1. Wolbachia Bioinformatics

2. Two 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

3. Advantages of BLAST No programming skills needed
Familiarity with personal computer and internet browser
Customizable and free

4. 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 understand phylogenies
To put your Wolbachia research in the context of what has been published

5. What are the specific goals of this lab?
(Eventually) to look for brand new Wolbachia strains using new sequence data.
To be able to make a phylogenetic tree of Wolbachia spp.
To contribute to a national “student” sequence database on the genetic diversity of Wolbachia 16S rRNA gene
EXTENSION:
To compare the Wolbachia tree to an insect phylogeny to infer lateral vs. vertical transmission of your Wolbachia strains.

6. Wolbachia – Host Interactions: Mutualism and Reproductive Parasitism
Required for insect oogenesis (Dedeine et al. 2001)
Mutualism
Parthenogenesis in wasps
Reproductive parasitism
Male-killing in insects
Required for nematode fertility and larval development
Feminization in isopods
Cytoplasmic incompatibility in arthropods

7. What we see here is a phylogenetic tree of the three Domains of life
What we see here is a phylogenetic tree of the three Domains of life. The three domain system is a biological classification system introduced by Carl Woese in 1977 that divides cellular life forms into archaea, bacteria and eukarya.
This system is different from the 5 kingdom system in that instead of relying only on physical characteristics and physiological processes (how the organism obtains energy) it looks at genetic relationships by comparing the Small Subunit rRNA gene (16S in prokaryotes and 18S in eukaryotes).
When we think about evolution, all life evolved from a common ancestor, often referred to as LUCA (Last Universal Common Ancestor). The earliest organisms on the planet were the bacteria, which evolved ~ 3.8 BYA, before the Archaea. About 1 BYA animals evolved.
One of the things that I want you to notice is that everything, except what is outlined in this blue circle is microbial…..single cellular organisms, or microorganisms. So these organisms that were the last to evolve, evolved alongside microbes. This means that they evolved with the challenges of dealing with MOs as parasites or they developed long term relationships with these organisms as beneficial or mutualistic symbionts.
The bottom line is that MOs will be here long after any animal is, and we can not live with out these microorganisms.

8. Application of Bioinformatics to Wolbachia
Alpha Proteobacteria
Wolbachia –Anaplasma Split
Ehrlichia
Anaplasma
Wolbachia
Neorickettsia
Obligatory
Intracellulars
in Arthropods
Rickettsia
Wins-Wnem
Split (~120MY)
Dunning-Hottop et al 2006

9. Wolbachia:
Mutualist
Parasite

10. Outcomes: A New Wolbachia Species?
Bioinformatics Lab - Dr. Seth Bordenstein
How do do this? BLAST to find out if your sequence is divergent?

11. Your Wolbachia Sequence: What do you do with it?
Bioinformatics Lab - Dr. Seth Bordenstein
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
1/10,000 bp error ratio

12. BLAST:
Bioinformatics Lab - Dr. Seth Bordenstein
Interrogate 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.

13. BLAST is like using Google for DNA Sequences

14. Bioinformatics Lab - Dr. Seth Bordenstein
National Center for Biotechnology Information (NCBI)
NCBI homepage. Logo will take you back to home page.
About NCBI provides introduction to the NCBI and contains basic information on genetics and bioinformatics.

15. Release 2008: 99 billion base pairs
99 million sequences

16. Target YOUR database: Adjustable using the pull-down menu
Bioinformatics Lab - Dr. Seth Bordenstein

20. A Traditional GenBank Record
Bioinformatics Lab - Dr. Seth Bordenstein
A Traditional GenBank Record
LOCUS AY bp mRNA linear PLN 04-MAY-2004
DEFINITION Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
complete cds.
ACCESSION AY182241
VERSION AY GI:
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, (2004)
REFERENCE 2 (bases 1 to 1931)
TITLE Direct Submission
JOURNAL Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REFERENCE 3 (bases 1 to 1931)
JOURNAL Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
REMARK Sequence update by submitter
COMMENT On Jun 26, 2003 this sequence version replaced gi:
FEATURES Location/Qualifiers
source
/organism="Malus x domestica"
/mol_type="mRNA"
/cultivar="'Law Rome'"
/db_xref="taxon:3750"
/tissue_type="peel"
gene
/gene="AFS1"
CDS
/note="terpene synthase"
/codon_start=1
/product="(E,E)-alpha-farnesene synthase"
/protein_id="AAO "
/db_xref="GI: "
/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 //
The Flatfile Format
Header
Line-type identifier format.
Feature Table
Sequence

