From Isolate to Whole Genome Sequencing · Genomic DNA extraction . Any DNA extraction (e.g.,...
Transcript of 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