Updated Reference Genome Sequence and Annotation of Mycobacterium bovis AF2122/97.

We report here an update to the reference genome sequence of the bovine tuberculosis bacillus Mycobacterium bovis AF2122/97, generated using an integrative multiomics approach. The update includes 42 new coding sequences (CDSs), 14 modified annotations, 26 single-nucleotide polymorphism (SNP) corrections, and disclosure that the RD900 locus, previously described as absent from the genome, is in fact present.

GENOME ANNOUNCEMENT

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is the most widely studied animal-adapted member of the Mycobacterium tuberculosis complex (MTBC). bTB exacts a tremendous global economic toll through productivity losses and disease control costs, while zoonotic transmission of M. bovis infection is a threat to human health (1–6).

M. bovis AF2122/97 was the first M. bovis strain to be sequenced and serves as the reference genome (7), and it was last updated in 2003. An updated reference M. bovis genome sequence will provide an essential resource for the tuberculosis (TB) research community and serve as a basis for comparative studies into animal- and human-adapted MTBC members.

To update the M. bovis AF2122/97 genome and annotation, a low-passage-number stock taken from the original M. bovis AF2122/97 seed stock was resequenced and reannotated using a combination of DNA sequencing, RNA sequencing, and proteomics data. All nucleic acid and protein samples were derived from exponentially grown cultures.

Short-read DNA sequencing libraries were prepared using the Nextera XT DNA library preparation kit (Illumina) and sequenced on the MiSeq system (Illumina), generating 250-bp paired-end reads that were trimmed using Sickle (Q > 30), with 60× reference coverage (8). For PacBio RSII sequencing, enzymatically extracted DNA was prepared using a large-insert library (6 kb to 8kb) size selection (9). Two single-molecule real-time (SMRT) cells were used for an output of 542,585,804 bases, a mean read length of 8,141, and 86× reference coverage. DNA sequencing data sets were analyzed using a combination of de novo assembly [short reads, SOAPdenovo (10); long reads, Canu (11)] and nucleotide variant identification methods [short reads, Stampy, SAMtools, and VCFtools (12–14); long reads, Pilon (15); and MUMmer (16)]. This allowed both an update of the genome nucleotide sequence and the identification of genomic regions that had been misassembled, or missed entirely, in the original sequencing project. Reannotation of the M. bovis AF2122/97 genome was achieved by automatic annotation transfer from M. tuberculosis H37Rv (17) and a proteogenomic analysis using both M. bovis AF2122/97 shotgun tandem mass spectrometry (MS/MS), sequential window acquisition of all theoretical mass spectra (SWATH MS) data sets, and M. tuberculosis H37Rv SWATH MS data sets (18).

Overall, 26 single nucleotide polymorphisms were identified. Strikingly, the large sequence polymorphism RD900, originally described as being deleted from M. bovis 2122/97 (19), was found to be present; recombination between repeat structures flanking the RD900 locus in clones used for the original shotgun sequencing genome project may have led to the loss of RD900. Furthermore, 42 novel coding sequences were identified, while 14 existing annotations were modified.

Accession number(s).

This whole-genome shotgun project had been deposited in DDBJ/ENA/GenBank under the accession no. LT708304. SWATH MS data can be found on PeptideAtlas under identifier PASS00932.