An imputed ancestral reference genome for the Mycobacterium tuberculosis complex better captures structural genomic diversity for reference-based alignment workflows

Reference-based alignment of short-reads is a widely used technique in genomic analysis of the Mycobacterium tuberculosis complex (MTBC) and the choice of reference sequence impacts the interpretation of analyses. The most widely used reference genomes include the ATCC type strain (H37Rv) and the putative MTBC ancestral sequence of Comas et al. both of which are based on a lineage 4 sequence. As such, these reference sequences do not capture all of the structural variation known to be present in the ancestor of the MTBC. To better represent the base of the MTBC, we generated an imputed ancestral genomic sequence, termed MTBC₀ from reference-free alignments of closed MTBC genomes. When used as a reference sequence in alignment workflows, MTBC₀ mapped more short sequencing reads and called more pairwise SNPs relative to the Comas et al. sequence while exhibiting minimal impact on the overall phylogeny of MTBC. The results also show that MTBC₀ provides greater fidelity in capturing genomic variation and allows for the inclusion of regions absent from H37Rv in standard MTBC workflows without additional steps. The use of MTBC₀ as an ancestral reference sequence in standard workflows modestly improved read mapping, SNP calling and intuitively facilitates the study of structural variation and evolution in MTBC.