Wolbachia Bioinformatics

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

Advantages of BLAST No programming skills needed Familiarity with personal computer and internet browser 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 understand phylogenies To put your Wolbachia research in the context of what has been published

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.

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

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.

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

Wolbachia: Mutualist Parasite

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

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

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.

BLAST is like using Google for DNA Sequences

Bioinformatics Lab - Dr. Seth Bordenstein National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov 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.

Release 2008: 99 billion base pairs 99 million sequences

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

A Traditional GenBank Record Bioinformatics Lab - Dr. Seth Bordenstein 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) 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) 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: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 /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 // The Flatfile Format Header Line-type identifier format. Feature Table Sequence

The Header LOCUS AY182241 1931 bp mRNA linear PLN 04-MAY-2004 Bioinformatics Lab - Dr. Seth Bordenstein 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) 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) 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:27804758.

Header: Locus Line Length Division Molecule type Locus name Bioinformatics Lab - Dr. Seth Bordenstein 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) 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) 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:27804758. LOCUS AY182241 1931 bp mRNA linear PLN 04-MAY-2004 Molecule type Division Modification Date Locus name Length Locus line.

Header: Database Identifiers Bioinformatics Lab - Dr. Seth Bordenstein 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) 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) 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:27804758. Accession Stable Reportable Universal ACCESSION AY182241 VERSION AY182241.2 GI:32265057

NCBI-controlled taxonomy Header: Organism Bioinformatics Lab - Dr. Seth Bordenstein 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) 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) 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:27804758. 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).

The Feature Table Coding sequence FEATURES Location/Qualifiers Bioinformatics Lab - Dr. Seth Bordenstein 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 /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" start (atg) stop (tag) Coding sequence Biologically interesting information.

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

Let’s Begin Our Bioinformatic Exercise Lab 5

https://digitalworldbiology.com/blast