The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of its 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. 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. We discuss how our results change the picture of virulence attenuation and the power of SMRT sequencing for producing high-quality reference genomes. * Equal contribution † Corresponding author: faramarz@sdsu.edu 1 .
CC-BY-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/064840 doi: bioRxiv preprint first posted online Jul. 19, 2016; Tuberculosis is a serious and pervasive public health problem [1]. It is a disease 1 caused by infection of bacteria from the Mycobacterium tuberculosis complex 2 (MTBC). The reference strain, Mycobacterium tuberculosis H37Rv, has an at-3 tenuated counterpart known as H37Ra that is available for studies where facil-4 ities to handle virulent samples are lacking. H37Ra exhibits a distinct colony 5 morphology, an absence of cord formation, decreased resistance to stress and 6 hypoxia, and attenuated virulence in mammalian models [2][3][4]. The H37Ra
22In this study, we sequenced and assembled the genome of M. [14, 15]. This insertion was the heterogeneous inser-56 tion responsible for the discrepant contig ends in our raw genome assembly.
57Such heterogeneity implies either a lack of selection pressure on the insertion in 58 culture, a recent emergence of the insertion, or both.
59The 3456bp insertion in ppe54 with respect to H37RaJH incidentally corre-60 sponds to a tandem duplication of a 1728bp sequence at the same site in H37Rv H37Rv and CDC1551 as "H37Ra-specific". These mutations fall within or ad-95 jacent to (which we term "affecting") 56 genes in H37Rv, which we refer to as The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/064840 doi: bioRxiv preprint first posted online Jul. 19, 2016; colleagues also discovered sequencing errors in the H37Rv reference sequence [5], 98 a number of which were corrected in NC 000962.3 [9], the version used in our 99 study.
100To see how well the HC genes are supported by our assembly of H37Ra, we 101 determined variants with respect to H37Rv ...