A prerequisite to enhancing AMR surveillance with WGS is a robust phenotypic testing setup. Four units – in Colombia, India, Nigeria, and the Philippines – were experienced with conventional bacterial typing and had rigorous protocols in place. This included an operational setup that verified the identity of all organisms referred from collection sites, tested their susceptibility against a panel of antimicrobials, and determined their virulence through intra-species typing [5, 13]. The resulting sample metadata were collected and integrated [28]. Figure 1 illustrates a typical WGS lab workflow when implemented downstream of phenotypic testing.
Embedding quality-assured genome sequencing requires careful planning, long before the purchase of a sequencer. In addition to the initial investment in equipment and reagents, there are crucial challenges and considerations for any new laboratory to ensure reliable, cost-effective, and reproducible quality of genomes. A pragmatic roadmap for prospective labs looking to assemble WGS processing power has been compiled, based on our experience (Fig. 2).
WIJ Roadmap
Typically, laboratories with finite resources and budgetary constraints, and therefore diminished purchasing power, prioritize low assay costs and ease of sample processing over other factors. Bacterial WGS supports this, as it: 1) simplifies lab workflows by utilizing the same protocol across all organisms of interest; 2) drastically decreases time-to-result, especially against lengthy Salmonella serotyping assays and for slow-growing bacteria like M. tuberculosis; and 3) benefits from extensive multiplexing opportunities that can help sequence several bacterial genomes on a single run [6, 14, 15, 16]. A general prerequisite to rapidly setting up WGS is the verified ability to perform high-quality PCR, since preparing WGS libraries involves similar techniques.
The journey to embedding WGS capacity can be split into four phases of activity: Assessment, Assembly, Optimization, and Reassessment.
Assessment
Considerations: The assessment phase serves to evaluate existing infrastructure for the potential to expand routine phenotypic AMR typing to WGS. Additionally, it allows for extensive engagement with laboratory scientists and microbiologists on-site to understand their existing lab-based workflows and proficiencies.
Challenges: Firstly, the equipment, footprint and safety guidelines required for routine microbiology differ greatly from those needed to run WGS. Like any molecular method that utilizes DNA as starting material, genome sequencing is performed on benches separate to bacterial culture. In reality, this cannot always be achieved at nascent labs due to extremely limited space or workbenches being shared among several research groups. Secondly, in our case, performing individual on-site assessments was not always practical because the partner locations were located around the world.
Solutions: Laboratories were presented with a checklist questionnaire designed in-house to evaluate WGS setup potential. Its purpose was to rapidly examine the premises and personnel skills, and to provide recommendations on infrastructure reconfiguration, i.e. dedicated workspaces, sterility measures, and controlled environments in which to perform sequencing. Labs were free to flexibly modify these suggestions based on their situation. Our partner sites in Colombia, Nigeria, and the Philippines reorganized or extended their lab footprint, while our partner site in India remodeled from the ground-up. A representative WGS-ready checklist is available (Supplementary Information).
Based on the checklist responses, our partners undertook an Internal Process Review (IPR) to identify potential gaps in practical lab expertise. This exercise verified each lab’s familiarity with DNA isolation and quantification, PCR amplification, and sample transport between sites (all necessities upstream to sequencing). Supplementary Fig. 1 illustrates the IPR exercise.
Assembly
Considerations: WGS is characterized by specialized capital equipment and consumables and hence reinforces the need to build strong relationships with local suppliers to run a sustainable operation. The assembly phase leans heavily on experimental planning, and the procurement of equipment (Capex) and reagents/labware (Opex). The aim with the GHRU on Genomic Surveillance of Antimicrobial Resistance initiative was to assemble a cost-effective short-read WGS pipeline that could rapidly and routinely sequence genomes from Gram positive and negative bacterial species to a minimum coverage depth of 50x per nucleotide base position.
