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

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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
-
-
-
-

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19. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

20. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Standard Settings

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Comparing All
Data

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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”