TABLE 2.

Next generation sequencing (NGS) strategies for drug-resistant Mycobacterium tuberculosis.

NGS approaches and applications	Advantages	Limitations
WGS • Species/strain identification. • Epidemiological studies. • TB diagnostics. • Molecular determinants of DR-M.tb.	• Nucleotide-level resolution: identification of single DR-M.tb conferring mutations. • Sequence directly from clinical samples (e.g., sputum). • Reveals M.tb genomic diversity and drug resistance microevolution events within the host.	• Only describe the genetic basis of DR-TB. • Limited information regarding DR-M.tb adaptation during disease progression and/or interactions with the host immune system.
RNA-Seq • Study DR-M.tb responses to drug treatment. • Study DR-M.tb metabolic/physiologic changes in different environment/conditions.	• Unbiased whole transcriptome approach. • High-throughput and relatively cost-effective. • Higher sensitivity and specificity compared to other gene expression approaches.	• Unable to distinguish DR-M.tb expression profiles of different strains in mixed infections. • Transcriptomic profile of either host or DR-M.tb in infected cells/tissues.
Dual RNA-seq • Study transcriptomic changes of both host and DR-M.tb simultaneously in infected cells/tissues.	• Global approach to study both host and DR-M.tb. • Establish causal host and DR-M.tb interactions: genome-wide association studies.	• Needs previous pathogen enrichment, and/or; • Increased sequencing depth to capture bacterial transcriptome (higher costs)
scRNA-seq • Study transcriptional responses of individual host cells or DR-M.tb bacillus during different physiological states (e.g., infection and disease progression). • Study DR-M.tb persister bacilli subpopulations during drug treatment. • Characterize individual responses in mixed infections.	• Unprecedented level of resolution and technological innovation (ability to barcode transcripts from a single cell). • Study host cell or DR-M.tb bacillus population heterogeneity. • Identify rare host cell or DR-M.tb bacillus populations. • Better understanding of tissue architecture.	• Lower throughput. • Requires effective isolation of viable M.tb bacilli or host cells. • Study either the host cell or M.tb, but mostly optimized for eukaryotes. • Need to overcome extra challenges in M.tb scRNA-seq [e.g., efficient lysis of the thick M.tb cell envelope, capture non-poly(A) mRNA]. • Complex bioinformatics analyses. • High sequencing costs.
Dual scRNA-seq • Study simultaneously host and pathogen at a single-cell resolution.	• Combined advantages of dual and scRNA-seq. • Define spatial-time relationships between host and pathogen. populations during different stages of the infection.	• Same as dual RNA-seq and scRNA-seq. • Difficult to extract enough bacterial information due to the low coverage and DR-M.tb bacilli:host ratio. • Complex interpretation of results.
Single-cell multi-Omics • Global understanding of complex DR-M.tb and host interactions through the integration of different “Omics” strategies, at a single-cell resolution.	• Systematic approach. • Dissect complex biological networks within a single-cell. • Integrate information from numerous “Omics.” • Highest level of information.	• Need for optimized and standardized protocols and bioinformatics approaches. • Difficult to integrate and interpret the data. • Single-cell resolution not easily achievable for some “Omics” approaches.

DR-M.tb, drug-resistant Mycobacterium tuberculosis; scRNA-seq, single-cell RNA sequencing; TB, tuberculosis; WGS, whole-genome sequencing.