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Bacterial functional genomics at Joint Genome Institute

Bacterial functional genomics at Joint Genome Institute. Pacific Biosciences User Meeting 09/18/13 Matthew Blow. DNA base modifications can be directly detected by PacBio sequencing. U nmodified DNA template. Time. Delay in base incorporation opposite methyl adenine.

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Bacterial functional genomics at Joint Genome Institute

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  1. Bacterial functional genomics at Joint Genome Institute Pacific Biosciences User Meeting 09/18/13 Matthew Blow

  2. DNA base modifications can be directly detected by PacBio sequencing Unmodified DNA template Time Delay in base incorporation opposite methyl adenine. Modified DNA template Time

  3. Detection of DNA base modifications is coverage dependent Require high coverage (100s x) for detection Detectable with ~50x coverage These modifications are important in microbial biology ~50x generated for microbe genome assembly

  4. DNA base modification sequencing at JGI x 2 Sufficient for >500 bacterial epigenomes / year - CSP call for bacterial epigenome projects (2 accepted) - JGI ‘Grand Challenge’ project: “Epigenomic landscape of Bacteria”

  5. Functional roles of DNA modification in Bacteria Cell cycle regulation GANTC in α-proteobacteria 1. Restriction systems 2. Regulation Many Others? Previously no easy way to look! Gene regulation GATC in E.Coli and other γ-proteobacteria Me 5’…GAATTC…3’ 3’…CTTAAG…5’ Me Me 5’…GAATTC…3’ Me Me Methylase (MT) protects host DNA RE MT 3’…CTTAAG…5’ Me ‘Orphan’ MT Me Me Me Me Me Me Me Me Me Challenge: Sequence specificities of restriction systems are hard to read

  6. The Epigenomic Landscape of Bacteria Rich Roberts (NEB) Jonas Korlach, Khailuong, Luke Hickey (Pacific Biosciences) Microbes with ‘interesting’ methylases Microbial genome program 100+ Diverse microbes 50x PacBio sequencing 100+ Methylomes Modified motifs Results 1. Descriptive tables of ‘Methylation landscape’ 3. Identification and characterization of regulatory DNA modifications 2. New insights into roles of methylation in Restriction systems Novel Known (GATC, GANTC)

  7. Global patterns of DNA modifications in bacteria Total analyzed genomes = 198 Genomes with modified motifs = 169 (>90%) Types of modification = 6mA (80%), 4/5mC (19%), ?T(1%) Total modified motifs = ~500 Average # modified motifs / genome = 3 (Max = 12) Novel motifs = ~20%

  8. New insights into restriction systems

  9. New insights into restriction systems • Analysis in progress by Rich Roberts at NEB • Many new patterns and specificities (e.g. novel protection of typeIIG and typeIII enzymes with m4C rather than 6mA) • Deep data may enable predictive models of DNA methylase specificity for type I enzymes

  10. 1) Are there any widely evolutionarily conserved modifications? 2) Are there unusual clusters of motifs in any organisms? 3) Are there unmodified sites in the genome? Identification of putative regulatory DNA modifications

  11. Evolutionarily conserved DNA modifications

  12. Evolutionarily conserved DNA modifications 2/2 thermococci Broad smattering of GATC modifications in other bacteria 2/2 cyanobacteria 5/11 clostridia The known GATC regulatory modification in γ-proteobacteria

  13. Evolutionarily conserved DNA modifications No GANTC outside of α-proteobacteria The known GANTC regulatory modification in α-proteobacteria No GANTC outside of α-proteobacteria

  14. Evolutionarily conserved DNA modifications 2/2 Halobacteria species 2/6 δ-proteobacteria species

  15. Evolutionarily conserved DNA modifications Conclusions: -No new major class of widely conserved modification -Several modifications conserved across more restricted sets of organisms -Need further evidence that they have some regulatory function All other modifications observed in 4 or less organisms

  16. Known example: gAtc sites in E.coli Are there unusual clusters of motifs in any organisms? Origin of replication GATC motif cluster Competition between methylase (dam) and other proteins for binding to GATC controls DNA replication

  17. Most enriched 1kb regions across all modifications / genomes Ctag not gAtc

  18. Conserved cluster of modified CTAG at replication origin in H. turkmenica orc1/cdc6 family replication initiation protein CTAG motifs Genes Conservation Halopigerxanaduensis Natrialbamagadii Conclusions: -Several genomes contain enriched clusters of modified motifs -May be indicators of novel regulatory functions for methylation -E.g Putative Ctag methylation control of DNA replication Conserved CTAG in non-coding sequence

  19. Are there unmodified sites in the genome? Non-methylated sites pose problem if part of restriction systems! Methylated motif Unmethylated motif

  20. Are there unmodified sites in the genome? Non-methylated sites may be due to ‘protection’ by competing binding proteins Methylated motif Unmethylated motif DNA-binding protein Methylase 1. Non methylated is just footprint of DNA binding protein (no regulation) 2. Competition between Methylase and DNA binding protein forms regulatory mechanism (multiple published examples support this)

  21. Can we detect known non-methylated GATC sites in E. Coli? Me Me GATC GATC GATC GATC Includes 3 / 3 known regulatory sites upstream of antigen 43! Unmethylated regulatory? Novel cluster of 3 unmodified sites upstream of tonB Antigen43 (Waldron et al Molmicrobiol 2002, Wallecha et al J. bacteriol 2002)

  22. Reproducible evidence for non-methylated GATC in gamma-proteobacteria Four γ-proteobacteria with regulatory GATC modification E.coli S.oneidensis Alteromonas sp. S.bongori What are the properties of non-methylated sites? -Enriched at 5’ end of genes / operons -One example reproducible across 2 genomes (tonB dependent receptor) -Multiple sites upstream of transcriptional regulators Four clostridia with restriction systemassociated GATC modification P.ferrophilus (b) (Archaea) C.thermocellum (a) C.thermocellum (b) C.clariflavum (b)

  23. Evidence for non-methylated GANTC sites in alpha-proteobacteria Bradyrhizobiumelkani Rhizobium etli Agrobacterium tumefaciens Methylobacteriumextorquens

  24. Are there examples of regulatory signatures at novel motifs? Spirochaetasmaragdinae raAtty Conclusions: -Analyses of non-methylated sites identifies known regulatory sites in gamma-proteobacteria -Candidate novel regulatory sites associated with known regulatory methylases -Evidence for regulatory sites involving ‘novel’ methylases 4/9 unmodified sites clustered upstream of GntRtranscriptional regulator

  25. Bacterial Epigenomics Summary We have generated ~200 bacterial DNA base modification datasets on the PacBio platform New insights into role of methylation in restriction systems and gene regulation JGI is soliciting additional projects to explore the functions of DNA base modification (CSP call)

  26. Acknowledgements JGI Jean Zhao Katy Munson Feng Chen Chris Daum Christa Pennacchio Matt Bendall Rex Malmstrom Len Pennacchio NEB Rich Roberts Dana Macelis Pac Bio Jonas Korlach KhaiLuong Luke Hickey PacBio Jonas Korlach KhaiLuong Luke Hickey NEB

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