21. The Header LOCUS AY182241 1931 bp mRNA linear PLN 04-MAY-2004
Bioinformatics Lab - Dr. Seth Bordenstein
LOCUS AY bp mRNA linear PLN 04-MAY-2004
DEFINITION Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
complete cds.
ACCESSION AY182241
VERSION AY GI:
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, (2004)
REFERENCE 2 (bases 1 to 1931)
TITLE Direct Submission
JOURNAL Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REFERENCE 3 (bases 1 to 1931)
JOURNAL Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
REMARK Sequence update by submitter
COMMENT On Jun 26, 2003 this sequence version replaced gi:

22. Header: Locus Line Length Division Molecule type Locus name
Bioinformatics Lab - Dr. Seth Bordenstein
LOCUS AY bp mRNA linear PLN 04-MAY-2004
DEFINITION Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
complete cds.
ACCESSION AY182241
VERSION AY GI:
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, (2004)
REFERENCE 2 (bases 1 to 1931)
TITLE Direct Submission
JOURNAL Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REFERENCE 3 (bases 1 to 1931)
JOURNAL Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
REMARK Sequence update by submitter
COMMENT On Jun 26, 2003 this sequence version replaced gi:
LOCUS AY bp mRNA linear PLN 04-MAY-2004
Molecule type
Division
Modification Date
Locus name
Length
Locus line.

23. Header: Database Identifiers
Bioinformatics Lab - Dr. Seth Bordenstein
LOCUS AY bp mRNA linear PLN 04-MAY-2004
DEFINITION Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
complete cds.
ACCESSION AY182241
VERSION AY GI:
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, (2004)
REFERENCE 2 (bases 1 to 1931)
TITLE Direct Submission
JOURNAL Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REFERENCE 3 (bases 1 to 1931)
JOURNAL Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
REMARK Sequence update by submitter
COMMENT On Jun 26, 2003 this sequence version replaced gi:
Accession
Stable
Reportable
Universal
ACCESSION AY182241
VERSION AY GI:

24. NCBI-controlled taxonomy
Header: Organism
Bioinformatics Lab - Dr. Seth Bordenstein
LOCUS AY bp mRNA linear PLN 04-MAY-2004
DEFINITION Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
complete cds.
ACCESSION AY182241
VERSION AY GI:
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, (2004)
REFERENCE 2 (bases 1 to 1931)
TITLE Direct Submission
JOURNAL Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REFERENCE 3 (bases 1 to 1931)
JOURNAL Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
REMARK Sequence update by submitter
COMMENT On Jun 26, 2003 this sequence version replaced gi:
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
Portion of the record that NCBI controls. Retrieving sequences in precise and accurate way (useful for Entrez searching).

25. The Feature Table Coding sequence FEATURES Location/Qualifiers
Bioinformatics Lab - Dr. Seth Bordenstein
FEATURES Location/Qualifiers
source
/organism="Malus x domestica"
/mol_type="mRNA"
/cultivar="'Law Rome'"
/db_xref="taxon:3750"
/tissue_type="peel"
gene
/gene="AFS1"
CDS
/note="terpene synthase"
/codon_start=1
/product="(E,E)-alpha-farnesene synthase"
/protein_id="AAO "
/db_xref="GI: "
/translation="MEFRVHLQADNEQKIFQNQMKPEPEASYLINQRRSANYKPNIWK
NDFLDQSLISKYDGDEYRKLSEKLIEEVKIYISAETMDLVAKLELIDSVRKLGLANLF
EKEIKEALDSIAAIESDNLGTRDDLYGTALHFKILRQHGYKVSQDIFGRFMDEKGTLE
NHHFAHLKGMLELFEASNLGFEGEDILDEAKASLTLALRDSGHICYPDSNLSRDVVHS
LELPSHRRVQWFDVKWQINAYEKDICRVNATLLELAKLNFNVVQAQLQKNLREASRWW
ANLGIADNLKFARDRLVECFACAVGVAFEPEHSSFRICLTKVINLVLIIDDVYDIYGS
EEELKHFTNAVDRWDSRETEQLPECMKMCFQVLYNTTCEIAREIEEENGWNQVLPQLT
KVWADFCKALLVEAEWYNKSHIPTLEEYLRNGCISSSVSVLLVHSFFSITHEGTKEMA
DFLHKNEDLLYNISLIVRLNNDLGTSAAEQERGDSPSSIVCYMREVNASEETARKNIK
GMIDNAWKKVNGKCFTTNQVPFLSSFMNNATNMARVAHSLYKDGDGFGDQEKGPRTHI
LSLLFQPLVN"
start (atg)
stop (tag)
Coding sequence
Biologically interesting information.

26. Bioinformatics is NOT just information technology.
Bioinformatics Lab - Dr. Seth Bordenstein
Bioinformatics is NOT just information technology.
It can teach the central dogmas of molecular biology
DNA
RNA
protein
phenotype
protein
sequence
databases
cDNA
DNA sequences
genomes

27. Let’s Begin Our Bioinformatic Exercise Lab 5

28. https://digitalworldbiology.com/blast