Introduction to cluster analysis purpose and parameters...

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

http://eurl-listeria.anses.fr

French Agency for Food, Environmental and Occupational Health Safety – Food Safety Laboratory Maisons-Alfort location and Boulogne-sur-Mer location

14, rue Pierre et Marie Curie - F94701 Maisons-Alfort Cedex - Telephone: + 33 149 771 350 - Fax: + 33 149 771 380 Boulevard du Bassin Napoléon - F62200 Boulogne-sur-Mer - Téléphone : +33 321 992 500 – Télécopie : + 33 321 991 725

www.anses.fr - @Anses_fr

Laboratory for Food Safety

Maisons-Alfort location

Introduction to cluster analysis purpose and parameters and Global

Database Management

Benjamin FELIX Joint training course on the use of BioNumerics software to analyse PFGE data

Rome 3-4 July 2017

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

2

Clustering

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Cluster Analysis

PFGE Profiles

Set of band-based profiles

Similarity matrix

Dendrogram/clustering

Coefficient calculation: Algorithm « Dice »

Binary: Band absence or presence

Clustering: Algorithm « UPGMA »

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Dice coefficient: Band-based similarities

Dice:

SD= 2NAB

NA+ NB

SD= 2Ncommon

NTotal

NA

NB

NAB

SD= 2x7

8+9

= 82%

3 bands of difference

A

B

A

82 100

100

72 100 C

B

90

C

…

…

Similarity matrix

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Dice coefficient: Optimization and tolerance

Optimization

1%

Tolerance

1%

Band Matching

Matching?

Optimal settings according to EURL Lm SOP

% < %

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

UPGMA: Unweighted Pair Group Method using Arithmetic average

A

B

A

82 100

100

72 100 C

B

98

C

…

…

Similarity matrix

B+C

C

B

77 100

98

70 100 D

A

95

D

…

…

A

90 100 (82+72)/2

B+C

C

B

95

98

D

A+D

…

A

90 100

B+C+A+D

C

B

73.5

D

…

A

70 80 90 100

73.5

…

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

7

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Database management

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Communication between databases

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

TIF Image

Database exchange

- Permant import of fingerprint, need image

processing, normalization and analysis

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Bundles

Database exchange

- Profile are temporally available in the database

(profiles, sequences and fields) until the software shut

down

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

XML

Database exchange

- Permanent import of data (profiles, sequences and

fields) without any analysis.

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Bundles XML

TIF Image

Database exchange

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Database compatibility

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

15

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Reference system

Reference system included in the gel:

- Salmonella Braenderup H9812 XbaI restriction

profile

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Reference system

Database main reference system included in the

BioNumerics experiment:

- Salmonella Braenderup H9812 XbaI restriction

profile

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Normalization process

Metric =

molecular

size kbp

Percentage of

migration

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Normalization process

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Normalization process

Balance zoom

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Normalization process

Decale

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Normalization process

Distortion

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Normalization process

Normalized gel

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Reference system

Database main reference parameters

Internal reference system

Active zone

Calibration

curve

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

How to proceed when the reference systems are

different ?

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Several reference systems can work together

Re-mapping

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Re-mapping

The re-mapping can be used for bundles and XML

import

XML import implement automatically parallel

reference system

Bundles need re-mapping to be set

beforehand

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Re-mapping

The calibration curve is used to perform the re-

mapping process

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Re-mapping

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Re-mapping is implemented

EURL Lm European Union Reference Laboratory for

Listeria monocytogenes

Re-normalization

- Specific script provided by Applied Maths

- Convert_refsys.BNS

- The script car re-analysed automatically all fingerprint into another

internal reference system

- However this script only to convert

similar reference system e.g. SB01

into SB02

- The script works by batch of 20

gels

- Beware to backup your system

before to proceed

- Beware to verify the output of the

re-normalisation