from isolate to whole genome sequencing · genomic dna extraction . any dna extraction (e.g.,...

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

DTU Food, Technical University of Denmark

3

Who is speaking:

A molecular microbiologist with experience in host-microbiome interaction.

Microbial community analyst.

New technology enthusiast, not a promotor!

Lab scientist.

DTU Food, Technical University of Denmark

4

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

DTU Food, Technical University of Denmark

19 September 2018 5

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

DTU Food, Technical University of Denmark

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

DTU Food, Technical University of Denmark

19 September 2018 7

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

(Hinchliff., et al., 2015)

DTU Food, Technical University of Denmark

8

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

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)

DTU Food, Technical University of Denmark

various antibiotics

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

Traditional workflow Tomorrow

DTU Food, Technical University of Denmark

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

A sample

NGS workflow

DNA extraction Library preparation

Sequence

Analyses & interpretation:

Resistance gene profile

DTU Food, Technical University of Denmark

19 September 2018 11

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

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)

DTU Food, Technical University of Denmark

19 September 2018

(Simner., et al., 2017)

Comparison between NGS and traditional methods as a diagnostic tool

DTU Food, Technical University of Denmark

19 September 2018

(Simner., et al., 2017)

Comparison between NGS and traditional methods as a diagnostic tool

DTU Food, Technical University of Denmark

14

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

DTU Food, Technical University of Denmark

19 September 2018 15

Day 1

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

Sample selection.

DNA extraction.

Sequencing technology selection.

Library preparation.

An example: Nextera XT First-timer mishaps

Illumina NextSeq

DTU Food, Technical University of Denmark

19 September 2018 16

Critically choose sample suitable for sequencing

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

Whole-Genome sequencing (pure culture)

DTU Food, Technical University of Denmark

19 September 2018 17

Critically choose sample suitable for sequencing

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

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

Streak plate method.

DTU Food, Technical University of Denmark

19 September 2018 18

Critically choose sample suitable for sequencing

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

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

Streak plate method.

Suspension dilutions.

DTU Food, Technical University of Denmark

19 September 2018 19

Critically choose sample suitable for sequencing

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

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

Streak plate method.

Suspension dilutions.

FACS (fluorescence-activated cell sorting).

DTU Food, Technical University of Denmark

19 September 2018 20

Critically choose bacterial cultures suitable for sequencing

1

3

2

4

DTU Food, Technical University of Denmark

19 September 2018 21

Critically choose bacterial cultures suitable for sequencing

1

3

2

4

DTU Food, Technical University of Denmark

19 September 2018 22

Day 1

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

Sample selection.

DNA extraction.

Sequencing technology selection.

Library preparation.

An example: Nextera XT First-timer mishaps

Illumina NextSeq

DTU Food, Technical University of Denmark

19 September 2018 23

Day 1

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

Sample selection.

DNA extraction.

Sequencing technology selection.

Library preparation.

An example: Nextera XT First-timer mishaps

Illumina NextSeq

DTU Food, Technical University of Denmark

19 September 2018 24

Genomic DNA extraction

The science behind DNA extraction.

An example.

Trouble shooting.

DTU Food, Technical University of Denmark

19 September 2018 25

Genomic DNA extraction

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.

DTU Food, Technical University of Denmark

19 September 2018 26

Genomic DNA extraction

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.

DTU Food, Technical University of Denmark

19 September 2018 27

Genomic DNA extraction

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.

DTU Food, Technical University of Denmark

19 September 2018 28

Genomic DNA extraction

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

1- Lysis.

2- Precipitation.

3- Clean up.

DTU Food, Technical University of Denmark

Genomic DNA extraction

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

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

DTU Food, Technical University of Denmark

Genomic DNA extraction

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

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)

DTU Food, Technical University of Denmark

19 September 2018 31

Genomic DNA extraction

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

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.

DTU Food, Technical University of Denmark

19 September 2018 32

Genomic DNA extraction

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

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

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

collect pellet by centrifugation.

