Transcript Bioinformatics Lab - NCBI, BLAST, and Phylogeny

Phage?
Molecular
Evolution
New
Sequence
Horizontal
Transfer
2 Interrelated Modules on Bioinformatics
Module 1: To show the ways in which the NCBI
online database classifies and organizes
information on DNA sequences, evolutionary
relationships, and scientific publications.
Module 2: To identify an unknown nucleotide
sequence from the Wolbachia endosymbiont by
using the NCBI search tool BLAST
Teaching Time – 45 minutes
OH NO!
Advantages
1. No programming skills needed
2.Familiarity with personal computer
and internet browser
3.Customizable and free
What are the broad goals of this lab?
 To provide an introduction to bioinformatics
(NCBI)
 To introduce you to searching for articles,
sequences, scientists (perhaps yourself)
 To use phylogenies
To put your Wolbachia research in the
context of what’s been published
What are the specific goals of this lab?
 To look for brand new W strains
 To make a phylogenetic tree of W
 To ultimately compare the W tree to an
insect phylogeny to infer lateral vs. vertical
transmission of your W strains
 To contribute to a national “student”
sequence database on the genetic diversity of
W 16S rRNA gene
Wolbachia – Host Interactions:
Mutualism and Reproductive Parasitism
Required for insect
oogenesis (Dedeine et al. 2001)
Parthenogenesis in
wasps
Male-killing in insects
Required for nematode
fertility and larval
development
Feminization in isopods
Cytoplasmic incompatibility in
arthropods
Application of Bioinformatics to Wolbachia
Alpha Proteobacteria
Wolbachia –
Anaplasma Split
Ehrlichia
Anaplasma
Wolbachia
Neorickettsia
Rickettsia
Obligatory
Intracellulars
in Arthropods
Wins-Wnem
Split (~120MY)
Dunning-Hottop et al 2006
Wolbachia:
Mutualist
Parasite
Outcomes: A New Wolbachia Species?
Wolbachia: Complete genomic sequences from a
related parasite and mutualist
wMel
wBm
(Wu et al. 2004)
(Foster et al. 2005)
696
shared
genes
Foster et al 2005
Wu et al 2004
1.27 Mb
1270 genes
1.08 Mb
806 genes
Your Wolbachia Sequence:
What do you do with it?
ORIGIN
1
61
121
181
ttcttgtatc
tcagagttca
aacctgaagc
ggaagaacga
ccaaacatct
cttgcaagct
ctcttacttg
tttcctagat
cgagcttctt
gataatgagc
attaatcaaa
caatctctta
gtacaccaaa
agaaaatttt
gacggtctgc
tcagcaaata
ttaggtattc
tcaaaaccag
aaattacaag
cgatggagat
actatggaat
atgaaacccg
ccaaatattt
gagtatcgga
BLAST:
Query a database for sequences homologous to
an input (ie, query) sequence.
GATGCCATAGAGCTGTAGTCGTACCCT <—
—>
CTAGAGAGC-GTAGTCAGAGTGTCTTTGAGTTCC
•
•
•
Compare new genes to old ones
Compare genes from different species or
hosts
Identify possible functions based on
similarities to known sequences.
BLAST is like using ‘Google’ for DNA sequences
National Center for Biotechnology Information (NCBI)
http://www.ncbi.nlm.nih.gov
Release 2008: 99 billion base pairs
99 million sequences
Target database: Adjustable using the pull-down menu
LOCUS
DEFINITION
AY182241
1931 bp
mRNA
linear
PLN 04-MAY-2004
Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
complete cds.
ACCESSION
AY182241
VERSION
AY182241.2 GI:32265057
KEYWORDS
.
SOURCE
Malus x domestica (cultivated apple)
ORGANISM Malus x domestica
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots;
rosids; eurosids I; Rosales; Rosaceae; Maloideae; Malus.
REFERENCE
1 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Cloning and functional expression of an (E,E)-alpha-farnesene
synthase cDNA from peel tissue of apple fruit
JOURNAL
Planta 219, 84-94 (2004)
REFERENCE
2 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Direct Submission
JOURNAL
Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REFERENCE
3 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Direct Submission
JOURNAL
Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REMARK
Sequence update by submitter
COMMENT
On Jun 26, 2003 this sequence version replaced gi:27804758.
