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

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”Obtaining a representative overview of the microbial community is important for

understanding the ecology of a system”

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Sampling SequencingExtraction Sample prep Bioinformatics

16S Amplicon Sequencing

Sampling Extraction Sample prep

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”Obtaining a representative overview of the microbial community is important for

understanding the ecology of a system”

What is representative?

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DNA extraction independent comparison

Nielsen et al., 2012. Current opinion in biotechnology 23, 452–9.

Commonsense

Explore parameters

StandardLow High

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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

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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

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Sampling SequencingExtraction Sample prep Bioinformatics

Standardize Protocol SettingsPrimer Selection

Ashelford et al.2005. Appl. Environ. Microbiol. 71, 7724-36

• Input DNA• PCR Cycles

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DNA extraction independent assesment using 28 qFISH probes which cover most of the known bacterial diversity in Danish EBPR plants

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Time series

0 2 4 8 13 23Weeks

Activated sludge from 2 different WWTPs

Aalborg WestWWTP

Aalborg EastWWTP

Put Parameters Variations into Context

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Primer Selection

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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

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Bead Beating

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4 6 Intensity (ms-1)Duration (s)40

016

0804020804020

10 kbp

6 kbp

3 kbp

1 kbp

-

-

-

-

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CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYStandard Settings

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Comparing All Data

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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

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Thank you for your attention

Acknowledgements

Mads AlbertsenAnja Sloth ZieglerPer Halkjær NielsenStaff at “Center for Microbial Communties”