DNA Extraction and
Primer Selection
Søren M. Karst, Mads Albertsen,
Anja S. Ziegler and Per H. Nielsen
MEWE 2013
CENTER FO...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Agenda
Why do we need to care about DNA extraction and
primer select...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
”Obtaining a representative overview of the
microbial community is i...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sampling SequencingExtraction Sample prep Bioinformatics
16S Amplico...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
”Obtaining a representative overview of the
microbial community is i...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
DNA extraction
independent
comparison
Nielsen et al., 2012. Current ...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Agenda
Why do we need to care about DNA extraction and
primer select...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sampling SequencingExtraction Sample prep Bioinformatics
Storage
• F...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sampling SequencingExtraction Sample prep Bioinformatics
Biomass
Inp...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sampling SequencingExtraction Sample prep Bioinformatics
Standardize...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
DNA extraction independent assesment using 28 qFISH probes which
cov...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Time series
0 2 4 8 13 23
Weeks
Activated sludge from 2
different WW...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Primer
Selection
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Bp
Meanfrequencyof
mostcommonresidue
in50bpwindow
0 500 1000 1500
1....
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Bead
Beating
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
4 6 Intensity (ms-1)
Duration (s)
400
160
80
40
20
80
40
20
10 kbp
6...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Standard Settings
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Comparing All
Data
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Activated Sludge
Recommendations
1. Standardize
2. Intense Bead Beat...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
1. Standardize
2. Test on own Ecosystem
3. qFISH reference
General
R...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Thank you for your attention
Acknowledgements
Mads Albertsen
Anja Sl...
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[13.07.07] karst mewe13 dna_extraction_nonotes

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[13.07.07] karst mewe13 dna_extraction_nonotes

  1. 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. 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. 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. 4. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Sampling SequencingExtraction Sample prep Bioinformatics 16S Amplicon Sequencing Sampling Extraction Sample prep
  5. 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. 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. 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. 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. 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. 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. 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. 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. 13. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Primer Selection
  14. 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. 15. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  16. 16. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Bead Beating
  17. 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. 18. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  19. 19. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  20. 20. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Standard Settings
  21. 21. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Comparing All Data
  22. 22. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  23. 23. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  24. 24. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  25. 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. 26. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY 1. Standardize 2. Test on own Ecosystem 3. qFISH reference General Recommendations
  27. 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”
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