This was my master thesis presentation for my thesis: Developing methods for on-site DNA sequencing

1. Developing Methods for
On-site DNA Sequencing
Peter Rendbæk
Rasmus H. Kirkegaard, Per H. Nielsen &
Mads Albertsen
CENTER FOR MICROBIAL COMMUNITIES

2. Background
Aim
Description of the methods developed
Results
Conclusion
Further development
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 West Wastewater Treatment Plant

5. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
16S rRNA amplicon sequencing
DNA
Gene
Might code for an
enzyme that
degrades fat.
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 to identify microbes?
Foaming caused by Candidatus Microthrix Nitrogen removal by Thauera

7. SequencingExtraction Sample prep Bioinformatics
DNA
Extraction Sample prep.
>50 min 150 min
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Now 2880 min
Identification by 16S rRNA amplicon sequencing
>50 min 150 min 2880 min Total time: 7 days
Sampling

8. SequencingExtraction Sample prep BioinformaticsSampling
DNA
Extraction Sample prep.
>50 min 150 min
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Now 2880 min
10 min 10 min 10 min Total time: 30 min
SequencingSample prep BioinformaticsSampling Sample prep.
DNA
Extraction
Soon
Identification by 16S rRNA amplicon sequencing
Total time: 7 days

9. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Novel technologies are portable, fast and
enables real-time data
VolTRAX: automated sample preparation MinION: USB-sized DNA
sequencer

10. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
The importance of portability
Real time sequencing and portable
genomic surveillance of Ebola
DNA sequencing in microgravity on
the International Space Station (ISS)
And more…
Genomic survalianse of Zika virus in brazil

11. From laboratory identification to on-site
identification
On-site microfluidic
sample preparation and
DNA Sequencing
Sampling
Cloud-based
bioinformatic
processing
Data generation
Microbe
identification
Operational
decisions
Functional
information
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

12. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Problem

13. The objective of this project is to develop a mobile,
fast, robust and easy to use DNA extraction protocol
to enable close to real-time and on-site identification
of microbes and compare it to current state-of-the-art
methods.
Specific objectives:
• Design and evaluate a cheap and mobile bead
beating tool
• Develop and evaluate a fast DNA extraction protocol
• Compare all results to the golden standards within
the field of DNA extraction
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Aims

14. The golden standard vs. a new mobile method
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Step 1: Cell lysis
Step 2: Removal of cell debris
Step 3: DNA isolation and elution
3 principle steps of DNA extraction:

15. Step 1: Cell lysis
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Standard bead beater
Price: 83,000 kr
Weight: 20 kg
Replacement part:
Not 3D printable
Mobile bead beater
Price: 500 kr
Weight: 2kg
Replacement part:
3D printable

16. 3D printable replacement parts
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
www.thingiverse.com/Peter161
MBB adaptor

17. Step 2: Removal of cell debris
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Centrifugation at 2000 xG for 1 min
Time: 20 mins Time: 1 min

18. Step 3: DNA isolation and elution
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Time: 25 mins Time: 8 mins

19. Time comparison
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Standard method Steps Mobile Method
10 min Cell lysis 1 min
20 min Removal of cell debris 1 min
25 min DNA isolation and
elution
8 min
60 min Total time 11:30 min

20. GoPro video of the method
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

21. Strategy for evaluating the DNA extraction
method
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Heatmap of top genera in the
microbial communities
Standard
activated sludge
Standard
method
DNA quality
control
16S rRNA gene amplification
of region V1-3
Mobile
method
DNA Yield
DNA fragment
length
DNA purity
Principal components analysis
of the microbial communities

22. Optimization of the mobile method
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Bead beating time
Evaluate the 3D-printed
bead beater and get faster
extraction time
Settings tested
• 4x 40sec (160sec total)
• 60 sec
AMPure XP beads used
To test lowest amount of
beads can be used
Settings tested
• 170uL
• 80uL
From the original solution
diluted into cheap buffer
Centrifugation
speed and time
To test for faster DNA
extraction time and smaller
equipment
Settings tested
• 10min at 14000xG
• 1min at 14000xG
• 1min at 2000xG