Challenges: Laboratories onboarding WGS for AMR surveillance need to carefully consider the affordability of both Capex and the recurring Opex. Emerging WGS territories are serviced by local distributors of the parent provider (sometimes called subsidiaries or channel partners). Here, product pricing is entirely demand-driven, poorly regulated, heavily taxed upon import, and controlled almost exclusively by the local distributor. This leads to highly inflated device and reagent costs [6]. Due to lower demand in our partner countries, we observed that distributors never manufactured locally and were often forced to deliver perishable stock that is closer to expiry. Fresh reagent stocks were only imported periodically with shipping delays, and usually had a longer turnaround time that, in turn, required laboratory managers to purchase months in advance. Paradoxically, we also found that LMIC labs such as our partner Units spent significantly more on Capex and Opex items than their counterparts in established NGS markets did. Additionally, after-sales support from equipment/reagent suppliers does not hit the expected level of standard, with severe delays in customer service and a lack of promptness and accuracy with technical troubleshooting.
Moreover, there are several sequencing platform types available to perform microbial genomics. Selecting the optimal technology depends on factors like eventual application, current and expected sample turnover, sequence read lengths, required depth of coverage, time to result, and the acceptable assay running cost.
Solutions: Mapping out essential Capex and Opex helps address process gaps and negotiate fit-for-purpose products from vendors. To accelerate time-to-purchase and instrument validation, we compiled a WGS Lab Toolkit of the main equipment, labware, and reagents that partner labs could use as a guide to navigate the plethora of procurement options (Supplementary Table 1). It is worth noting that this list represents just one of several ways to assemble a functional WGS suite – one that was well suited to our budget and applications.
Preferred suppliers (specially in LMICs) were chosen based on the following criteria:
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Technology relevance (best-in-class or equivalent).
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Ease of access, convenient format for routine use.
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Previous validation on bacterial isolates or bacterial WGS protocols.
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Low operational assay costs and maintenance premiums (if applicable).
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Global presence with reliable supply chains and delivery dispatch networks.
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Swift, efficient technical support and issue escalation that is regionally available.
Using the WGS-ready checklist, transformational changes were enforced upon the arrival of equipment. Supplementary Fig. 2 illustrates a representative reconfiguration of a laboratory to accommodate WGS alongside microbiology.
Optimization
Considerations: This phase involved refining lab protocols that would enable WGS of collected bacterial isolates (Fig. 1). This consisted of:
1) validation of instruments; 2) hands-on training in DNA isolation and quantification (if needed), library preparation and QC, initiation and assessment of a short-read Illumina sequencing run; and 3) development of standard operating procedures (SOP) or protocols for routine use.
Challenges: Practical WGS training courses offered online, at a university or as part of joint HIC-LMIC research initiatives, are generally organized within typical, non-challenging environments. For scientists in low- and middle-income countries, these are almost always organized abroad and cost a significant amount to attend. Most workshops go into great depth about specific techniques, yet they ignore key aspects in laboratory management. Methods are taught using an ‘ideal recipe sequence’ style that does not prepare beginners for real-world scenarios. We also therefore found that the newly acquired skills could not easily be transferred to one’s home laboratory upon return due to a different ambience and work dynamic. Moreover, courses organized on-site offered little value if instructors did not fully understand local logistical nuances and complexities before initiating training.
Generally, scientific journals do not enable authors to provide a detailed list of protocols alongside scientific work, which can make it challenging for users to replicate or introduce adjustments to published work. Additionally, we observed that a lack of standardized annotation, formulation, and archiving of SOPs led to poor reproducibility of results, both between labs and between operators within a lab.
