As you sow so shall you reap sample to sequencer process · Presentation Title or Date via...
Transcript of As you sow so shall you reap sample to sequencer process · Presentation Title or Date via...
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As you sow so shall you reap
sample to sequencer process
Saria Otani, MRes., MSc., PhD
EURL-AR training course
Denmark, 2019
![Page 2: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/2.jpg)
DTU Food, Technical University of Denmark
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As you sow so shall you reap
The dress colour “enigma”
e.g., the light exposure, the observer, the device.
![Page 3: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/3.jpg)
DTU Food, Technical University of Denmark
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As you sow so shall you reap
(Thompson., et al., 2015)
e.g., the light exposure, the observer, the device (ID’ing).
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DTU Food, Technical University of Denmark
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What do we want to achieve?
Bacterial profiling, isolates or metagenomes.
Functional analyses: gene identifications, AMR,
virulence, diet-decomposition, immunity-related, evolutionary traits.
From sample to sequencer
![Page 5: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/5.jpg)
DTU Food, Technical University of Denmark
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A project workflow, WGS or metagenome
Plan ahead: what exactly are you looking for? design the experiment
accordingly!
Sample.
DNA extraction.
Library preparation.
Sequencing platforms.
Analyses.
![Page 6: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/6.jpg)
DTU Food, Technical University of Denmark
Add or change Presentation Title or Date via ”Insert”; ”Header & Footer”
A project workflow, WGS or metagenome
Plan ahead: what exactly are you looking for? design the experiment
accordingly!
Sample.
DNA extraction.
Library preparation.
Sequencing.
Analyses. 1
2
3
4
![Page 7: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/7.jpg)
DTU Food, Technical University of Denmark
Add or change Presentation Title or Date via ”Insert”; ”Header & Footer”
A project workflow, WGS or metagenome
Plan ahead: what exactly are you looking for? design the experiment
accordingly!
Sample.
DNA extraction.
Library preparation.
Sequencing platforms.
Analyses.
![Page 8: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/8.jpg)
DTU Food, Technical University of Denmark
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Sequencing technology selection
Many classifications for the different methods:
![Page 9: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/9.jpg)
DTU Food, Technical University of Denmark
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Sequencing technology selection
![Page 10: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/10.jpg)
DTU Food, Technical University of Denmark
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This is commonly known
as NGS
Sequencing technology selection
![Page 11: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/11.jpg)
DTU Food, Technical University of Denmark
Add or change Presentation Title or Date via ”Insert”; ”Header & Footer” (Ronholm., et al., 2016)
This is commonly known
as NGS
Sequencing technology selection
![Page 12: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/12.jpg)
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Sequencing technology selection
A bit confusing!
What do I want it for? Gene detection (AMR)? Plasmids?
Evolutionary analyses? Microbiome?
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Sequencing technology selection
• Short read technologies
– Illumina (MiSeq, NextSeq, HiSeq, NovaSeq…)
– Ion Torrent
– 454
• Long read technologies
– Pacific Biosciences (PacBio)
– Oxford Nanopore Technologies (MinION)
![Page 14: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/14.jpg)
DTU Food, Technical University of Denmark
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Sequencing technology selection
• Short read technologies
– Illumina (MiSeq, NextSeq, HiSeq, NovaSeq…):
Average 300 bp reads
Good accuracy
Error rate ~0.1%
– Ion Torrent: Was the fastest runtime and work-flow in this category (until 2018)
Average 400 bp reads
Error rate ~1%
– 454: Out of market
![Page 15: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/15.jpg)
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Sequencing technology selection
• Long read technologies
– Pacific Biosciences (PacBio):
Long reads. (Max: 50 kb. Avg.: 10-15 kb)
Low throughput
– Oxford Nanopore Technologies (MinION):
Very long reads (up to 900 kb.)
Fast turnaround time (down to 1.5 hrs)
Portable and real-time sequencing
Large error rates (3-8%)
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16
Oxford Nanopore Technologies (MinION):
Long and ultra-long reads – Best high throughput available (100 Gbp) –
Portable, fast and real-time sequencing.
![Page 17: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/17.jpg)
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Illumina:
Nanopore:
DNA concentration ≃ 1 ng
DNA concentration > 1000 ng
Comparison example, differences in DNA input between 2
sequencing technologies:
![Page 18: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/18.jpg)
DTU Food, Technical University of Denmark
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A project workflow, WGS or metagenome
Plan ahead: what exactly are you looking for? design the experiment
accordingly!
Sample.
DNA extraction.
Library preparation.
Sequencing platforms.
Analyses.
![Page 19: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/19.jpg)
DTU Food, Technical University of Denmark
Add or change Presentation Title or Date via ”Insert”; ”Header & Footer”
A project workflow, WGS or metagenome
Plan ahead: what exactly are you looking for? design the experiment
accordingly!
Sample.
DNA extraction.
Library preparation.
Sequencing platforms.
Analyses.
![Page 20: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/20.jpg)
DTU Food, Technical University of Denmark
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DNA library preparation:
Collection of steps to
prepare DNA to be
read by a sequencing
machine.
![Page 21: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/21.jpg)
DTU Food, Technical University of Denmark
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DNA library preparation:
Collection of steps to
prepare DNA to be
read by a sequencing
machine.
This example is for
Illumina machines.
![Page 22: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/22.jpg)
DTU Food, Technical University of Denmark
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DNA library preparation:
Collection of steps to
prepare DNA to be
read by a sequencing
machine.
This example is for
Oxford Nanopore machines.
Same steps, yet different
ingredients and handling!
Clean up – magnetic beads
Clean up – magnetic beads
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DTU Food, Technical University of Denmark
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DNA library preparation Illumina vs MinIon:
Same steps, yet different ingredients and handling!
