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Unknown 13

Unknown 13. A Phage Mystery IT 227 – Purdue University Robert Agee 04/18/2013. Getting Started (Intro/Methods). Objective: Decide phage taxonomy prior to genomic sequencing Procedure: Protein production and isolation Orbitrap GC/MS SpectrumMill database comparisons PhagesDB , NCBInr

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Unknown 13

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  1. Unknown 13 A Phage Mystery IT 227 – Purdue University Robert Agee 04/18/2013

  2. Getting Started (Intro/Methods) • Objective: Decide phage taxonomy prior to genomic sequencing • Procedure: • Protein production and isolation • Orbitrap GC/MS • SpectrumMilldatabase comparisons • PhagesDB, NCBInr • Omics Discovery Pipeline comparison • Vs. Unknown 14 • Vs. Positive Control (Czyszczon1) • Vs. Negative Control

  3. Rounding up Suspects (Results) • PhagesDB, all species

  4. NCBInr, Mycobacterium species

  5. Top 20 Highest Scoring Proteins • Unknown 13 (PhagesDB)

  6. Top 20 Highest Scoring Proteins • Unknown 13 (NCBInr)

  7. Top 20 Highest Scoring Proteins • Unknown 13 vs. 14

  8. Top 20 Highest Scoring Proteins • Unknown 13 vs. Positive Control (Czyszczon1)

  9. Top 20 Highest Scoring Proteins • Positive Control (Czyszczon1)

  10. Top 20 Highest Scoring Proteins • Unknown 13 vs. Negative Control

  11. Exhibit A (Protein Comparison) • Expectations: • Smegmatis genes in Negative Control • Similar protein profile across comparisions • Surprises: • Most functions unknown • Phage proteins in Negative Control • Genomic overlap in database • Tuberculosis and Smegmatis • Diverse phams in phage samples

  12. Exhibit A (Protein Comparison) Pt2 • Unknown 13 vs. other phages: • Similar A cluster proteins and phams (genome ‘backbone’) • Bxz2 proteins • 271, 1842, 4284 • Different and specific proteins and phams in Uk13 • EpicPhail, BAKA proteins, BAKA_gp28 (major tail subunit) • 748, 2872

  13. Exhibit A (Protein Comparison) Pt3 • To summarize, Unknown 13: • Comparison against databases strongly suggests Cluster J. • Comparison against other samples strongly suggests Cluster J, particularly phams748 and 2872. • To summarize, Positive Control (Czyszczon1): • Comparison and individual protein profile are inconclusive.

  14. The Line-up (Morphology) • Cluster A suspects Bxz2 Unknown 13 web plate EpicPhail

  15. The Line-up (Morphology) Pt 2 • Cluster J suspects Ariel Unknown 13 web plate Omega

  16. Exhibit B (Morphology) • To summarize: • Unknown 13 - many small, temperate plaques 1-5mm in diameter. • Bxz2 – fewer, larger temperate plaques • EpicPhail – many small, temperate plaques • Ariel – small, temperate plaques • Omega – small, temperate plaques • Evidence is inconclusive, but suggests Cluster A or J are correct suspicions.

  17. Deliberation (Discussion/Thoughts) • Based on the protein profiles and morphology notes, Unknown 13 is mostly likely a member of Cluster J. • As an aside, the positive control remains unclustered. • Genomic sequencing is necessary to confirm both.

  18. Project Thoughts • Known/Discovered • Protein profile and morphology • Unknowns • Genomic sequence • Challenges • Genomic overlap in databases • Cluster vsSubcluster proteins • Fixed taxonomical assignments (highly fluid genome)

  19. References • http://www.phagesdb.org/ • http://www.omicsdp.org/ • http://chem.agilent.com/

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