title: Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates.
abstract: BACKGROUND
  Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains.
  
  
  RESULTS
  To validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal.
  
  
  CONCLUSIONS
  Based on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.
authors.affiliation: KIT Biomedical Research, Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands. s.sengstake@kit.nl.
authors.forename: Sarah
authors.initials: S
authors.lastname: Sengstake
authors.forename: Nino
authors.initials: N
authors.lastname: Bablishvili
authors.forename: Anja
authors.initials: A
authors.lastname: Schuitema
authors.forename: Nino
authors.initials: N
authors.lastname: Bzekalava
authors.forename: Edgar
authors.initials: E
authors.lastname: Abadia
authors.forename: Jessica
authors.initials: J
authors.lastname: de Beer
authors.forename: Nona
authors.initials: N
authors.lastname: Tadumadze
authors.forename: Maka
authors.initials: M
authors.lastname: Akhalaia
authors.forename: Kiki
authors.initials: K
authors.lastname: Tuin
authors.forename: Nestani
authors.initials: N
authors.lastname: Tukvadze
authors.forename: Rusudan
authors.initials: R
authors.lastname: Aspindzelashvili
authors.forename: Elizabeta
authors.initials: E
authors.lastname: Bachiyska
authors.forename: Stefan
authors.initials: S
authors.lastname: Panaiotov
authors.forename: Christophe
authors.initials: C
authors.lastname: Sola
authors.forename: Dick
authors.initials: D
authors.lastname: van Soolingen
authors.forename: Paul
authors.initials: P
authors.lastname: Klatser
authors.forename: Richard
authors.initials: R
authors.lastname: Anthony
authors.forename: Indra
authors.initials: I
authors.lastname: Bergval
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D008040:Genetic Linkage; D005838:Genotype; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000592:standards; D020641:Polymorphism, Single Nucleotide; D014376:Tuberculosis / Q000382:microbiology / Q000473:pathology
chemical_list: D004269:DNA, Bacterial