Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates

Abstract

BackgroundMultiplex 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.ResultsTo 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.ConclusionBased 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-572) contains supplementary material, which is available to authorized users.

Consequences of genomic diversity in Mycobacterium tuberculosis

“…More recently, accumulating WGS data have led to the development of novel SNP-typing methods that rely on a broader understanding of the global MTBC phylogenetic diversity [11]. Some of these methods have been built into highly multiplexed assays [37–40]. In addition to SNPs, other forms of genetic diversity have been incorporated in high-throughput genotyping schemes, including spoligotyping data [41,42], and drug resistance-conferring mutations [43].…”

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

“…Since the first report using MLPA as a promising alternative for TB genetic characterisation (Bergval et al 2008), few studies evaluating MLPA in comparison to other assays have been described (Bergval et al 2012, Sengstake et al 2014, Chaidir et al 2016). In this scenario, as EMB and PZA susceptibility testing show no reliable results, and having small sample size of drug-resistant M. tuberculosis strains, in our study we relied on the evaluation of MLPA performance on RIF and INH resistance.…”

Recent Advances in Tuberculosis Diagnosis: IGRAs and Molecular Biology

“…The aforementioned microbead-based multiplex method has been also used to classify isolates epidemiologically by spoligotyping, showing near 100 % correlation with classical spoligotyping results [75,76,81]. This same method also served to detect lineage-specific SNPs willing to improve the taxonomic, evolutionary, and epidemiological classification [82].…”