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

Minako Hijikata,Ikumi Matsushita,Shinji Maeda,Naoto Keicho,Seiya Kato,Le Van Duc,Nguyen Thi Le Hang,Igor Mokrousov

doi:10.1371/journal.pone.0186800

Abstract

BackgroundSpacer 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.ObjectivesWe investigated the genomic features of Vietnamese lineage 1 strains, comparing spoligotype patterns using whole-genome sequencing (WGS) data.MethodsM. 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.ResultsIndicating 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.ConclusionInformation 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.

Highlights

The methods of genotyping Mycobacterium tuberculosis complex (MTBC) facilitate many aspects of tuberculosis studies
M. tuberculosis strains isolated in Da Nang, Vietnam were subjected to conventional spoligotyping, followed by Whole-genome sequencing (WGS) analysis using a high-throughput sequencer
De novo assembly of the entire direct repeat (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

Summary

Introduction

The methods of genotyping Mycobacterium tuberculosis complex (MTBC) facilitate many aspects of tuberculosis studies. The direct repeat (DR) region of the M. tuberculosis genome consists of 36 base pair (bp) DR copies and 35 to 41 bp spacers. The original spoligotyping method uses 43 of 68 DVRs in the region and detects the presence or absence of these spacers by a PCR-based dot-blot hybridization technique [5, 7]. 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

Methods

Results

Conclusion