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DNA Extraction and Primer Selection
Søren M. Karst, Mads Albertsen,Anja S. Ziegler and Per H. Nielsen
MEWE 2013
CENTER FOR MICROBIAL COMMUNITIES
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
”Obtaining a representative overview of the microbial community is important for
understanding the ecology of a system”
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sampling SequencingExtraction Sample prep Bioinformatics
16S Amplicon Sequencing
Sampling Extraction Sample prep
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?
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
DNA extraction independent comparison
Nielsen et al., 2012. Current opinion in biotechnology 23, 452–9.
Commonsense
Explore parameters
StandardLow High
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sampling SequencingExtraction Sample prep Bioinformatics
Storage
• Fresh• 24 h @ 4°C• 24 h @ 20 °C
12
3
4
Standardization!!!!
Replicates
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sampling SequencingExtraction Sample prep Bioinformatics
BiomassInput (mg)Bead beating
Intensity (ms-1)
Dur
ation
(s)
4 6
400160
804020
1 2 4 9 22
eDNA removal
PMA650 W 10 min+ N+ CH3
NH2
N3
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
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Time series
0 2 4 8 13 23Weeks
Activated sludge from 2 different WWTPs
Aalborg WestWWTP
Aalborg EastWWTP
Put Parameters Variations into Context
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Primer Selection
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Bp
Mea
n fr
eque
ncy
of
mos
t com
mon
resi
due
in 5
0 bp
win
dow
0 500 1000 1500
1.0
0.8
0.6 V1 V2 V3V4 V5
V6
V7 V8V9
V1.3 V4V3.4
76.8% 81.6% 82.6%
V1.3 V3.4 V4
Coverage of SILVA SSU database
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)40
016
0804020804020
10 kbp
6 kbp
3 kbp
1 kbp
-
-
-
-
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYStandard 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. Standardize2. Intense Bead Beating3. V1-3 primers4. qFISH reference
www.midasfieldguide.org
M. Albertsen Poster“The influence of DNA extraction and primer choice on phylogenetic analysis inactivated sludge communities”
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
1. Standardize2. Test on own Ecosystem3. qFISH reference
GeneralRecommendations
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Thank you for your attention
Acknowledgements
Mads AlbertsenAnja Sloth ZieglerPer Halkjær NielsenStaff at “Center for Microbial Communties”
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