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[13.07.07] karst mewe13 dna_extraction_nonotes

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  • 1. DNA Extraction and Primer Selection Søren M. Karst, Mads Albertsen, Anja S. Ziegler and Per H. Nielsen MEWE 2013 CENTER FOR MICROBIAL COMMUNITIES
  • 2. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Agenda Why do we need to care about DNA extraction and primer selection? Exploring the influence of DNA extraction and primer selection in Activated Sludge Recommendations
  • 3. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY ”Obtaining a representative overview of the microbial community is important for understanding the ecology of a system”
  • 4. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Sampling SequencingExtraction Sample prep Bioinformatics 16S Amplicon Sequencing Sampling Extraction Sample prep
  • 5. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY ”Obtaining a representative overview of the microbial community is important for understanding the ecology of a system” What is representative?
  • 6. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY DNA extraction independent comparison Nielsen et al., 2012. Current opinion in biotechnology 23, 452–9. Common sense Explore parameters StandardLow High
  • 7. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Agenda Why do we need to care about DNA extraction and primer selection? Exploring the influence of DNA extraction and primer selection in Activated Sludge Recommendations
  • 8. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Sampling SequencingExtraction Sample prep Bioinformatics Storage • Fresh • 24 h @ 4°C • 24 h @ 20 °C 1 2 3 4 Standardization!!!! Replicates
  • 9. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Sampling SequencingExtraction Sample prep Bioinformatics Biomass Input (mg)Bead beating Intensity (ms-1) Duration(s) 4 6 400 160 80 40 20 1 2 4 9 22 eDNA removal PMA650 W 10 min + N+ CH3 NH2 N3
  • 10. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Sampling SequencingExtraction Sample prep Bioinformatics Standardize Protocol SettingsPrimer Selection Ashelford et al.2005. Appl. Environ. Microbiol. 71, 7724-36 • Input DNA • PCR Cycles
  • 11. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY DNA extraction independent assesment using 28 qFISH probes which cover most of the known bacterial diversity in Danish EBPR plants
  • 12. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Time series 0 2 4 8 13 23 Weeks Activated sludge from 2 different WWTPs Aalborg West WWTP Aalborg East WWTP Put Parameters Variations into Context
  • 13. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Primer Selection
  • 14. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Bp Meanfrequencyof mostcommonresidue in50bpwindow 0 500 1000 1500 1.0 0.8 0.6 V1 V2 V3 V4 V5 V6 V7 V8 V9 V1.3 V4 V3.4 76.8% 81.6% 82.6% V1.3 V3.4 V4 Coverage of SILVA SSU database
  • 15. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 16. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Bead Beating
  • 17. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY 4 6 Intensity (ms-1) Duration (s) 400 160 80 40 20 80 40 20 10 kbp 6 kbp 3 kbp 1 kbp - - - -
  • 18. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 19. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 20. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Standard Settings
  • 21. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Comparing All Data
  • 22. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 23. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 24. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 25. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Activated Sludge Recommendations 1. Standardize 2. Intense Bead Beating 3. V1-3 primers 4. qFISH reference www.midasfieldguide.org M. Albertsen Poster “The influence of DNA extraction and primer choice on phylogenetic analysis in activated sludge communities”
  • 26. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY 1. Standardize 2. Test on own Ecosystem 3. qFISH reference General Recommendations
  • 27. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Thank you for your attention Acknowledgements Mads Albertsen Anja Sloth Ziegler Per Halkjær Nielsen Staff at “Center for Microbial Communties”