Optimizing culture-free approaches to recover high quality M. tuberculosis genomic variation

Mycobacterium tuberculosis (Mtb) genomic epidemiology often relies on culturing patient sputum, a time and labor-intensive process. Hybrid capture approaches have been successfully used to enrich Mtb DNA from complex clinical samples, yet the accuracy of variant identification from captured samples has not been systematically evaluated.We created artificial strain mixtures of two well-characterized Mtb isolates such that the minor strain comprised 0-50% of Mtb DNA and serially diluted the Mtb DNA into human DNA to simulate diagnostic samples with different sputum smear burdens (32 samples). We also prospectively collected paired Mtb diagnostic cultures and sputum submitted to a national diagnostic laboratory (7 sample pairs). We performed hybrid capture and Illumina whole genome sequencing for all samples. For the artificial strain mixtures, we measured hybrid capture efficiency, the percentage of total reads mapping to Mtb, and performance of fixed and minority variant identification. For the diagnostic samples, we compared the number, identity of, and minor allele frequencies of minority variants identified in the cultured and hybrid captured samples.