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

Background. Mycobacterium tuberculosis (Mtb) genomic epidemiology often relies on culturing patient sputum, a time- and labour-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.Methods. 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 (seven 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 and minor allele frequencies of minority variants identified in the cultured and hybrid-captured samples.Results. In the artificial strain mixture experiment, hybrid capture efficiency was 97% when Mtb comprised 0.01% of the input DNA. SNP identification via hybrid capture had a sensitivity ≥91% and precision ≥97% for Mtb lineages 4.1.2.1 and 4.9, excluding PE/PPE genes, when Mtb comprised 0.01% of input DNA. Observed minor allele frequencies were closely correlated (r=0.62 to r=0.79, P<0.001) with input minor allele frequencies across all dilutions. Among paired diagnostic samples, hybrid capture efficiency was high, 95%. However, four of the seven captured sputum samples were overwhelmed with Pseudomonas contamination, which comprised >25% of sequence reads. We did not detect a significant difference in the number of minority variants identified in cultured (median, 14; range, 10-18) and hybrid-captured samples (median: 17 variants, range 9-47, P=0.25) and minor allele frequencies were correlated (r=0.87, P<0.001) outside of PE/PPE genes.Conclusions. Hybrid capture of diagnostic sputum samples efficiently recovers accurate Mtb whole-genome sequences. Minority variant detection is robust in controlled mixtures but shows reduced concordance in clinical samples, highlighting the need for further validation in real-world settings.