iterated BLAST
Example: Confirming relationships of purine
nucleotide metabolism proteins

103. PSI-BLAST E value cutoff for PSSM
>gi|113340|sp|P03958|ADA_MOUSE ADENOSINE DEAMINASE (ADENOSINE
MAQTPAFNKPKVELHVHLDGAIKPETILYFGKKRGIALPADTVEELRNIIGMDKPLSLPGF
VIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVD
EQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAMDLAGDETIEGSSLFPGHVEAY
RTVHAGEVGSPEVVREAVDILKTERVGHGYHTIEDEALYNRLLKENMHFEVCPWSSYLTGA
VRFKNDKANYSLNTDDPLIFKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKK
0.005
E value cutoff for PSSM

104. RESULTS: Initial BLASTP
Same results as protein-protein BLAST; different format

105. Results of First PSSM Search
Other purine nucleotide metabolizing enzymes not found by ordinary BLAST

106. Tenth PSSM Search: Convergence
Just below threshold, another
nucleotide metabolism enzyme
Check to add to PSSM

107. PHI-BLAST [GA]xxxxGK[ST]
>gi|231729|sp|P30429|CED4_CAEEL CELL DEATH PROTEIN 4
MLCEIECRALSTAHTRLIHDFEPRDALTYLEGKNIFTEDHSELISKMSTRLERIANFLRIYRRQASE
LIDFFNYNNQSHLADFLEDYIDFAINEPDLLRPVVIAPQFSRQMLDRKLLLGNVPKQMTCYIREYHV
IKKLDEMCDLDSFFLFLHGRAGSGKSVIASQALSKSDQLIGINYDSIVWLKDSGTAPKSTFDLFTDI
LKSEDDLLNFPSVEHVTSVVLKRMICNALIDRPNTLFVFDDVVQEETIRWAQELRLRCLVTTRDVEI
ASQTCEFIEVTSLEIDECYDFLEAYGMPMPVGEKEEDVLNKTIELSSGNPATLMMFFKSCEPKTFEK
[GA]xxxxGK[ST]

108. What’s New?

109. BLAST Databases nr = nr Nucleotide refseq_rna = NM_*, XM_*
refseq_genomic = NC_*, NG_*
env_nt
environmental sample[filter], e.g., 16S rRNA
Protein
refseq = NP_*, XP_*
env_nr
nr = nr

110. New Formatter
Select lower case
Select red

111. BLAST Output: Alignments & Filter
low complexity sequence filtered

112. BLAST Output: CDS Feature

113. Advanced Options Limit to Organism Example Entrez Queries
all[filter] NOT ma
Example Entrez Queries
all[Filter] NOT mammalia[Organism]
ray finned fishes[Organism]
srcdb refseq[Properties]
Nucleotide only:
biomol mrna[Properties]
biomol genomic[Properties]
OtherAdvanced
–e expect value
-v descriptions
-b 2000 alignments
-e v 2000

114. Genome BLAST

115. Genome BLAST via Map Viewer

116. Example: Human Genome BLAST
TGCCTCCTTTGGTGAAGGTGACACATCATGTGACCTCTTCAGTGACCACTCTACGGTGTCGGGCCTTGAACTACTACCCCCAGAAC
ATCACCATGAAGTGGCTGAAGGATAAGCAGCCAATGGATGCCAAGGAGTTCGAACCTAAAGACGTATTGCCCAATGGGGATGGGAC
CTACCAGGGCTGGATAACCTTGGCTGTACCCCCTGGGGAAGAGC
Human EST

117. Human Genome BLAST: Results

118. Human Genome BLAST: MapViewer
Entrez Gene

119. Example: Mapping Oligos Onto a Genome?
>forward
CCATGGCGACCCTGGAAAAGC
>reverse
CAGCAGCGGCTGTGCCTGCGG ? ?

120. Map Oligos Onto Genome forward primer reverse primer -W 7 –e 1000
>CCATGGCGACCCTGGAAAAGCNNNNNNNNNNCAGCAGCGGCTGTGCCTGCGG
forward primer
reverse primer
-W 7 –e 1000

121. Genome BLAST Results

122. Primer Alignments
reverse primer
forward primer

123. MapViewer

124. MapViewer

125. Sequence View (sv)
forward
reverse

126. Service Addresses BLAST blast-help@ncbi.nlm.nih.gov
General Help
Wayne Matten