MLST versus MicrosatellitesMLST versus Microsatellites

AAA, Miami Beach, 2008AAA, Miami Beach, 2008

MLST versus Microsatellites

Corné HW KlaassenCanisius Wilhemina Hospital (CWZ)

Nijmegen, The Netherlands

CWZ, Nijmegen, The Netherlands. Contact: c.klaassen@cwz.nl Tel.: +31 (0)24 365 7514 Fax: +31 (0)24 365 7516

“Strain typing”The ability to discriminate between individuals of the same species

Genotypic methods

REA, RAPD, REP-PCR, IGS-PCR, RFLP, PCR-RFLP, AFLP, PFGE, RAMS, SSDP, RISC.

Genotypic methods

REA, RAPD, REP-PCR, IGS-PCR, RFLP, PCR-RFLP, AFLP, PFGE, RAMS, SSDP, RISC.

Fingerprints / genotypes are ill-defined !

>>> use numbers to distinguish between individuals ! <<<

Suggested back in the year 10 post Watson and Crick,one of the most popular journals in the world,

read by millions of subscribers !

Multi-Locus Sequence Typing(MLST)

versus

Microsatellites

CWZ, Nijmegen, The Netherlands. Contact: c.klaassen@cwz.nl Tel.: +31 (0)24 365 7514 Fax: +31 (0)24 365 7516

MicrosatellitesPrinciple

Multiplex options

Microsatellites

Microsatellites in real-life

De Valk et al. JCM 2005.

The STRAf assay:

Microsatellites in real-life

De Valk et al. JCM 2005.

The STRAf assay:

Compatible between labs

Poster by Hanneke de Valk

Multilocus sequence typingPrinciple

Multilocus sequence typingPrinciple cont’d

MLST in real-lifeA. fumigatus CAT1 forward sequence reaction

MLST in real-lifeA. fumigatus CAT1 reverse sequence reaction

MLST versus Microsatellites Pros and Cons

MLST Microsatellites

Throughput low highMultiplex option No YesCosts high lowTime to result long shortInterpretation easy easyReproducibility excellent excellentExchangeability excellent excellentDiscriminatory power low high

Discriminatory power A matter of stability

MLST depends on nucleotide changes.In general, DNA sequences are very stable.

Microsatellite variability is the result ofslipped strand mispairing (SSP),which occurs much more frequently.

Discriminatory power

Microsatellites

Marker Alleles

STRAf 2A 18STRAf 2B 19STRAf 2C 34

STRAf 3A 100+

STRAf 3B 37STRAf 3C 50

STRAf 4A 25STRAf 4B 28STRAf 4C 32

99 isolates – 96 genotypes

MLST

Marker ‘polymorphisms’

ANXC4 8BGT1 7CAT1 8LIP 8MAT1 3SODB 3ZRF2 4

100 isolates – 30 sequence types

de Valk et al., JCM 2005.Bain et al., JCM 2007.

Discriminatory power A matter of stability

Nice illustrations, but …What do they actually mean?

How to interpret a genotype ?

• Identical genotypes > identical isolates• Different genotypes > ???

– Related isolates ?– Possibly related isolates ?– Unrelated isolates ?

Not all differences are equal

STR data interpretationCategorical approach: markers are either identical or not

Distance matrixA B C D E F

A 0 2 1 1 1 2B 2 0 3 3 3 4C 1 3 0 2 2 3D 1 3 2 0 2 1E 1 3 2 2 0 3F 2 4 3 1 3 0

A BC

D

E

F

Typing resultsA B C D E F

1 10 11 10 10 10 102 6 5 6 6 6 63 8 8 8,2 8 8 84 50 50 50 53 50 535 42 42 42 42 45 426 8 8 8 8 8 7

Euclidian distance: absolute difference between 2 isolates

Distance matrixA B C D E F

A 0 2 0,2 3 3 4B 2 0 2,2 5 5 6C 0,2 2,2 0 3,2 3,2 4,2D 3 5 3,2 0 6 1E 3 5 3,2 6 0 7F 4 6 4,2 1 7 0

A BC

D

E

F

Typing resultsA B C D E F

1 10 11 10 10 10 102 6 5 6 6 6 63 8 8 8,2 8 8 84 50 50 50 53 50 535 42 42 42 42 45 426 8 8 8 8 8 7

Alternative algorithm: based on individual marker characteristics

A BC

D

E

FTyping results

A B C D E F1 10 11 10 10 10 102 6 5 6 6 6 63 8 8 8,2 8 8 84 50 50 50 53 50 535 42 42 42 42 45 426 8 8 8 8 8 7

Conclusions

• Microsatellites are the preferred targets for ultra high resolution strain typing, but …

• Not all microsatellites are equally suitable and …• Data interpretation needs to take the behavior of

individual markers into account

Acknowledgements

Canisius Wilhelmina Hospital,Nijmegen, The Netherlands:

Ilse CurfsHanneke de ValkJacques Meis

University of Aberdeen, UK:J.M. BainFrank Odds et al.