Systematic review and meta-analysis of protocols and sequencing yield for whole genome sequencing of Mycobacterium tuberculosis directly from sputum samples

Direct sputum whole genome sequencing (dsWGS) can revolutionize Mycobacterium tuberculosis (Mtb) diagnosis by enabling rapid detection clinically relevant resistance mutations and strain diversity without the biohazard of culture. We searched PubMed, Web of Science, and Google Scholar, identifying 8 studies meeting inclusion criteria for testing protocols for dsWGS. Utilizing meta-regression, we identified factors positively associated with dsWGS success, including higher Mtb bacillary load, mechanical disruption, enzymatic/chemical lysis and sequencing volume. Decontamination with sodium hydroxide (NaOH) was negatively associated with dsWGS success (OR = 0.00032, 95% CI: 1.33×10ˆ-6–0.077; p = 0.004), likely due to its harsh effects on Mtb cells. Mechanical lysis (OR = 6120, 95% CI: 7.23–5.18×10ˆ6; p = 0.011) and enzymatic/chemical lysis (OR = 131, 95% CI: 1.68–1.03×10ˆ4; p = 0.028) were positively associated with sequencing success, as was heat inactivation (OR = 4.66, 95% CI: 1.24–17.5; p = 0.023). Total sequencing volume was also strongly associated with dsWGS success (OR = 10.35, 95% CI: 4.43–24.2; p = 6.53×10ˆ-8). In addition to these effects, we also observed high variability in pre-processing approaches, highlighting the need for standardized practices and identified pre-processing steps including decontamination and DNA extraction as priorities for further optimisation. Considering the strong association between Mtb load and successful dsWGS, protocols for optimal sputum sample collection, handling, and storage could also further enhance the success rate of dsWGS. • Comprehensive synthesis of all published direct-from-sputum sequencing studies. • Identifies key factors influencing dsWGS success, including bacillary load and extraction. • Reveals wide variability in sputum processing and decontamination protocols. • Highlights need for standardized workflows to improve coverage and sequencing yield. • Provides first quantitative framework for benchmarking Mtb dsWGS performance.