Gel-gebaseerde typeringsmethoden

SMT 2013
Gel-gebaseerde typeringsmethoden
Genus Species Subspec. Strain
DNA-DNA reassociation
Speed Ease Platf. Typ. Flex. €
>>24h
16S rDNA sequencing
24h
RAPD
8h
PFGE
-
n.a.
n.a./I
H
+
S
D/I
H
+++
A
C/T
H
48h
++
A
D/IT
H
Diversilab (rep-PCR)
24h
+++
A
D/T
SS
AFLP
24h
+++
A/S
DC/IT
H
MLST
48h
++
S
D/IT
SS
MLVA
24h
+++
A/S
D/T
SS
Raman Spectrometry
48h
+++
n.a.
D/IT
SS
Optical mapping
48h
+
n.a.
D/IT
H
SNP analyse
24h
+
S
DT
SS
Pulse Field Gel Electrophoresis (PFGE)


 

Proteinase K
Restriction enzymes
Agarose embedded bacteria with enzymes
PFGE profile
Fragment separation
Embedded
digested
bacteria placed in
agarose gel wells
Electric Pulse Field separation
cursus Breda 2008
3
RAPD
Random amplified polymorphic DNA
CARRIED OUT WITH
ONLY ONE PRIMER
Amplification at low
annealings
temperature
with one primer
RAPD profile
Agarose gel separation
‘Quick & dirty’
cursus Breda 2008
4
rep-PCR
Geconserveerde repetitieve
elementen
BOX, ERIC, REP
verspreid aanwezig bij
de meeste bacteriën
Stringente vermenigvuldiging van
specifieke chromosomale regios
rep-PCR
profile
(agarose) Gelelectrophorese
Geautomatiseerd systeem: Diversilab
cursus Breda 2008
5
Amplified Fragment Length Polymorphism (AFLP)
CHROMOSOMAAL DNA
SIMULTANE knip en plak
(restrictie & ligatie)
AATTC
G
G
CTTAA
AATTC
G
T
AAT
CTCGTAGACTGCGTACCAATTC
CATCTGACGCATGGTTAAG
T
AAT
TAA
T
TTACTCAGGACTCAT
AATGAGTCCTGAGTAGCAG
STRINGENTE
AMPLIFICATIE
TTACTCAGGACTCAT
AATGAGTCCTGAGTAG
CTCGTAGACTGCGTACCAATTC
GACTGCGTACCAATTC
CATCTGACGCATGGTTAAG
AATGAGTCCTGAGTAGCAG
PAGE
cursus Breda 2008
6
Amplified Fragment Length Polymorphism (AFLP)
Combination of RFLP & rep-PCR:
restriction analysis combined with specific amplification
Presently there are several variations on the method
Two main variations:
High frequent-medium frequent DNA digestion on PAGE
Medium frequent DNA digestion on agarose
Selection of fragments analyzed on PAGE
All fragments analyzed on Agarose
Vos, et.al. NAR 1995
40 50 60 70 80 90
100
500bp
400bp
300bp
200bp
100bp 50bp
S. capitis
S. capitis
S. capitis
S. capitis
S. capitis
S. capitis
S. capitis
S. capitis
S. cap(ref
S. cap(ref
S. aureus
S.aureus
S. aureus
S. warneri
S. warneri
S. epiderm
S. epidermidis
S. epiderm
S. epiderm
S. haemolyticus
Window of identific.
S. haemo
typing
Agarose AFLP
Chromosomal DNA
Digestion & ligation
One or more enzymes can be used
Each enzyme with specific adaptor
Fragments can be isolated from gel
No expensive platform needed
All fragments are amplified
Specific PCR amplification
Fragment separation
Analyzing Band based typing methods
Some pitfalls:
- Number of bands too high!
-Important that quality of gels are high leading to reproducible results
- Band analysis requires specific pretreatments:
- Definition of lanes
- Calculation of curves
- Normalization of tracks
- Search for bands
In addition:
Background removal
Noise filtering
Band tolerance
Guest Lecture: prof. dr. Bruno Pot
Interpretation of gels:
Differences in band intensities
esp. random amplification techniques)
Overlapping bands
Better discrimination with polyacrylamide gels
(e.g. capillary sequencers)
Need for standardized protocols with quality controls
Some techniques suited for local database comparison
General steps
Data
Pictures, photographs  2D TIFF images
Sequence data  densitometric curves
One dimensional curves (mass spectroscopy)
Densiometric curve vs fingerprint
Example: TIFF File
General steps
Data processing
 Give the computer the basic information  define lanes
 Not all lanes migrate the same  normalize tracks
(against MW)
 Define what you are looking for  search bands OR
 Let the computer calculate curves
Example: Define lanes
Example: normalize tracks
Similarity coefficient
Definition: Creation of the “best guess” relation
between data according to a computer algorithm
Band based:
(binary data)
Dice
Jaccard
Jeffrey’s
Ochiai
Pattern/curve based:
Pearson correlation
Cosine coefficient
Example: calculate curves I
Example: calculate curves I
Pattern vs band matching
Band matching is a binary typing method
Subjective by nature
Suited for less complex fingerprints
Data format very suitable for database construction
Pattern/curve matching is a densitometric typing method
Objective by nature
Suited for complex fingerprints
Under analysis restrictions suited for database construction
Band matching I
Band matching II
Band matching III
Example: search bands
(your experiment)
Comparison techniques (grouping)
Clustering: Pairwise
UPGMA
WARD
Neighbor Joining
Nearest neighbor
Furthest neighbor
K-means partitioning
Global optimization
maximum parsimony
maximum likelyhood
Guest Lecture by Bruno Pot
Cluster validation
Bootstrap analysis
Repeated analysis, 100-1000x, gives indication of robustness
of the tree
