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On-site Identification
of Microorganisms by
Nanopore Technology
Peter Rendbæk
Rasmus H. Kirkegaard, Per H. Nielsen &
Mads ...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Background
Aim
Identification using
DNA sequencing
Experimental setu...
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
...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Why do we need the ID of microbes?
Foam formation by Microthrix Nitr...
From university to operation
On-site microfluidic
sample preparation and
DNA Sequencing
Sampling
Cloud-based
bioinformatic...
Develop “real time” and on-site identification of
microbes in activated sludge.
Specific objectives:
• Develop fast, porta...
SequencingExtraction Sample prep BioinformaticsSampling
DNA
Extraction Sample prep.
>50 min 150 min
CENTER FOR MICROBIAL C...
SequencingExtraction Sample prep BioinformaticsSampling
DNA
Extraction Sample prep.
>50 min 150 min
CENTER FOR MICROBIAL C...
Setup of the mobile lab
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
SequencingSample prep BioinformaticsSampling...
Strategy for developing a rapid DNA
extraction method
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Standard
activ...
Old vs. New method (3 principle steps)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Step 1: Cell lysis
Step 2: Re...
Step 1: Cell lysis
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Standard bead-beater Mobile bead-beater
Price: 83...
3D printable replacement part
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
www.thingiverse.com/Peter161
MBB adapt...
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...
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
Optimizat...
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. N...
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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.

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Presentation of master project at status seminar

  1. 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. 2. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Background Aim Identification using DNA sequencing Experimental setup Methods and results Future plans Agenda
  3. 3. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Microbes are everywhere!
  4. 4. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Microbes clean our wastewater Sewer system Aalborg Vest Renseanlæg
  5. 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. 6. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Why do we need the ID of microbes? Foam formation by Microthrix Nitrogen removal by Thauera
  7. 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. 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. 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. 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. 11. Setup of the mobile lab CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY SequencingSample prep BioinformaticsSampling Sample prep. DNA Extraction
  12. 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. 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. 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. 15. 3D printable replacement part CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY www.thingiverse.com/Peter161 MBB adaptor
  16. 16. Step 2: Remove cell debris CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Centrifuge at 2000 xG for 1 min
  17. 17. Step 3: DNA isolation and elution CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  18. 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. 19. Analyses of preliminary tests CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  20. 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. 21. The mobile lab is taking shape! CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  22. 22. Future plans CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  23. 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

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