Bioinformatics Lab

Phage?

New
Sequence

Horizontal
Transfer

Molecular
Evolution

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!


1.

No programming skills needed

2.
Familiarity with personal computer
and internet browser

3.
Customizable and free

Advantages

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

Parthenogenesis

in
wasps

Male
-
killing
in insects

Feminization
in isopods

Cytoplasmic incompatibility
in
arthropods

Required for nematode
fertility and larval
development

Required for insect
oogenesis
(Dedeine et al. 2001)

Dunning
-
Hottop et al 2006

Wolbachia

Anaplasma

Ehrlichia

Neorickettsia

Rickettsia

Alpha Proteobacteria

Obligatory

Intracellulars

in Arthropods

Wolbachia
–
Anaplasma
Split

W
ins
-
W
nem

Split (~120MY)

Application of Bioinformatics to
Wolbachia

Mutualist

Wolbachia
:

Parasite

Outcomes: A New
Wolbachia

Species?

Wolbachia
: Complete genomic sequences from a
related parasite and mutualist

wBm
(Foster et al. 2005)

wMel
(Wu et al. 2004)

1.08 Mb


806 genes

1.27 Mb


1270 genes

696
shared
genes

Wu et al 2004

Foster et al 2005

Your Wolbachia Sequence
:

What do you do with it?

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



BLAST:

•
Compare new genes to old ones

•
Compare genes from different species or
hosts

•
Identify possible functions based on
similarities to known sequences.

Query a database for sequences homologous to


an input (ie, query) sequence.


GATG
C
C
A
T
A
G
A
G
C
T
G
T
A
G
T
C
GT
A
CCC
T <
—


—
>
C
T
A
G
A
G
A
G
C
-
G
T
A
G
T
C
AG
A
GTG
T
CTTTGAGTTCC


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


A Traditional

GenBank Record

LOCUS AY182241 1931 bp mRNA linear PLN 04
-
MAY
-
2004

DEFINITION 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

//

Header

Feature Table

Sequence

The Flatfile Format

LOCUS AY182241 1931 bp mRNA linear PLN 04
-
MAY
-
2004

DEFINITION 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.

The Header

LOCUS AY182241 1931 bp mRNA linear PLN 04
-
MAY
-
2004

DEFINITION 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.

Header: Locus Line

LOCUS AY182241 1931 bp mRNA linear PLN 04
-
MAY
-
2004

Molecule type

Division

Modification Date

Locus name

Length

LOCUS AY182241 1931 bp mRNA linear PLN 04
-
MAY
-
2004

DEFINITION 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.

Header: Database Identifiers

ACCESSION AY182241

VERSION AY182241.2 GI:32265057

Accession

•
Stable

•
Reportable

•
Universal

LOCUS AY182241 1931 bp mRNA linear PLN 04
-
MAY
-
2004

DEFINITION 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.

Header: Organism

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.

NCBI
-
controlled taxonomy

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


NHHFAHLKGMLELFEASNLGFEGEDILDEAKASLTLALRDSGHICYPDSNLSRDVVHS


LELPSHRRVQWFDVKWQINAYEKDICRVNATLLELAKLNFNVVQAQLQKNLREASRWW


ANLGIADNLKFARDRLVECFACAVGVAFEPEHSSFRICLTKVINLVLIIDDVYDIYGS


EEELKHFTNAVDRWDSRETEQLPECMKMCFQVLYNTTCEIAREIEEENGWNQVLPQLT


KVWADFCKALLVEAEWYNKSHIPTLEEYLRNGCISSSVSVLLVHSFFSITHEGTKEMA


DFLHKNEDLLYNISLIVRLNNDLGTSAAEQERGDSPSSIVCYMREVNASEETARKNIK


GMIDNAWKKVNGKCFTTNQVPFLSSFMNNATNMARVAHSLYKDGDGFGDQEKGPRTHI


LSLLFQPLVN"

The Feature Table

Coding sequence

start (atg)

stop (tag)

DNA

RNA

cDNA

phenotype

DNA sequences

genomes

protein

sequence

databases

protein

Bioinformatics is NOT just information technology.

It can teach the central dogmas of molecular biology


GATG
C
C
A
T
A
G
A
G
C
T
G
T
A
G
T
C
GT
A
CCC
T <
-

100%



GATG
C
C
A
T
A
G
A
G
C
T
G
T
A
G
T
C
GT
A
CCC
T <
-

100%



GATG
C
C
A
T
A
G
A
G
C
T
G
T
A
G
T
C
GT
A
CCC
T <
-

100%



GATG
C
C
A
T
A
G
A
G
C
T
G
T
A
G
T
C
GT
A
CCC
T <
-

100%


Insect Phylogeny

Top 5 Wolbachia BLAST matches


GATG
C
C
A
T
A
G
A
G
C
T
G
T
A
G
T
C
GT
A
CCC
T <
-

100%


Outcomes:

Lateral Transfer ?






Let
’
猠䉥杩渠併爠䉩潩s景牭慴楣⁅硥i捩獥c
䱡戠L

HTTP://WWW.DIGITALWORLDBIOLO
GY.COM/BLAST