comparing typing methods : do's and don't's

João André Carriço, PhDMicrobiology Institute/Institute for Molecular Medicine

Faculty of Medicine, University of Lisbon

Portugal

How to compare typing techniques:

do’s and Don’t’s

http://im.fm.ul.pthttp://imm.fm.ul.pthttp://www.joaocarrico.info

WORKSHOP 24:NGS FOR MICROBIAL GENOMIC

SURVEILLANCE AND MORE - ONE TECHNOLOGY FITS ALL

CONFLICTS OF INTEREST

NOTHING TO DISCLOSE

MICROBIAL TYPING

“Crude classifications and False generalizations are the curse of organized life”

George Bernard Shaw (1856 – 1950)

Microbial Typing: discriminating strains within a species/subspecies

TYPING METHODS: TYPES / SUBTYPES

Street market, Florence, Italy

HOW TO COMPARE TYPING METHODS

Struelens, M.J. et al, 1996. Clinical microbiology and infection, 2(1), pp.2–11.

HOW TO COMPARE TYPING METHODS

Struelens, M.J. et al, 1996. Clinical microbiology and infection, 2(1), pp.2–11.

Performance Criteria:TypeabilityReproducibilityStabilityDiscriminatory powerEpidemiological concordanceTyping System concordance

Convenience Criteria

TYPING METHODS: TYPES / SUBTYPESPFGE :PFGE Type (cut-off 80% DICE/UPGMA)PFGE Subtype (cut-off 80% DICE/UPGMA)

PFT DMLST :Clonal Complex (goeBURST)Sequence Type

ST 239 : 2-3-1-1-4-4-3ST 8 : 3-3-1-1-4-4-3

Serotype :SerogroupSerotype

emm typing:emm typeemm subtypes

cgMLST/ wgMLST/ SNP / kmer :Any clustering done on a tree or graph

TRADITIONAL TYPING AND NGS

Chronicle of a Death Foretoldhttp://en.wikipedia.org/wiki/File:ChronicleOfADeathForetold.JPG

Whole Genome Sequencing in typing:

- Gene-by-gene: wgMLST, cgMLST

- SNP comparison approaches: comparison with reference strains

- k-mer distances

- Ability to recover most of the present sequence based typing information in a single experimental procedure

COMPARING TYPING METHODS

Weissman S J et al. Appl. Environ. Microbiol. 2012;78:1353-1360

Conc

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ated

MLS

T lo

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flmH sequences

The Hard way….

NEED FOR QUANTIFICATION AND STATISTICS

When you can measure what you are talking about and express it in numbers you know something about it. When you cannot measure it, when you cannot express it, your knowledge is of a meagre and unsatisfactory kind.

- Lord Kelvin 1861

POPULATION AND SAMPLE

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POPULATION AND SAMPLE

9

7

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Sampling introduces an error…. …. but this error can be quantified!

Confidence intervals allow for that quantification of sampling error and should be used instead of point estimates!

COMPARING PARTITIONS FRAMEWORK

Three Coefficients :

1)Simpson’s Index of Diversity

2)Adjusted Rand

3)Adjusted Wallace

And the respective 95% confidence intervals

COMPARINGPARTITIONS WEBSITE

http://www.comparingpartitions.info

COMPARINGPARTITIONS WEBSITE

Copy/Paste from Excel

MEASURING DIVERSITY: SIDSimpson’s Index of Diversity

This index indicates the probability of two strains sampled randomly from a population belonging to two different types

Since it is a probability varies between 0 – 1.

Highly discriminatory methods are desired…

..but are they always needed?

Confidence intervals were defined for SID and should be used.

NGS methods: Increased discrimination but what if every individual is a type ?? Simpson, 1948

Hunter and Gaston, 1988Grundmann et al ,2001

Comparing SID’s 95% CIs

Null Hypothesis: The values under comparison are the same

COMPARING METHODS RESULTS

PFG

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s2

s3

s4

s5

s6

s7

Same Sequence Type?

Same PFGE cluster?

Y

N

Y N

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For each pair of isolates:

Seq

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ADJUSTED RAND

Overall concordance of two methods taking into account that the agreement between results could arise by chance alone.

Bi-directional agreement measureConfidence intervals by jackknife pseudo-values method.

CHANCE AGREEMENT ILLUSTRATION

Two possible random rearrangements…

CHANCE AGREEMENT: RAND VS ADJUSTED RAND

Min: 0.629Max: 0.826

Test created by generating 6 to 11 random classifications for 237 cases (strains)

ADJUSTED WALLACE

Probability that if two strains share the same classification by a Method A they also share the same classification by Method B, corrected by chance agreement

Analytical confidence intervals.Jackknife pseudo values confidence intervals

ADJUSTED WALLACE

COMPARING AR AND AW 95% CI

Null Hypothesis: The values under comparison are the same

COMPARINGPARTITIONS WEBSITE

Scripts

Bionumerics™ Partition Mapping module (http://www.applied-maths.com/features/partition-mapping)

OTHER APPLICATIONS FOR SID,AR AND AW

• Determination of the best set of markers for typing purposes : given dozens to hundreds or thousands of possible loci or SNPs is there a subset with enough discrimination to produce the same results as other typing method?

http://www.cidmpublichealth.org/pages/ausetts.html.

OTHER APPLICATIONS FOR SID,AR AND AW

OTHER APPLICATIONS FOR SID,AR AND AW

• Determination of the best set of markers /typing methods for typing purposes for predicting a specific outcome or any associated metadata. Examples:• Using AW to determine the which typing method

better predicts a clinical outcome or prognosis.• Using AW to determine association between

alleles and Clonal Complexes (Weissman S J et al. Appl. Environ. Microbiol. 2012;78:1353-1360)

• Determining association between alleles or types and geographical location of sampling

Conclusions: Do’s and Don’t’sDO’s

• The larger the sample size the more accurate can be the conclusions

• Always use SID, Adjusted Rand and Adjusted Wallace

• Confidence intervals give more information than the point estimates because they intrinsically take the sample size into consideration

• Understand the algorithm before making conclusions about the results

• Assess the biological meaning of the results

Conclusions: Do’s and Don’t’s

DON’T’s

• Make comparisons using small number of isolates. Usually >50 is enough but >100 is better to get statistically significant results

• Don’t use coefficients that not corrected by chance agreement when comparing typing methods

TO KNOW MORE:

For examples of usage see the list of references in:http://darwin.phyloviz.net/ComparingPartitions/index.php?link=References

ACKNOWLEDGEMENTS

Mário RamirezFrancisco Pinto Ana Severiano

UMMI Members

Funding from Fundação para a Ciência e TecnologiaEU 7th Framework programme

Dag Harmsen, for the invitation to participate in the workshop

www.comparingpartitions.info