Can we read TB’s genetic code directly from a patient's blood without growing the bacteria?

Project: Developing a culture-free, blood-to-genome workflow for tuberculosis identification and genotypic antimicrobial resistance profiling.

Research status: Proof of concept.

Research objective
ActiWGS TB aims to integrate mycobacterial DNA recovery, next generation sequencing (NGS) and genomic analysis into a streamlined research workflow.

The primary research objective is to establish an assay-specific criterion that predicts successful whole-genome sequencing (WGS) and sufficient genomic coverage for reliable resistance-associated variant assessment.

The project will initially evaluate the integrated workflow using characterised reference DNA, including BCG-derived DNA, before progressing towards more complex sample matrices and subsequent clinical validation.

The longer-term development target is a culture-free, blood-to-genotypic-resistance-profile workflow completed within 72 hours. This turnaround target remains to be demonstrated.

Milestone M1: Demonstrate successful processing of BCG-derived DNA through the integrated pipeline.

The first phase establishes whether the individual components can function together as one reproducible analytical workflow.

Research activities
1. Assess reference-material identity and suitability against predefined acceptance criteria.

2. Use the partner laboratory's approved procedures to assess MTBC DNA using qPCR, recording assay identity, amplification validity, Ct values, and control outcomes.

3. Evaluate DNA quantity and quality for their potential influence on downstream sequencing performance.

4. Perform sequencing-library preparation and Oxford Nanopore sequencing.

5. Process the resulting sequencing data through the ActiWGS TB bioinformatics pipeline.

6. Compare reference-material identity, genome coverage, and sequencing quality against predefined study acceptance criteria.

Data collected
Reference material and sample identifiers
DNA quality measurements
MTBC qPCR results and Ct values
Sequencing run and library identifiers
Total sequence output and read quality
MTBC-associated read assignments
Genome breadth and depth of coverage
Bioinformatics processing outcomes
Time required for each analytical stage

Expected deliverable: A documented demonstration of the integrated DNA-to-genome pipeline, identifying which stages work reliably and which require further optimisation.

Milestone M2: Establish the analytical detection characteristics of the workflow.

This phase investigates how reference DNA abundance and quality influence the likelihood of obtaining usable whole-genome sequencing data.

Research activities
1. Define a study panel representing different target-DNA abundance levels.

2. Establish appropriate negative controls, reference controls, and analytical replicates in the study design.

3. Assess qPCR performance and document valid amplification results.

4. Compare qPCR Ct values with sequencing outcomes from the corresponding materials.

5. Evaluate the relationship between target abundance, genome coverage, and sequence quality.

6. Identify where sufficient evidence exists to support reliable genomic analysis and where results become inconclusive.

7. Apply statistical analysis to estimate detection performance and uncertainty across the assessed reference-material range.

Data analysis
Determine whether an assay-specific criterion can predict successful WGS according to predefined genome-coverage and quality requirements.

Expected deliverable: A preliminary analytical detection-performance profile and an evidence-based candidate criterion for sequencing success.

Milestone M3: Demonstrate compatibility with multiplex sequencing.

Multiplexing offers the potential to analyse several specimens during a sequencing run, improving future operational efficiency.

Research activities
1. Define the intended multiplex study design using characterised reference materials.

2. Specify analytical acceptance criteria for individual sample identification and sequence assignment.

3. Evaluate sequencing output for each reference material using approved sequencing procedures.

4. Assess the accuracy of assigning sequence data to the corresponding sample identifier.

5. Compare sample-specific coverage and sequence quality with the initial single-sample evaluation.

6. Identify unequal sequence allocation, barcode misassignment, or other data-quality issues.

7. Document the influence of multiplexing on analytical turnaround time and sequencing-resource utilisation.

Data analysis
The resulting data will be assessed for sample assignment accuracy, distribution of sequencing output, genome coverage, quality, and reproducibility.

Expected deliverable: A documented assessment of whether multiplexing can be incorporated into ActiWGS TB without compromising the predefined analytical quality requirements.