DTU Food, Technical University of Denmark

DTU Food, Technical University of Denmark

DTU Food, Technical University of Denmark

19 September 2018 35

Genomic DNA extraction

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

3- Clean up.

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

Clean DNA is resuspended in a TRIS buffer.

DTU Food, Technical University of Denmark

19 September 2018 36

Genomic DNA extraction

Quality/integrity of DNA

•Qubit

•Nanodrop

•Bioanalyzer

DTU Food, Technical University of Denmark

19 September 2018 37

Genomic DNA extraction

A B C D E Ladder

DTU Food, Technical University of Denmark

19 September 2018 38

Genomic DNA extraction

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.

Plant: grinding

G+: lysozyme

Faeces: beads Organic Salt

Cold temp. treatment

Isopropanol

Methanol

DTU Food, Technical University of Denmark

19 September 2018 39

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

DTU Food, Technical University of Denmark

19 September 2018 40

Genomic DNA extraction, examples:

1- Lysis.

2- Precipitation.

3- Clean up.

DTU Food, Technical University of Denmark

19 September 2018 41

Day 1

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

Sample selection.

DNA extraction.

Sequencing technology selection.

Library preparation.

An example: Nextera XT First-timer mishaps

Illumina NextSeq

DTU Food, Technical University of Denmark

19 September 2018 42

Day 1

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

Sample selection.

DNA extraction.

Sequencing technology selection.

Library preparation.

An example: Nextera XT First-timer mishaps

Illumina NextSeq

DTU Food, Technical University of Denmark

19 September 2018 43

Sequencing technology selection

Many classifications for the different methods:

DTU Food, Technical University of Denmark

19 September 2018 44

(Ronholm., et al., 2016)

Sequencing technology selection

DTU Food, Technical University of Denmark

19 September 2018 45

(Ronholm., et al., 2016)

This is commonly known as NGS

Sequencing technology selection

DTU Food, Technical University of Denmark

19 September 2018 46

(Ronholm., et al., 2016)

This is commonly known as NGS

Sequencing technology selection

DTU Food, Technical University of Denmark

19 September 2018 47

Sequencing technology selection

A bit confusing!

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

DTU Food, Technical University of Denmark

19 September 2018 48

Sequencing technology selection

• Short read technologies

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

– Ion Torrent

– 454

• Long read technologies

– Pacific Biosciences (PacBio)

– Oxford Nanopore Technologies (MinION)

DTU Food, Technical University of Denmark

19 September 2018 49

Sequencing technology selection

• Short read technologies

– 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

DTU Food, Technical University of Denmark

19 September 2018 50

Sequencing technology selection

• Long read technologies

– 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%)

DTU Food, Technical University of Denmark

19 September 2018 51

Oxford Nanopore Technologies (MinION):

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

DTU Food, Technical University of Denmark

19 September 2018 52

Oxford Nanopore Technologies (MinION):

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

DTU Food, Technical University of Denmark

19 September 2018 53

Sequencing technology selection

• Short read technologies

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

– Ion Torrent

• Long read technologies

– Pacific Biosciences (PacBio)

– Oxford Nanopore Technologies (MinION)

DTU Food, Technical University of Denmark

19 September 2018 54

Genomic DNA extraction

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

1- Lysis.

2- Precipitation.

3- Clean up.

DTU Food, Technical University of Denmark

19 September 2018 55

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.

DTU Food, Technical University of Denmark

19 September 2018 56

Let’s give it a try

Case 2:

Diabetes patient microbiomes.

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

Lysis steps?

Which NGS: Bacterial community. long reads not needed.

DTU Food, Technical University of Denmark

19 September 2018 57

Let’s give it a try

Case 3:

Plant contamination in herbal products.

Plant – Plant taxon identification – turnaround 72 hrs.

Lysis steps? Tissue nature?

Which NGS: Plant community. Big genomes!

DTU Food, Technical University of Denmark

19 September 2018 58

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.

DTU Food, Technical University of Denmark

19 September 2018 59

To remember

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

This is commonly known as NGS

DTU Food, Technical University of Denmark

19 September 2018 60

Thank you