FEATURES
Location/Qualifiers
source
1..1931
/organism="Malus x domestica"
/mol_type="mRNA"
/cultivar="'Law Rome'"
/db_xref="taxon:3750"
/tissue_type="peel"
gene
1..1931
/gene="AFS1"
CDS
54..1784
/gene="AFS1"
/note="terpene synthase"
/codon_start=1
/product="(E,E)-alpha-farnesene synthase"
/protein_id="AAO22848.2"
/db_xref="GI:32265058"
/translation="MEFRVHLQADNEQKIFQNQMKPEPEASYLINQRRSANYKPNIWK
NDFLDQSLISKYDGDEYRKLSEKLIEEVKIYISAETMDLVAKLELIDSVRKLGLANLF
EKEIKEALDSIAAIESDNLGTRDDLYGTALHFKILRQHGYKVSQDIFGRFMDEKGTLE
DFLHKNEDLLYNISLIVRLNNDLGTSAAEQERGDSPSSIVCYMREVNASEETARKNIK
GMIDNAWKKVNGKCFTTNQVPFLSSFMNNATNMARVAHSLYKDGDGFGDQEKGPRTHI
LSLLFQPLVN"
ORIGIN
1 ttcttgtatc ccaaacatct cgagcttctt gtacaccaaa ttaggtattc actatggaat
61 tcagagttca cttgcaagct gataatgagc agaaaatttt tcaaaaccag atgaaacccg
121 aacctgaagc ctcttacttg attaatcaaa gacggtctgc aaattacaag ccaaatattt
181 ggaagaacga tttcctagat caatctctta tcagcaaata cgatggagat gagtatcgga
241 agctgtctga gaagttaata gaagaagtta agatttatat atctgctgaa acaatggatt
//
A Traditional
GenBank Record
Header
The Flatfile Format
Feature Table
Sequence
The Header
LOCUS
DEFINITION
ACCESSION
VERSION
KEYWORDS
SOURCE
ORGANISM
REFERENCE
AUTHORS
TITLE
JOURNAL
REFERENCE
AUTHORS
TITLE
JOURNAL
REFERENCE
AUTHORS
TITLE
JOURNAL
REMARK
COMMENT
AY182241
1931 bp
mRNA
linear
PLN 04-MAY-2004
Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
complete cds.
AY182241
AY182241.2 GI:32265057
.
Malus x domestica (cultivated apple)
Malus x domestica
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots;
rosids; eurosids I; Rosales; Rosaceae; Maloideae; Malus.
1 (bases 1 to 1931)
Pechous,S.W. and Whitaker,B.D.
Cloning and functional expression of an (E,E)-alpha-farnesene
synthase cDNA from peel tissue of apple fruit
Planta 219, 84-94 (2004)
2 (bases 1 to 1931)
Pechous,S.W. and Whitaker,B.D.
Direct Submission
Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
3 (bases 1 to 1931)
Pechous,S.W. and Whitaker,B.D.
Direct Submission
Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
Sequence update by submitter
On Jun 26, 2003 this sequence version replaced gi:27804758.
Header: Locus Line
LOCUS
AY182241
1931 bp
mRNA
linear
PLN 04-MAY-2004
DEFINITION
Malus x domestica
synthase (AFS1)
mRNA,
LOCUS
AY182241
1931 (E,E)-alpha-farnesene
bp
mRNA
linear
PLN 04-MAY-2004
complete cds.
ACCESSION
AY182241
VERSION
AY182241.2 GI:32265057
KEYWORDS
.
SOURCE
Malus x domestica (cultivated apple)
ORGANISM Malus x domestica
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots;
rosids; eurosids I; Rosales; Rosaceae; Maloideae; Malus.
REFERENCE
1 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Cloning and functional expression of an (E,E)-alpha-farnesene
synthase cDNA from peel tissue of apple fruit
JOURNAL
Planta 219, 84-94 (2004)
REFERENCE
2 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Direct Submission
JOURNAL
Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REFERENCE
3 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Direct Submission
JOURNAL
Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REMARK
Sequence update by submitter
COMMENT
On Jun 26, 2003 this sequence version replaced gi:27804758.
Length
Locus name
Molecule type
Division
Modification Date
Header: Database Identifiers
LOCUS
DEFINITION
ACCESSION
VERSION
KEYWORDS
SOURCE
ORGANISM
AY182241
1931 bp
mRNA
linear
PLN 04-MAY-2004
Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
Accession
complete cds.
AY182241
•Stable
AY182241.2 GI:32265057
•Reportable
.
•Universal
Malus x domestica (cultivated apple)
Malus x domestica
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots;
rosids; eurosids I; Rosales; Rosaceae; Maloideae; Malus.
1 (bases 1 to 1931)
Pechous,S.W. and Whitaker,B.D.
Cloning and functional expression of an (E,E)-alpha-farnesene
synthase cDNA from peel tissue of apple fruit
Planta 219, 84-94 (2004)
2 (bases 1 to 1931)
Pechous,S.W. and Whitaker,B.D.
Direct Submission
Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
3 (bases 1 to 1931)
Pechous,S.W. and Whitaker,B.D.