Milestone M4: Determine the effect of multiplexing on analytical detection and sequencing success.

This phase extends the findings from Milestones M2 and M3.

Research activities
1. Apply the established performance-assessment framework to multiplex sequencing data generated using characterised reference materials.

2. Compare detection success and genomic coverage against results from the earlier non-multiplex evaluation.

3. Examine whether multiplexing changes the relationship between qPCR Ct and sequencing success.

4. Quantify differences in usable sequencing output, incomplete genome coverage, and inconclusive results.

5. Evaluate whether a common criterion is supported or whether separate operational criteria require investigation.

6. Document uncertainty and limitations before proposing any performance claims.

Data analysis
The resulting data will be assessed as was for M2 and M3.

Expected deliverable: A comparative assessment of analytical detection performance under multiplex and non-multiplex conditions, including a preliminary determination of whether the candidate criterion remains applicable.

Aditional scope:

Bioinformatics and genotypic resistance analysis
The bioinformatics component will investigate whether sequencing data provide sufficient genomic evidence to support interpretable MTBC identification and resistance-associated variant assessment.

Analysis workflow
Sequence quality assessment: Examine sequence-read quality, data integrity, and sequencing-run metrics.
Sequence identification: Assess correspondence with appropriate MTBC reference sequences and investigate potentially ambiguous assignments.
Genome coverage assessment: Determine genome breadth, depth, and coverage consistency.
Variant analysis: Evaluate sequence differences against appropriate reference genomes where analytical quality permits.
Resistance-associated variant interpretation: Compare adequately supported findings with recognised resistance-mutation catalogues, including relevant WHO resources.
Locus-level quality control: Document whether each relevant resistance-associated locus meets its analysis requirements.
Research reporting: Categorise results based on the strength and completeness of genomic evidence.

Interpretation categories
Resistance-associated variant detected: A recognised variant is identified with sufficient supporting sequence evidence.
No resistance-associated variant detected: No recognised variant is found within the adequately assessed regions. This does not independently establish drug susceptibility.
No call: Coverage, quality, or other analytical criteria are insufficient for a reliable interpretation.
Uncertain significance: A variant is identified, but its association with drug resistance is not established.
Findings will be reported only within the demonstrated analytical capabilities of the research workflow.

Data recording, statistical analysis, and research reporting
A consistent data-management framework will be applied throughout the project.

Each research sample will receive a unique identifier linking its reference-material characteristics, qPCR result, sequencing output, quality metrics, and analytical conclusions.

Planned analyses
Descriptive analysis of qPCR and sequencing performance
Ct-versus-genome-coverage relationships
Estimation of analytical detection probability
Assessment of sequencing-success rates
Comparison of multiplex and non-multiplex performance
Analysis of analytical repeatability and reproducibility
Assessment of workflow completion times and individual processing-stage contributions

Results will include unsuccessful experiments and inconclusive outcomes to minimise selective reporting.

Quality assurance
All activities involving biological materials will be conducted by authorised research personnel under approved institutional biosafety procedures and established laboratory SOPs.

Clinical specimens will not be included without the required ethical, governance, and biosafety approvals.

Any subsequent clinical evaluation will require separately defined clinical-performance endpoints and appropriate reference-method comparisons.

Final project deliverables
At completion, the project aims to provide:

1. An integrated ActiWGS TB research workflow demonstrated using characterised reference material.

2. An initial analytical detection-performance assessment.

3. A candidate criterion associated with successful WGS.

4. An assessment of multiplex sequencing compatibility.

5. A comparison of sequencing performance under multiplex and non-multiplex conditions.

6. A documented bioinformatics quality-control and genotypic resistance-reporting framework.

7. An evidence-based development plan for future clinical evaluation and end-to-end workflow validation.

Important research limitation: A negative blood-based assay result cannot exclude tuberculosis. Detection of MTBC DNA does not guarantee successful WGS, and a successful identification result does not automatically establish a reliable genotypic resistance profile.

ActiWGS TB remains a proposed research platform until the relevant analytical and clinical performance characteristics have been established.