From Isolate to Whole Genome Sequencing · Genomic DNA extraction . Any DNA extraction (e.g., commercial kit, in-house protocol, paper methods) is 3 steps: 1- Lysis. 2- Precipitation

From Isolate to Whole Genome Sequencing

Saria Otani, MRes., MSc., PhD

EURL-AR training course

Denmark, 2018

DTU Food, Technical University of Denmark

2

Day 1

Do I need to change! A transition from conventional to NGS methods.

Sample selection.

DNA extraction.

Sequencing technology selection.

Library preparation.

An example: Nextera XT First-timer mishaps

Illumina NextSeq


3

Who is speaking:

A molecular microbiologist with experience in host-microbiome interaction.

Microbial community analyst.

New technology enthusiast, not a promotor!

Lab scientist.


4

Day 1


Sample selection.

DNA extraction.




Illumina NextSeq


19 September 2018 5

Day 1


Sample selection.

DNA extraction.




Illumina NextSeq


6

Next-Generation sequencing as a diagnostic tool, why bother!

“One day this training course will consist of hands-on and theoretical teaching focusing at NGS only.”

Hasman, EURL workshop, Denmark 2014


19 September 2018 7


(Hinchliff., et al., 2015)


8


Until 2018, no bacterial isolate has been detected.

NGS was used in 2017:

> 300 bacterial genera have been detected. A good deal is pathogenic

(Raghupathi., et al., 2018)


various antibiotics

Next-Generation sequencing as a diagnostic tool for surveillance of antimicrobial resistance

Traditional workflow Tomorrow



A sample

NGS workflow

DNA extraction Library preparation

Sequence

Analyses & interpretation:

Resistance gene profile


19 September 2018 11


Turnaround time for NGS between 6 hours to 7 days (average of 48 hours) from specimen receipt

depending on the sequencing technology, methods, and bioinformatics programs exploited.

(Simner., et al., 2017)


19 September 2018


Comparison between NGS and traditional methods as a diagnostic tool


19 September 2018


Comparison between NGS and traditional methods as a diagnostic tool


14

Day 1


Sample selection.

DNA extraction.




Illumina NextSeq



Day 1

Do I need to change! An easy transition from conventional to NGS methods.

Sample selection.

DNA extraction.




Illumina NextSeq



Critically choose sample suitable for sequencing

Metagenomics (e.g., soil, meat, faeces)

Whole-Genome sequencing (pure culture)





Whole-Genome sequencing (pure culture) Critically choose bacterial cultures suitable for sequencing

Streak plate method.







Suspension dilutions.







Suspension dilutions.

FACS (fluorescence-activated cell sorting).



Critically choose bacterial cultures suitable for sequencing

1

3

2

4



Critically choose bacterial cultures suitable for sequencing

1

3

2

4



Day 1


Sample selection.

DNA extraction.




Illumina NextSeq



Day 1


Sample selection.

DNA extraction.




Illumina NextSeq



Genomic DNA extraction

The science behind DNA extraction.

An example.

Trouble shooting.




Must know:

The starting material.

The downstream application.

Please keep in mind:

No commercially available kit is optimal for all.

A method that works for all, yes.




Any DNA extraction (e.g., commercial kit, in-house protocol, paper methods) is 3 steps:

1- Lysis.

2- Precipitation.

3- Clean up.

Differences in how these 3 steps are carried out.

Because

We have different:

Starting material.

Downstream application.





1- Lysis.

2- Precipitation.

3- Clean up.


Because

We have different:

Starting material.






1- Lysis.

2- Precipitation.

3- Clean up.




1- Lysis. Tissues:

Homogenization: mechanical or chemical.

Cells: 60-70 C degree temperature treatment

Alkaline/Detergents: (e.g., SDS, triton X-100, CTAB) breaks down cell membrane

Proteinases: e.g., Proteinase K




1- Lysis. Tissues:

Homogenization: mechanical or chemical.

Cells:

Alkaline/Detergents: (e.g., SDS, triton X-100, CTAB) breaks down cell membrane

Proteinases: e.g., Proteinase K

Left after the lysis:

DNA, protein (denatured and not)





2- Precipitation: separates DNA from the remaining cellular components.