Indicates the significance (stability) of the different clusters.
Applicable to: character sets, bandmatching & sequences
Jackknife & Group isolation techniques
Shows the stability of the group
Indicates the separation between any set of groups defined
by the user
Error flags
Indicates the uncertainty (based on the standard deviation)
of the branch linkage positions
ONLY UPGMA
Software packages
Bionumerics (Applied Maths)
Dendron (Solltech)
Taxotron (Inst. Pasteur)
Quantar Suite (Keygene Int.)
Phoretix 1D (Phoretix Int.)
RFLP analysis (T-rex)
Clustal
RAPDistance
NJTREE
PAUP
PHYLIP
Freeware
Final remarks
You may choose a very good software package
You may analyse your data in the correct way
You may include all possible controls
Your result may look perfect
It’s always a matter of choices and results may differ
Between different typing methods
“In silico prediction
of band based restriction
methods”
e.g. AFLP
“in silico” points at a theoretical prediction
of the results of a band based typing method
The procedure is technically carried out by the software
 More genomes become available
 Reference strains for technique
 Controls for performance
 Predictive value
 Omitting laborious testing
Also applicable to other restriction based techniques
like PFGE
Step 1
Enter the internet and go to the NCBI site
www.ncbi.nlm.nih.gov/
Look for: complete genomes
Get the accession numbers and download
the sequence to your computer
Standard DNA programs or specialised analysis software
(e.g. Bionumerics, Applied Maths)
Resulting database: e.g. adenovirus
Select one sequence (NC_003266)
Request restriction enzyme analysis
(all annotations are kept)
Perform a restriction analysis of your choice
(EcoRI: 2 cuts; PstI: 17 cuts; MseI: 57 cuts)
Fragment list displayed on the lower part
Show ‘synthetic gel’ in Bionumerics
Use multiple RE’s simultaneously; paste result(s) in the same text
file; write .txt file to disk
Calculate dendrogram for the given selection
Select coefficient desired
Dendrogram / Profile / Information (groupings in color) /similarity Matrix
based on Dice
Final
For AFLP normally between 20-35 fragments are optimal
When your results are satisfying, you can start your
practical work to verify this
When you’re not satisfied you can repeat the digestion with
other enzymes or (in the case of AFLP) use other selective
nucleotides
Verification of “in silico” typing results
 Check with same strain if In silico AFLP corresponds to practical
results
 In silico AFLP is applicable on genomic sequence data for new
typing procedures (e.g. no previous experience with species
requested).
 Cheap, rapid verification method becoming more predictive when
more genome sequences are present
 Very good quality control on your own procedure
Only applicable on restriction based typing methods
http://insilico.ehu.es/AFLP/
Dice (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%]
100
isAFLP
50
0
isAFLP
Type 2; PstI-PstI
type C; PstI-PstI
type 5; pstI-pstI
type 40; EcoRI-Ec.
type 5; ecorI-ecorI
type F; EcoRI-EcoRI
Type 2; EcoRI-MseI
type C; MseI-EcoRI
type 5; ecoRI-MseI
type 12; EcoRI-MseI
type A; MseI-EcoRI
type 40; PstI-MseI .
type F; MseI-PstI
type 40; EcoRI-Mse.
type F; MseI-EcoRI
Type 2; PstI-MseI
type 5; pstI-MseI
type C; MseI-PstI
type E; MseI-PstI
type 12; PstI-MseI
type A; MseI-PstI
Type 2; MseI-MseI
type C; MseI-MseI
type 5; mseI-mseI
type 40; MseI-MseI .
type F; MseI-MseI
type 12; MseI-MseI
type A; MseI-MseI
type E; MseI-MseI
type 12; PstI-PstI
type A; PstI-PstI
type E; PstI-PstI
type 40; PstI-PstI (.
type F; PstI-PstI
type E; MseI-EcoRI
type 12; EcorI-Eco.
Type 2; EcoRI-Eco.
type A; EcoRI-Eco.
type C; EcoRI-Eco.
type E; EcoRI-Eco.
SMT 2013
50
Wat gaan jullie praktisch doen
Van 6 stammen (Staphyloccen) is na kweek DNA geïsoleerd
Kies uit de 6 DNA’s 3 verschillende om te typeren met Agarose AFLP
De methode wordt in twee fasen uitgevoerd en is in praktijk geheel identiek aan
standaard AFLP met fluorescende primers en capillair analyse
Fase1: digestie en ligatie van DNA
Hiervoor worden alle ingrediënten bij elkaar gevoegd en bij 37°C geplaatst.
Fase 2: de PCR
Hiervoor is een kan en klaar mix gemaakt die toegevoegd wordt aan het
Restrictie/ligatie mengsel (na verdunning).
De PCR vindt overnacht plaats.
Morgen gel electroforese (gelen zijn al klaar)
Woensdag band based gel analysis
Ref:2013
LM-PCR
Staphylococcen AFLP
100.00
110.00
120.00
130.00
140.00
150.00
160.00
170.00
180.00
190.00
200.00
210.00
220.00
230.00
240.00
250.00
100
90
80
70
60
50
LM-PCR
40
30
20
LM-PCR
. 9349
TY
MRSA
.
AT12135
A
. 9436
TY
MRSA
.
AT12135
B
. 9351
TY
MRSA
.
AT12137
C
. 9636Q
TY
MRSA
.
AT12203
D
. 8132
TY
S. epidermidis
.
E
. 5887
TY
S. schleifferi
.
F
De afkapwaarde voor identieke stammen resp. identieke species ligt op 90% en 35% en wordt weergegeven
d.m.v. stippellijnen.