Illumina MinIon
Clean up –
magnetic beads
handling the
DNA
![Page 24: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/24.jpg)
DTU Food, Technical University of Denmark
Add or change Presentation Title or Date via ”Insert”; ”Header & Footer”
A project workflow, WGS or metagenome
Plan ahead: what exactly are you looking for? design the experiment
accordingly!
Sample.
DNA extraction.
Library preparation.
Sequencing platforms.
Analyses.
![Page 25: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/25.jpg)
DTU Food, Technical University of Denmark
Add or change Presentation Title or Date via ”Insert”; ”Header & Footer”
A project workflow, WGS or metagenome
Plan ahead: what exactly are you looking for? design the experiment
accordingly!
Sample.
DNA extraction.
Library preparation.
Sequencing platforms.
Analyses.
![Page 26: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/26.jpg)
DTU Food, Technical University of Denmark
Add or change Presentation Title or Date via ”Insert”; ”Header & Footer”
A project workflow, WGS or metagenome
Plan ahead: what exactly are you looking for? design the experiment
accordingly!
Sample.
DNA extraction.
Library preparation.
Sequencing platforms.
Analyses.
![Page 27: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/27.jpg)
DTU Food, Technical University of Denmark
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DNA extraction
Must know:
The starting material.
The downstream application.
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DNA extraction
Starting material
Modified from Printerest.com
![Page 29: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/29.jpg)
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DNA extraction
Starting material
Cell lysis
Modified from Printerest.com
Left after the lysis:
DNA, RNA, protein
![Page 30: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/30.jpg)
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DNA extraction
Starting material
Cell lysis
Protein precipitation
Clean up
Modified from Printerest.com
![Page 31: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/31.jpg)
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DNA extraction, what could be different for long reads!
Starting material
Cell lysis
Protein precipitation
Clean up
Modified from Printerest.com
Different for long reads
Different for long reads
![Page 32: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/32.jpg)
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DNA extraction, examples:
1- Lysis.
2- Precipitation.
3- Clean up.
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DNA extraction
Quality/integrity of DNA
•Qubit
•Nanodrop
•Bioanalyzer
![Page 36: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/36.jpg)
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DNA extraction
A B C D E Ladder
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DNA extraction
Quality/integrity of DNA
•Qubit
•Nanodrop
•Bioanalyzer
Why is it important to check
DNA quality?
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As you sow so shall you reap
The dress colour “enigma”
e.g., the light exposure, the observer, the device.
![Page 39: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/39.jpg)
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Illumina seq. example:
Sensitivity of AMR detection in microbiomes.
As you sow so shall you reap, act 1
Nanopore seq. example:
Plasmidome characterisation.
As you sow so shall you reap, act 2
![Page 40: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/40.jpg)
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Illumina seq. example:
Sensitivity of AMR detection in microbiomes.
As you sow so shall you reap, act 1
Poultry faeces Pig faeces
1:1000
1:100
1:10
1:1
1:0
0:1
10:1
100:1
1000:1
volume volume
![Page 41: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/41.jpg)
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As you sow so shall you reap, act 1
Stepwise pattern of AMR profiles
follows the gradual mix of the two
microbiomes.
![Page 42: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/42.jpg)
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Nanopore seq. example:
Plasmidome characterisation.
As you sow so shall you reap, act 2
![Page 43: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/43.jpg)
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DNA QC is a key for Nanopore sequencing.
Pure - high concentration - long fragments DNA.
~ 10 Kbp ~ 20 Kbp
Nanopore seq. example: Plasmidome characterisation.
As you sow so shall you reap, act 2
Magnetic beads
(e.g., ZymoBIOMICS)
Modified for increased yield
Gravity flow colums
(e.g., Qiagen Genomic tips)
![Page 44: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/44.jpg)
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Sewage Plasmid DNA
> 10 plasmid extraction
protocols were tested
To select and enrich for circular plasmids:
• Linear DNA was digested
• Rolling circle enrichment
Nanopore seq. example:
Plasmidome characterisation. Pre-sequencing treatment:
As you sow so shall you reap, act 2
![Page 45: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/45.jpg)
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Plasmidome characterisation
Comparing plasmidome
throughputs
![Page 46: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/46.jpg)
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Comparing plasmidome
throughputs
Plasmidome characterisation
> 4000 contigs
> 2000 potenial circular plasmids
![Page 47: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/47.jpg)
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As you sow so shall you reap, act 2
Nanopore from complex samples
Very dependent on DNA and library preparation.
Easy from single isolates
![Page 48: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/48.jpg)
DTU Food, Technical University of Denmark
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A project workflow, WGS or metagenome
Plan ahead: what exactly are you looking for? design the experiment
accordingly!
Sample.
DNA extraction.
Library preparation.
Sequencing platforms.
Analyses. 1
2
3
4
To remember
![Page 49: As you sow so shall you reap sample to sequencer process · Presentation Title or Date via ”Insert”; ”Header & Footer” To remember Choose your sequencing technology that serves](https://reader034.fdocuments.us/reader034/viewer/2022042319/5f08277e7e708231d4209bab/html5/thumbnails/49.jpg)
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To remember
Choose your sequencing technology that serves your analysis best (e.g.,
gene detection, identification).
No commercially available kit is optimal for all DNA extraction or
library prep.
A method that works for all, yes.
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DTU Food, Technical University of Denmark
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We have come a long way
Next-Generation sequencing as a diagnostic tool
(Microbiology)
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DTU Food, Technical University of Denmark
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Thank you
Twitter: @SariaOtani
LinkedIn: Saria Otani
Email: [email protected]