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Background  The genetic basis of virulence in  Mycobacterium tuberculosis  has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication.    Results  Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome.    Conclusions  Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.    Electronic supplementary material  The online version of this article (doi:10.1186/s12864-017-3687-5) contains supplementary material, which is available to authorized users.

SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis