1) The document analyzes genetic diversity in the 'Haarlem' collection of Mycobacterium tuberculosis strains using AFLP and MIRU-VNTR polymorphism analysis. 2) The objectives are to validate AFLP analysis for strain identification and compare it to other typing systems like MIRU-VNTR. 3) Guidelines will be formulated for interpreting chromosomal patterns for epidemiological evidence of relatedness between strains.

1. Genetic analysis of ‘Haarlem’ collection of strains of M. tuberculosis using AFLP and MIRU-VNTR Polymorphism analysis Shuja S Malik University of Jammu

2. Genetic Diversity of MTB Complex - a tool for Epidemiology Inser-Del, Single base changes, Mobility of IS elements Mycobacteriophages These can be exploited for strain idnetification

5. Objectives: 1) To validate the technique of AFLP analysis for Mycobacterial genomes for epidemiology and outbreak investigation 2) To compare it with other typing systems based on polymorphic markers such as MIRU-VNTRs 3) To formulate guidelines for interpretation of chromosomal patterns for epidemiological evidence of clonality, relatedness and heterogeneity

6. What is so special about Haarlem strains? A blinded international reference set of 90 strains from 38 different countries 10 non-Mtb strains and 30 duplicate strains included Have been already tested with IS6110, PGRS, DR, MIRU & Spoligotyping analyses

8. EcoRI MseI Forward Primer Dye Reverse Primer Eco Adapter MseI adapter G A C EcoRI adapter No prior genome information is needed Produces hundreds of bands Can be automated and made portable Can be juxtaposed to genome sequence derived data Markers can be used as signatures Can be archived successfully At CDFD: Rich experience of typing strains from national and international collections Good collaborative network exists Electronic database is present Protocols established

9. FAFLP Output Coded samples N=135 Number of loci analyzed = 42

11. Why to compare this with MIRU-VNTR typing? Strains can be typed by a numerical Code corresponding to the numbers of VNTRs in different loci Evolutionarily stable markers Databases can be generated

13. Allele diversity of the 14 MIRU Loci

14. Standardization of multiplex priming for repetitive sequences is one of the most difficult assignments

15. Multiplex Mixture A Multiplex Mixture A M N P N T1 T2 T3

16. What remains to be done: Decoding of strains for FAFLP data analysis and development of consensus trees with MIRU-VNTR data Inter-laboratory reproducibility between CDFD and the Central Public Health Laboratory, London [Haarlem strains were sent by RIVM (a Dutch public health lab) to both the labs as blind coded DNAs] MIRU-VNTR analysis of Indian samples