23. Method settings tested
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Name Bead beating
time
Centrifuge time SPRI
volume
Replicas
Mobile 1 4x 40 sec. 10min 14000xG 170 µL 3
Mobile 2 4x 40 sec. 1min 14000xG 170 µL 3
Mobile 3 4x 40 sec. 1min 2000xG 170 µL 3
Mobile 4 60 sec. 1min 2000xG 170 µL 3
Mobile 5 60 sec. 1min 2000xG 80 µL 3

24. The effect of bead beating on DNA yield,
purity and quality
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
DNA yield
DNA quality DNA purity
Method Beating time
Mobile3 60 sec
Mobile4 4x 40 sec
Standard 4x 40 sec
Method overview
Method A260/280 A260/230
Contamination
type
Protein Organic
compounds
Pure DNA >1,8 >2.0
Standard 1.88 0.34
4x 40 sec 1.71 1.12
60 sec 1.88 1.49
L 4x 40 60 Standard
11.46µg
7.62µg
7.168µg
0 2 4 6 8 10 12 14
60 sec
4x40 sec
Standard

25. The effect of bead beating on the
microbial community of top 15 genera
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
4x 40sec 60 sec Standard

26. The effect of centrifugation on DNA yield,
purity and quality
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
DNA quality DNA purity
Method A260/280 A260/230
Contamination
type
Protein Organic
compounds
Pure DNA >1,8 >2.0
Mobile 1 1.93 1.88
Mobile 2 1.91 1.86
Mobile 3 1.71 1.12
Standard 1.88 0.34
L 1 2 3 Standard
Method Centrifuge speed
Mobile 1 10min 14000xG
Mobile 2 1min 14000xG
Mobile 3 1min 2000xG
Standard 10min 14000xG
Method overview
11.46µg
7.62µg
7.17µg
8.88µg
0 2 4 6 8 10 12 14
Mobile 1
Mobile 2
Mobile 3
Standard
DNA yield

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10minX14000xg 1minX14000xg 1minX2000xg Standard
The effect of centrifugation on the
microbial community of top 15 genera

28. The effect of SPRI beads on DNA yield,
purity and quality
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
DNA yield
DNA quality DNA purity
Method A260/280 A260/230
Contamination
type
Protein Organic
compounds
Pure DNA >1,8 >2.0
170 uL 1.84 1.49
80 uL 1.79 1.72
Standard 1.88 0.34
11.46 µg
7.168 µg
2.568 µg
0 2 4 6 8 10 12 14
80 uL
170 uL
Standard
L 170uL 70uL Standard
Method SPRI beads used
Mobile 4 170uL
Mobile 5 80uL
Standard none
Method overview

29. The effect of SPRI beads on the microbial
community of top 15 genera
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
170µL 70µL Standard

30. PCA - Comparison of the methods
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

31. Mobility:
Easy to use:
No dangerous chemicals:
Conclusion
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Bead beating time
60 sec
AMPure XP beads used
80 µL
Centrifugation
1 min 2000xG
Method with highest purity, fragment length, fastest speed: Mobile 5
Price: 30 kr. per sample - 48% reduction compared to gold standard
Time: 11:30 min - 80% reduction compared to gold standard

32. Mobile lab assembled and ready for field test
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

33. Automation of the mobile lab
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
VolTRAX = automated sample preparation system
Almost fully automated DNA sequencing

34. Overview of on-site identification of microbes
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Microbialcommunity
Alert
Alert
Time

35. Thank you for your attention
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Albertsen Lab
www.albertsenlab.org
Per H. Nielsen
Mads AlbertsenRasmus H. Kirkegaard
+ The EB group