ABSTRACTWhole genome sequencing (WGS) has become the main tool for studying the transmission ofMycobacterium tuberculosiscomplex (MTBC) strains; however, the clonal expansion of one strain often limits its application in local MTBC outbreaks. The use of an alternative reference genome and the inclusion of repetitive regions in the analysis could potentially increase the resolution, but the added value has not yet been defined. Here, we leveraged short and long WGS read data of a previously reported MTBC outbreak in the Colombian Amazon Region to analyze possible transmission chains among 74 patients in the indigenous setting of Puerto Nariño (March to October 2016). In total, 90.5% (67/74) of the patients were infected with one distinct MTBC strain belonging to lineage 4.3.3. Employing a reference genome from an outbreak strain and highly confident single nucleotide polymorphisms (SNPs) in repetitive genomic regions, e.g., the proline-glutamic acid/proline-proline-glutamic-acid (PE/PPE) gene family, increased the phylogenetic resolution compared to a classical H37Rv reference mapping approach. Specifically, the number of differentiating SNPs increased from 890 to 1,094, which resulted in a more granular transmission network as judged by an increasing number of individual nodes in a maximum parsimony tree, i.e., 5 versus 9 nodes. We also found in 29.9% (20/67) of the outbreak isolates, heterogenous alleles at phylogenetically informative sites, suggesting that these patients are infected with more than one clone. In conclusion, customized SNP calling thresholds and employment of a local reference genome for a mapping approach can improve the phylogenetic resolution in highly clonal MTBC populations and help elucidate within-host MTBC diversity.IMPORTANCEThe Colombian Amazon around Puerto Nariño has a high tuberculosis burden with a prevalence of 1,267/100,000 people in 2016. Recently, an outbreak ofMycobacterium tuberculosiscomplex (MTBC) bacteria among the indigenous populations was identified with classical MTBC genotyping methods. Here, we employed a whole-genome sequencing-based outbreak investigation in order to improve the phylogenetic resolution and gain new insights into the transmission dynamics in this remote Colombian Amazon Region. The inclusion of well-supported single nucleotide polymorphisms in repetitive regions and ade novo-assembled local reference genome provided a more granular picture of the circulating outbreak strain and revealed new transmission chains. Multiple patients from different settlements were possibly infected with at least two different clones in this high-incidence setting. Thus, our results have the potential to improve molecular surveillance studies in other high-burden settings, especially regions with few clonal multidrug-resistant (MDR) MTBC lineages/clades.