Host Depletion Validation for Public Health TB Labs
Host Depletion Validation for Public Health Mycobacteria Labs
Public health laboratories running whole genome sequencing (WGS) for mycobacteria surveillance face a stubborn operational problem: host DNA contamination from liquid culture specimens drives sequencing failure rates above 10%, wasting reagents, delaying turnaround times, and forcing costly repeat runs. At the Pathogen Genomics Unit of Public Health Wales, host depletion for public health laboratory workflows was validated using a simple pre-extraction filtration step that reduced human DNA contamination and improved sequencing pass rates from 77% to 85% — with minimal disruption to an ISO-accredited process. In this symposium presentation, Joanne Watkins shared the validation data and operational rationale behind integrating the Devin filter into routine mycobacteria sequencing.
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Key Takeaways
- Mycobacteria whole genome sequencing had failure rates of 10%+ compared to 1–2% for all other pathogens at Public Health Wales — driven by host DNA in liquid culture specimens.
- Parallel testing showed the Devin filter significantly reduced human DNA reads in sequencing output while maintaining or improving taxonomic identification accuracy.
- Sequencing pass rates improved from 77% (pre-filter, 2023 baseline) to 85% (post-filter, January–October 2024 average).
- The filter was selected specifically because it had minimal impact on existing ISO-accredited extraction and library preparation workflows.
- Remaining failures shifted from host contamination to insufficient starting DNA — a separate upstream issue in specimen handling, not a filtration limitation.
Pathogen Genomics at Public Health Wales — Lab Architecture and Services
The Pathogen Genomics Unit at Public Health Wales operates as a next-generation sequencing hub for reference laboratories across Wales. The unit is structured with a deliberate separation between wet lab (sequencing) and dry lab (bioinformatics) teams, each with specialized expertise. End users include hospital reference units, public health surveillance teams, local infection control epidemiologists, NHS trusts, and national and global surveillance databases.
The unit runs ISO-accredited WGS services for a diverse pathogen portfolio: COVID-19 (scaled to 7,000 genomes per week during the pandemic), HIV resistance testing (repatriated from England with turnaround times reduced from 15 days to 5–7 days), tuberculosis and other mycobacteria, C. difficile for resistance and outbreak monitoring, Fusobacterium, influenza A and B surveillance, RSV monitoring for vaccine effectiveness, and antimicrobial-resistant bacteria from the Specialist Antimicrobial Chemotherapy Unit.
A key operational principle is multi-loading: the unit pools different organisms (viral and bacterial) onto the same flow cell, balancing by genome size and quantification to maintain turnaround times and maximize cost efficiency. This approach means that any pathogen class with elevated failure rates disproportionately impacts the entire workflow.
The Mycobacteria Sequencing Failure Problem
While all other pathogen sequencing services maintained failure rates of 1–2%, mycobacteria consistently failed at 10% or higher. The root cause was straightforward: unlike other specimen types processed by the unit (which are either PCR-amplified viral extracts or selectively cultured bacterial isolates), mycobacteria specimens arrive as heat-killed liquid culture broth. The original patient sample material — including substantial human cellular content — remains in the broth.
This host DNA contamination causes problems at multiple workflow stages. During extraction, high DNA concentrations create viscous samples that interfere with automated extraction platforms using fine aspirating needles. On the flow cell, human reads consume sequencing capacity that should be generating microbial coverage. The result: insufficient data for reliable species identification and resistance profiling, triggering repeat sequencing at additional cost and delayed reporting.
Devin Filter Validation — Parallel Testing Results
Phase 1: Controlled Comparison
Watkins designed a two-phase validation approach. Phase 1 ran a small set of parallel tests: each mycobacteria specimen was split, with one aliquot processed through a Devin host depletion filter before extraction and the paired aliquot processed via the standard protocol.
Results showed a consistent and significant reduction in human DNA reads in filtered samples. Taxonomic identification was maintained — filtered samples produced the expected species identification without loss of target organism data. The number of reads matching the target taxon and total base pairs attributed to the taxon generally increased in filtered samples, with no cases showing a decrease. Mean library fragment length also increased slightly in filtered samples, which improves downstream assembly quality.
Critically, filtered samples that had previously been unable to report (insufficient data for confident identification) could now produce reportable results. The unfiltered parallels for those same specimens remained unreportable.
Phase 2: Month-Long Service Trial
Phase 2 integrated the Devin filter into the routine service workflow for all mycobacteria specimens over a month-long trial. Comparing 2023 pre-filter baseline data against January–October 2024 post-filter data, sequencing pass rates improved from 77% to 85%.
Analysis of the remaining failures revealed a shift in failure mode: post-filter failures were primarily due to insufficient starting DNA rather than host contamination. This means the filter successfully eliminated the host DNA problem, exposing a separate upstream issue — the amount of mycobacterial growth in liquid culture broth before specimens are sent for sequencing. Resolving this requires coordination with the clinical mycobacteriology laboratory on culture handling and specimen allocation, not further sequencing optimization.
Why Host Depletion Fits ISO-Accredited Workflows
A central consideration for Watkins in selecting a host depletion method was ISO accreditation impact. Public Health Wales holds ISO accreditation across its sequencing services, and any process change triggers a formal audit and re-accreditation cycle. The Devin filter was selected specifically because it integrates as a pre-extraction step with minimal changes to downstream protocols — extraction, library preparation, pooling, and sequencing remain identical.
This is a practical reality that laboratory-developed test evaluations often overlook: in regulated environments, the cost of adopting a new method includes not just reagent price and validation labor, but the regulatory burden of process change. A method that achieves host depletion within the existing workflow architecture has a fundamentally lower adoption barrier than one requiring library preparation or bioinformatic pipeline modifications.
Watkins noted that double filtering is being considered for specimens with very high host content, and that the unit continues to investigate why filtered samples produce slightly longer library fragments — an unexpected but beneficial finding.
About the Speaker
Joanne Watkins is the Deputy Head of the Pathogen Genomics Unit at Public Health Wales. With nearly 35 years of experience in microbiology spanning serology, molecular diagnostics, and pathogen genomics, she leads wet lab operations for one of the UK’s national reference sequencing hubs, managing ISO-accredited WGS services across a broad pathogen portfolio.
