Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine

Abstract

Background Mycobacterium bovis bacillus Calmette-Guérin ( M. bovis BCG) is the only vaccine available against tuberculosis (TB). In an effort to standardize the vaccine production, three substrains, i.e. BCG Danish 1331, Tokyo 172–1 and Russia BCG-1 were established as the WHO reference strains. Both for BCG Tokyo 172–1 as Russia BCG-1, reference genomes exist, not for BCG Danish. In this study, we set out to determine the completely assembled genome sequence for BCG Danish and to establish a workflow for genome characterization of engineering-derived vaccine candidate strains. Results By combining second (Illumina) and third (PacBio) generation sequencing in an integrated genome analysis workflow for BCG, we could construct the completely assembled genome sequence of BCG Danish 1331 (07/270) (and an engineered derivative that is studied as an improved vaccine candidate, a SapM KO), including the resolution of the analytically challenging long duplication regions. We report the presence of a DU1-like duplication in BCG Danish 1331, while this tandem duplication was previously thought to be exclusively restricted to BCG Pasteur. Furthermore, comparative genome analyses of publicly available data for BCG substrains showed the absence of a DU1 in certain BCG Pasteur substrains and the presence of a DU1-like duplication in some BCG China substrains. By integrating publicly available data, we provide an update to the genome features of the commonly used BCG strains. Conclusions We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of the BCG Danish vaccine strain. The BCG Danish WHO reference genome will serve as a reference for future engineered strains and the established workflow can be used to enhance BCG vaccine standardization. Electronic supplementary material The online version of this article (10.1186/s12864-019-5909-5) contains supplementary material, which is available to authorized users.

First insight into the whole-genome sequence variations in Mycobacterium bovis BCG-1 (Russia) vaccine seed lots and their progeny clinical isolates from children with BCG-induced adverse events

“…During the last decades, the whole-genome sequencing (WGS) was applied to assess the genome stability of BCG vaccine strains from different culture collections and to evaluate the impact of minor sequence variations occurring in seed and commercial lots on the efficacy of BCG-vaccines used in different countries [ 30 – 34 ]. Recently, sequence variations were identified in clinical isolates from two infants with BCG-induced disease compared with the sub-cultured M. bovis BCG Moscow strain (Serum Institute of India, India) in South Africa [ 15 ].…”

Phylogenomic and epidemiological insights into two clinical Mycobacterium bovis BCG strains circulating in South Africa

“…The draft genome sequences of the two isolates, M1_S48 and M2_S49, were 2 767 203 bp and 1 432 828 bp, respectively. These genome sizes are significantly smaller than the previously sequenced BCG genomes (Borgers et al, 2019); the reasons for this are not known, although it may suggest that some genes are missing in these isolates, making their genomes smaller. Annotation with the NCBI Prokaryotic Genome Annotation Pipeline identified 3999 coding genes, 3999 coding CDS, 51 RNA (3 rRNA, 45 tRNA, 3 ncRNA), 187 pseudogenes, and three CRISPR arrays in the genome of M1_48.…”

Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource

“…The 192 96-well plates containing the transposon mutants were processed in two sets of 96- by 96-well plates. We recently established a reference genome sequence for M. bovis BCG Danish 1331 (NIBSC 07/270 [ https://doi.org/10.6084/m9.figshare.c.4489496 ] [ 40 , 41 ]), which was used to map the sequencing reads. A detailed procedure for the construction of the archived mutant library can be found in Method S7 at https://doi.org/10.6084/m9.figshare.c.4507472 .…”

“…Supplemental material accompanies this paper at the Figshare repository at https://doi.org/10.6084/m9.figshare.c.4507472, also including the Galaxy workflows for the bioinformatics data analysis and the updated BioPerl Script for the automated mutant location assignment (Zip File S1 at https://doi.org/10.6084/m9.figshare.c.4507472). The BCG Danish 1331 genome assembly as used during the data analysis can be found on Figshare at https://doi.org/10.6084/m9.figshare.c.4489496 (40). The novel sequencing data (raw Illumina reads) generated in this study have been deposited at NCBI's Sequence Read Archive under project code PRJNA528037.…”