Direct Submission
Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
Sequence update by submitter
On Jun 26, 2003 this sequence version replaced gi:27804758.
ACCESSION
AY182241
VERSION
AY182241.2
REFERENCE
AUTHORS
TITLE
JOURNAL
REFERENCE
AUTHORS
TITLE
JOURNAL
REFERENCE
AUTHORS
TITLE
JOURNAL
REMARK
COMMENT
GI:32265057
Header: Organism
LOCUS
DEFINITION
AY182241
1931 bp
mRNA
linear
PLN 04-MAY-2004
Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
complete cds.
ACCESSION
AY182241
VERSION
AY182241.2 GI:32265057
KEYWORDS
.
SOURCE
Malus x domestica (cultivated apple)
SOURCE
(cultivated apple)
ORGANISMMalus
Malusxx domestica
domestica
ORGANISM Malus
x domestica
Eukaryota;
Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta;
Magnoliophyta; Streptophyta;
eudicotyledons; core
eudicots;
Eukaryota;
Viridiplantae;
Embryophyta;
rosids; eurosids I; Rosales; Rosaceae; Maloideae; Malus.
Tracheophyta;
Spermatophyta; Magnoliophyta; eudicotyledons;
REFERENCE
1 (bases 1 to 1931)
eudicots;
eurosids I; Rosales; Rosaceae;
AUTHORS core
Pechous,S.W.
androsids;
Whitaker,B.D.
TITLE Maloideae;
Cloning andMalus.
functional expression of an (E,E)-alpha-farnesene
synthase cDNA from peel tissue of apple fruit
JOURNAL
Planta 219, 84-94 (2004)
REFERENCE
2 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.NCBI-controlled taxonomy
TITLE
Direct Submission
JOURNAL
Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REFERENCE
3 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Direct Submission
JOURNAL
Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REMARK
Sequence update by submitter
COMMENT
On Jun 26, 2003 this sequence version replaced gi:27804758.
The Feature Table
FEATURES
source
gene
CDS
start (atg)
Coding sequence
Location/Qualifiers
1..1931
/organism="Malus x domestica"
/mol_type="mRNA"
/cultivar="'Law Rome'"
/db_xref="taxon:3750"
/tissue_type="peel"
1..1931
/gene="AFS1"
stop (tag)
54..1784
/gene="AFS1"
/note="terpene synthase"
/codon_start=1
/product="(E,E)-alpha-farnesene synthase"
/protein_id="AAO22848.2"
/db_xref="GI:32265058"
/translation="MEFRVHLQADNEQKIFQNQMKPEPEASYLINQRRSANYKPNIWK
NDFLDQSLISKYDGDEYRKLSEKLIEEVKIYISAETMDLVAKLELIDSVRKLGLANLF
EKEIKEALDSIAAIESDNLGTRDDLYGTALHFKILRQHGYKVSQDIFGRFMDEKGTLE
NHHFAHLKGMLELFEASNLGFEGEDILDEAKASLTLALRDSGHICYPDSNLSRDVVHS
LELPSHRRVQWFDVKWQINAYEKDICRVNATLLELAKLNFNVVQAQLQKNLREASRWW
ANLGIADNLKFARDRLVECFACAVGVAFEPEHSSFRICLTKVINLVLIIDDVYDIYGS
EEELKHFTNAVDRWDSRETEQLPECMKMCFQVLYNTTCEIAREIEEENGWNQVLPQLT
KVWADFCKALLVEAEWYNKSHIPTLEEYLRNGCISSSVSVLLVHSFFSITHEGTKEMA
DFLHKNEDLLYNISLIVRLNNDLGTSAAEQERGDSPSSIVCYMREVNASEETARKNIK
GMIDNAWKKVNGKCFTTNQVPFLSSFMNNATNMARVAHSLYKDGDGFGDQEKGPRTHI
Bioinformatics is NOT just information technology.
It can teach the central dogmas of molecular biology
DNA
RNA
cDNA
DNA sequences
genomes
protein
protein
sequence
databases
phenotype
Insect Phylogeny
Outcomes:
Lateral Transfer ?
Top 5 Wolbachia BLAST matches
GATGCCATAGAGCTGTAGTCGTACCCT <-
100%
GATGCCATAGAGCTGTAGTCGTACCCT <-
100%
GATGCCATAGAGCTGTAGTCGTACCCT <- 100%
GATGCCATAGAGCTGTAGTCGTACCCT <- 100%
GATGCCATAGAGCTGTAGTCGTACCCT <- 100%
Let’s Begin Our Bioinformatic Exercise
Lab 5
HTTP://WWW.DIGITALWORLDBIOLO
GY.COM/BLAST