Solutions: Once the equipment was fully validated, local on-site training could commence. Fully-integrated workshops were delivered free-of-charge at the partner’s laboratory site within the exact working environment and layout required. Hands-on coordination was needed to ensure the advanced preparation and timely arrival of the appropriate reagents, labware, and course handbooks. The instructor-trainee ratio was maintained at a maximum of 1:3 to encourage healthy levels of interaction and engagement. This also ensured that the instructor was able to stock each participant’s workstation adequately. Hands-on ‘recipe-style’ protocol repetition was blended with interactive discussions and coursework on the principles of WGS, current technology, and applications in clinical microbiology. This empowered participants with no prior sequencing experience to understand the benefits, limitations, and scalability of laboratory genomics in a public health context. Alongside technical competence, we trained team managers in project planning, lab biosafety, quality assurance, procurement, supply chain, SOP compilation, and sample data management.
The train-the-trainer model addressed the scalability of lab training through improved onward training coverage, thereby reducing operational costs [17, 27]. The aim was to work with a cohort of trainees knowledgeable about regional challenges to build technical proficiency in AMR and WGS techniques, and pedagogical skills to efficiently share expertise with neighboring or collaborating staff and researchers. The overall goal was to ensure organic growth of a network of regional trainers who will teach, mentor and share lessons with further audiences locally.
To enhance reproducibility and consistency of results across sites, a consolidated catalogue of optimized lab SOPs to perform bacterial WGS was made available on protocols.io [19, 20].
Reassessment
Considerations: Reviews were initiated 6–9 months after end-to-end WGS lab expertise was delivered. This allowed each lab to retrain, practice, and optimize methods taught during the optimization phase. Reassessment served to review and improve throughput of sample processing to meet any increase in demand, and to evaluate batch-to-batch genomic data quality, which in turn indicated the degree of training success.
Challenges: Firstly, when the demand for routine WGS grows, manual DNA and library preparation becomes increasingly expensive, time-consuming, and error-prone. Currently, staff are limited to processing smaller batches, due to restrictions imposed by lab equipment or benchtop sample degradation.
Secondly, quality assurance (QA) schemes are carried out extensively for microbiological laboratories that wish to challenge and accredit their protocols [13]. Previous attempts to recreate this for WGS have failed due to lack of resources and globally recognized QC standards for genomic data [18]. This is further complicated by the need to tightly control DNA extraction outputs, library preparation methods, and sequencing chemistry within a preferred platform.
Solutions: To address the first challenge, liquid-handling robotics help realize more efficient sample processing, while reducing consumable costs and turnaround times [21, 22]. It enables the processing of batch sizes about 2-3-fold larger in the same timeframe as protocols done by hand, while drastically improving batch to batch quality [21, 22]. A reassessment exercise often helps labs transform manual SOPs into automated workflows. Besides pipetting accuracy and versatility, we found that sustainable liquid handlers that addressed the challenges at resource-limited labs shared the following salient features: 1) Affordably priced hardware (with fixed pricing for most global regions); 2) Easily installable hardware and software setup; 3) Protocol development included within instrument cost; 4) Flexible, drag-and-drop API for method development; 5) Online repository of pre-validated methods scripts for DNA extraction, DNA quantification, PCR master mix preparation, WGS library construction, and DNA and library cherry-pick/dilution; 6) Self-repairable hardware and virtual service support (where possible); 7) Reagent/kit agnostic liquid dispensing; 8) Compatibility with low-cost, non-proprietary (generic) labware on deck.
To address the second challenge, we piloted an external QA exercise for bacterial whole genomes, which provided an independent verification of lab standards with a panel of blinded, phenotypically and genotypically well-characterized test isolates. This will be useful as WGS becomes more widely used for AMR surveillance. It is important that such QA schemes include traditional phenotypic ID/AST testing alongside wet/dry lab pathogen genome sequencing, to help maintain backwards compatibility between genomic results and traditional ‘gold-standard’ counterparts [4, 14, 15]. For wet lab, this includes reporting phenotypic AMR, quality metrics for DNA and library preparation, and performance metrics like pass filters and Q30 scores for the sequencing run.
WIJ Vignettes and Experiences with GHRU
A catalogue of laboratory setup challenges and solutions implemented as part of the GHRU project in Colombia, India, Nigeria, and the Philippines are documented in Supplementary Table 2.