Spoligotyping and whole-genome sequencing analysis of lineage 1 strains of Mycobacterium tuberculosis in Da Nang, Vietnam

BACKGROUND Spacer oligonucleotide typing (spoligotyping), a widely used, classical genotyping method for Mycobacterium tuberculosis complex (MTBC), is a PCR-based dot-blot hybridization technique to detect the genetic diversity of the direct repeat (DR) region. Of the seven major MTBC lineages in the world, lineage 1 (Indo-Oceanic) mostly corresponds to the East African-Indian (EAI) spoligotype family in East Africa and Southeast Asia. OBJECTIVES We investigated the genomic features of Vietnamese lineage 1 strains, comparing spoligotype patterns using whole-genome sequencing (WGS) data. METHODS M. tuberculosis strains isolated in Da Nang, Vietnam were subjected to conventional spoligotyping, followed by WGS analysis using a high-throughput sequencer. Vietnamese lineage 1 strains were further analyzed with other lineage 1 strains obtained from a public database. RESULTS Indicating a major spoligotype in Da Nang, 86 (46.2%) of the 186 isolates belonged to the EAI family or lineage 1. Although typical EAI4-VNM strains are characterized by the deletion of spacers 26 and 27, 65 (75.6%) showed ambiguous signals on spacer 26. De novo assembly of the entire DR region and in silico spoligotyping analysis suggested the absence of spacer 26, and direct sequencing revealed that the 17th spacer sequence not used for conventional typing, was cross-hybridized to the spacer 26 probe. Vietnamese EAI4-VNM, other EAI-like strains, and those showing a non-EAI pattern lacking many spacers formed a monophyletic group separate from other EAI families in the world. CONCLUSION Information about the alignment of spacers in the entire DR region obtained from WGS data provides a clue for the determination of experimentally ambiguous spoligo patterns. WGS data also helped to analyze the hidden relationships between apparently distinct spoligo patterns.