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.
