Presentation of master project at status seminar

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My presentation at the status seminar. The status seminar is all the master students at AAU Biotechnology department presenting their master thesis after four months of work.

On-site Identification
of Microorganisms by
Nanopore Technology
Peter Rendbæk
Rasmus H. Kirkegaard, Per H. Nielsen &
Mads Albertsen
CENTER FOR MICROBIAL COMMUNITIES
Statusseminar 2017
@PeterRendbaek

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Background
Aim
Identification using
DNA sequencing
Experimental setup
Methods and results
Future plans
Agenda

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Microbes are everywhere!

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Microbes clean our wastewater
Sewer
system
Aalborg Vest Renseanlæg

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Identification using 16S rRNA sequencing
DNA
Gene
Might code for an
enzyme that
degrades fat.
(Bacteria have 3000-5000 genes)
Genome
16S rRNA
Used as a fingerprint
for bacteria.
Unique for different
bacteria.
“16S rRNA amplicon sequencing”

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Why do we need the ID of microbes?
Foam formation by Microthrix Nitrogen removal by Thauera

From university to operation
On-site microfluidic
sample preparation and
DNA Sequencing
Sampling
Cloud-based
bioinformatic
processing
Data generation
Microbe
identification
Decisions in
operation
Functional
information
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Develop “real time” and on-site identification of
microbes in activated sludge.
Specific objectives:
• Develop fast, portable, robust and easy to use DNA
extraction.
• Perform on-site DNA sequencing.
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Aim

SequencingExtraction Sample prep BioinformaticsSampling
DNA
Extraction Sample prep.
>50 min 150 min
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Now 2880 min Total time: 7 days
Identification using DNA sequencing

Setup of the mobile lab
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
SequencingSample prep BioinformaticsSampling Sample prep.
DNA
Extraction

Strategy for developing a rapid DNA
extraction method
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Standard
active sludge
Standard
protocol
Yield
Microbial
community
16SrRNA v1-3
New Method

Old vs. New method (3 principle steps)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Step 1: Cell lysis
Step 2: Remove cell debris
Step 3: DNA isolation and elution

Step 1: Cell lysis
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Standard bead-beater Mobile bead-beater
Price: 83,000 kr Price: 700 kr
Weight: 20 kg Weight: 2kg
Replacement part:
3d printabel
Replacement part:
Not 3d printabel

3D printable replacement part
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
www.thingiverse.com/Peter161
MBB adaptor

Step 2: Remove cell debris
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Centrifuge at 2000 xG for 1 min

Step 3: DNA isolation and elution
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Does it work?
22
15
18
10
Standard
MiDAS
Midas AMPpure 1
Midas AMPpure 2
Midas AMPpure 3
0.0 5.0 10.0 15.0 20.0 25.0
Yield: Total DNA output/biomas input [ng/µL]
DNA fragments
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
New Protocol : 15 min
Standard Protocol: >50 min
1 2 3Ladder Standard
Standard
New 1
New 2
New 3
DNA Yield

Analyses of preliminary tests
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Steps to optimize and steps left
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Step 1: Bead-beading time
Optimization
Step 2: Centrifuge time and speed
Step left to chose DNA extraction method for Mobile lab
Sequencing the 16S rRNA of samples
Analyse the composition of microbes community
by use of Principal component analyses(PCA)
Step 3: Concentration of AMPure beads
Step Time [min]
Bead beading 1.00
Centrifuge 2000XG 1.00
Incubation of AMPure beads 5.00
Magnetic separation of beads 2.30
Washing of bead with ethanol 1.30
Drying of bead 0.30
Resuspendere of bead 1.00
Magnetic separation of beads 2.30
Total time 15.00
Name of step Time [min]
Bead-beading 10.00
centrifuge 10000XG 10.00
Protein Precipitation 7.00
Bindning of DNA 7.00
Ethanol wasning 4.00
Drying of sample 7.00
Elution af DNA 3.00
Total time 48.00
Current protocol
Standard protocol

The mobile lab is taking shape!
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Future plans
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Thanks for the attention
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Albertsen Lab
www.albertsenlab.org
Per H. Nielsen
Mads AlbertsenRasmus H. Kirkegaard
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Presentation of master project at status seminar

1. On-site Identification of Microorganisms by Nanopore Technology Peter Rendbæk Rasmus H. Kirkegaard, Per H. Nielsen & Mads Albertsen CENTER FOR MICROBIAL COMMUNITIES Statusseminar 2017 @PeterRendbaek

2. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Background Aim Identification using DNA sequencing Experimental setup Methods and results Future plans Agenda

3. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Microbes are everywhere!

4. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Microbes clean our wastewater Sewer system Aalborg Vest Renseanlæg

5. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Identification using 16S rRNA sequencing DNA Gene Might code for an enzyme that degrades fat. (Bacteria have 3000-5000 genes) Genome 16S rRNA Used as a fingerprint for bacteria. Unique for different bacteria. “16S rRNA amplicon sequencing”

6. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Why do we need the ID of microbes? Foam formation by Microthrix Nitrogen removal by Thauera

7. From university to operation On-site microfluidic sample preparation and DNA Sequencing Sampling Cloud-based bioinformatic processing Data generation Microbe identification Decisions in operation Functional information CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

8. Develop “real time” and on-site identification of microbes in activated sludge. Specific objectives: • Develop fast, portable, robust and easy to use DNA extraction. • Perform on-site DNA sequencing. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Aim

9. SequencingExtraction Sample prep BioinformaticsSampling DNA Extraction Sample prep. >50 min 150 min CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Now 2880 min Total time: 7 days Identification using DNA sequencing

10. SequencingExtraction Sample prep BioinformaticsSampling DNA Extraction Sample prep. >50 min 150 min CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Now 2880 min Total time: 7 days Identification using DNA sequencing 15 min 10 min 10 min Total time: 40 min SequencingSample prep BioinformaticsSampling Sample prep. DNA Extraction Soon

11. Setup of the mobile lab CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY SequencingSample prep BioinformaticsSampling Sample prep. DNA Extraction

12. Strategy for developing a rapid DNA extraction method CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Standard active sludge Standard protocol Yield Microbial community 16SrRNA v1-3 New Method

13. Old vs. New method (3 principle steps) CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Step 1: Cell lysis Step 2: Remove cell debris Step 3: DNA isolation and elution

14. Step 1: Cell lysis CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Standard bead-beater Mobile bead-beater Price: 83,000 kr Price: 700 kr Weight: 20 kg Weight: 2kg Replacement part: 3d printabel Replacement part: Not 3d printabel

15. 3D printable replacement part CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY www.thingiverse.com/Peter161 MBB adaptor

16. Step 2: Remove cell debris CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Centrifuge at 2000 xG for 1 min

17. Step 3: DNA isolation and elution CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

18. Does it work? 22 15 18 10 Standard MiDAS Midas AMPpure 1 Midas AMPpure 2 Midas AMPpure 3 0.0 5.0 10.0 15.0 20.0 25.0 Yield: Total DNA output/biomas input [ng/µL] DNA fragments CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY New Protocol : 15 min Standard Protocol: >50 min 1 2 3Ladder Standard Standard New 1 New 2 New 3 DNA Yield

19. Analyses of preliminary tests CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

20. Steps to optimize and steps left CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Step 1: Bead-beading time Optimization Step 2: Centrifuge time and speed Step left to chose DNA extraction method for Mobile lab Sequencing the 16S rRNA of samples Analyse the composition of microbes community by use of Principal component analyses(PCA) Step 3: Concentration of AMPure beads Step Time [min] Bead beading 1.00 Centrifuge 2000XG 1.00 Incubation of AMPure beads 5.00 Magnetic separation of beads 2.30 Washing of bead with ethanol 1.30 Drying of bead 0.30 Resuspendere of bead 1.00 Magnetic separation of beads 2.30 Total time 15.00 Name of step Time [min] Bead-beading 10.00 centrifuge 10000XG 10.00 Protein Precipitation 7.00 Bindning of DNA 7.00 Ethanol wasning 4.00 Drying of sample 7.00 Elution af DNA 3.00 Total time 48.00 Current protocol Standard protocol

21. The mobile lab is taking shape! CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

22. Future plans CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

23. Thanks for the attention CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Albertsen Lab www.albertsenlab.org Per H. Nielsen Mads AlbertsenRasmus H. Kirkegaard + The EB group

Editor's Notes

behøves ikke snakket om billedet
Bakterier er vigtig alle steder og det vigtig og ID dem, dermed kigge på reslingasluse. Tildte bedre Problem-> løsning For meget på sildes, bedre orden Nogen barktrier dårlig, hurtig skeverign Del op I to.
Bakterier er vigtig alle steder og det vigtig og ID dem, dermed kigge på reslingasluse. Tildte bedre Problem-> løsning For meget på sildes, bedre orden Nogen barktrier dårlig, hurtig skeverign Del op I to.
Bakterier er vigtig alle steder og det vigtig og ID dem, dermed kigge på reslingasluse. Tildte bedre Problem-> løsning For meget på sildes, bedre orden Nogen barktrier dårlig, hurtig skeverign Del op I to.
Lave total tid rød ‘ Bruge som billed som efter Brug same billed
Lave total tid rød ‘ Bruge som billed som efter Brug same billed
0
Forskillend på illumeme og nanopore.
Samling med midas protokolen.
Vedio of 3d printening
Vedio of 3d printening
Vedio of 3d printening
Vedio of 3d printening
Ikke brug minion, DNA fragment=how good quliterty MiDAS= standart Rød stregn på nok yield
PCA forkalr kort hvad der er, akser og heatmap Pænere, bedre skaler Sæt min værdig op, Nævn farved efter abundens Mere hamoniske
Total tid Samling med miDAs Igen tid

Presentation of master project at status seminar

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Editor's Notes