Organic: (e.g., Phenol-chloroform, Trizol) denatures and dissolves proteins

Salt: (e.g., NaCl, ammonium acetate) releases proteins

Both allow the protein to precipitate with centrifugation.





2- Precipitation: separates DNA from the remaining cellular components.

DNA is left. Precipitated with, alcohol (isopropanol or ethanol),

collect pellet by centrifugation.







3- Clean up.

Alcohol: wash with 70% ethanol to remove salts and other impurities.

Clean DNA is resuspended in a TRIS buffer.




Quality/integrity of DNA

•Qubit

•Nanodrop

•Bioanalyzer




A B C D E Ladder





1- Lysis.

2- Precipitation.

3- Clean up.


Because

We have different:

Starting material.


Plant: grinding

G+: lysozyme

Faeces: beads Organic Salt

Cold temp. treatment

Isopropanol

Methanol



Genomic DNA extraction, examples:

1- Lysis.

2- Precipitation.

3- Clean up.

ATL buffer

Proteinase K

AL buffer

Detergent

Percipitate protein

Collect the DNA on the column



Genomic DNA extraction, examples:

1- Lysis.

2- Precipitation.

3- Clean up.



Day 1


Sample selection.

DNA extraction.




Illumina NextSeq



Day 1


Sample selection.

DNA extraction.




Illumina NextSeq



Sequencing technology selection

Many classifications for the different methods:



(Ronholm., et al., 2016)





This is commonly known as NGS










A bit confusing!

What do I want it for? Gene detection (AMR)? Plasmids? Evolutionary analyses? Microbiome?




• Short read technologies

– Illumina (MiSeq, NextSeq, HiSeq, NovaSeq…)

– Ion Torrent

– 454

• Long read technologies

– Pacific Biosciences (PacBio)

– Oxford Nanopore Technologies (MinION)





– Illumina (MiSeq, NextSeq, HiSeq, NovaSeq…):

Average 300 bp reads

Good accuracy

Error rate ~0.1%

– Ion Torrent: Fastest runtime and work-flow in this category

Average 400 bp reads

Error rate ~1%

– 454: Out of market





– Pacific Biosciences (PacBio):

Long reads. (Max: 50 kb. Avg.: 10-15 kb)

Error rate ~15% (single pass)

Low throughput

– Oxford Nanopore Technologies (MinION):

Very long reads (up to 900 kb.)

Fast turnaround time (2 hrs)

Portable and real-time sequencing

Large error rates (3-8%)



Oxford Nanopore Technologies (MinION):

Long and ultra-long reads – Best high throughput available (100 Gbp) – Portable, fast and real-time sequencing.



Oxford Nanopore Technologies (MinION):

Long and ultra-long reads – Best high throughput available (100 Gbp) – Portable, fast and real-time sequencing.





– Illumina (MiSeq, NextSeq, HiSeq, NovaSeq…)

– Ion Torrent


– Pacific Biosciences (PacBio)

– Oxford Nanopore Technologies (MinION)





1- Lysis.

2- Precipitation.

3- Clean up.



Let’s give it a try

Case 1:

Salmonella contamination from a poultry.

Chicken sample – Salmonella identification - turnaround time 48 hrs.

Lysis step? Chemical or mechanical? Do you need enzymes?

Which NGS? ID’ing of Salmonella: long reads not needed, but quick results are.




Case 2:

Diabetes patient microbiomes.

Faeces – Microbiome profile (bacterial taxa) – turnaround time a month.

Lysis steps?

Which NGS: Bacterial community. long reads not needed.




Case 3:

Plant contamination in herbal products.

Plant – Plant taxon identification – turnaround 72 hrs.

Lysis steps? Tissue nature?

Which NGS: Plant community. Big genomes!



To remember

Know your starting material and downstream application.

No commercially available kit is optimal for all.

A method that works for all, yes.

Any DNA extraction is 3 steps:

1- Lysis: Tissues and cells.

2- Precipitation: Protein and DNA.

3- Clean up: The DNA.



To remember

Choose your sequencing technology that serves your analysis best (e.g., gene detection, identification).




Thank you

Documents

From Isolate to Whole Genome Sequencing · Genomic DNA extraction . Any DNA extraction (e.g., commercial kit, in-house protocol, paper methods) is 3 steps: 1- Lysis. 2- Precipitation