Rescue ofMycobacterium bovisDNA obtained from cultured strains during official surveillance of animal TB: key steps for robust whole genome sequence data generation

Abstract

Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of Mycobacterium bovis has recently gained track due to its high resolution to identify infection sources, characterize the pathogen population structure, and for contact tracing. However, the workflow from bacterial isolation to sequence data analyses has several technical challenges that may severely impact the power to understand the epidemiological scenario and inform outbreak response. While trying to use archived DNA from cultured strains obtained during routine official surveillance of animal TB in Portugal, we struggled against three major challenges: the low amount of M. bovis DNA obtained from routinely processed animal samples; the lack of purity of M. bovis DNA, i.e. high levels of contamination with DNA from other organisms; and the co-occurrence of more than one M. bovis strain per sample (within-host mixed infection). The loss of an isolate genome is a missed link in transmission chain reconstruction, hampering the biological and epidemiological interpretation of data as a whole. Upon identification of these challenges, we implemented an integrated solution framework, based on whole genome amplification and a dedicated computational pipeline, to minimize their effects and recover as many genomes as possible. With the approaches described herein, we were able to recover 62 out of 100 samples that would have otherwise been lost. Based on these results, we discuss adjustments that should be made in official and research laboratories to facilitate sequential implementation of bacteriological culture, PCR, downstream genomics and computational-based methods. All of this in a time frame supporting data-driven intervention.

Genomic epidemiology sheds light on the emergence and spread of Mycobacterium bovis Eu2 Clonal Complex in Portugal

“…in which the defining SNPs had ambiguous calls. In these cases, strain separation was performed with SplitStrains [ 21 ] using as a reference the genome of M. bovis AF2122/27 (LT708304.1), as described in [ 21 ] and [ 22 ]. Each of these two samples was shown to have two different strains, that were successfully split, originating information corresponding to four different strains.…”