Customer Spotlight
Supporting Malaria Genomic Surveillance with the CleanPlex® Malaria NGS Panel and Illumina MiSeq™ i100 Plus
Advancing Malaria Surveillance in Malawi
Genomic surveillance has become increasingly important for monitoring drug resistance and tracking pathogen evolution. Laboratories around the world are adopting targeted next-generation sequencing (NGS) workflows to support public health initiatives.
Recently, the Public Health Institute of Malawi implemented the Illumina MiSeq™ i100 Plus together with the Paragon Genomics CleanPlex® Malaria NGS Panel as part of a training and validation initiative focused on malaria genomic surveillance. Using dried blood spot (DBS) samples from previous malaria indicator surveys, the team evaluated workflow performance while training researchers on sequencing and downstream analysis workflows.
1. Research focus and specific goals
Focus: Improving the Malaria surveillance using targeted amplicon genotyping of Plasmodium falciparum resistance and diagnostic loci using a CleanPlex-derived panel on an Illumina MiSeq i100.
Goals: To use the training opportunity and the new Illumina Miseq i100 and Paragon Genomics CleanPlex Malaria NGS Panel to identify and classify nucleotide changes at key resistance positions, quantify locus coverage, and produce reproducible, shareable bioinformatics outputs for routine surveillance.
Malawi had no machine capable of genomically analyse the Plasmodium species that cause high morbidity and mortality in sub Saharan African countries. The rescue came from……. who funded the procurement of an Illumina MiSeq i100, which we used, and the idea of randomly selecting a Dry Blood Spot sample from the biobank of the Public Health Institute of Malawi to test its function. The primary objective was to identify and classify nucleotide changes at key resistance positions, quantify locus coverage, and produce reproducible, shareable bioinformatics outputs for routine comparison with the manufacturer of the Paragon kit and the Illumina MiSeq i100. The objective was successfully achieved by the quality training that was offered by Separations Company. The training was conducted in 5 days including theory and practical sessions.
2. Experience using the Paragon Genomics CleanPlex Malaria NGS Panel
Overall experience: The team used the MAD4HaTTeR assay, a CleanPlex-derived modular amplicon panel, to amplify resistance and diversity targets prior to MiSeq sequencing, and the workflow integrated well with existing extraction and QC steps.
The MAD4HaTTeR assay, a CleanPlex-derived modular amplicon panel, was used to amplify resistance and diversity targets before high-throughput sequencing on an Illumina MiSeq i100.
MAD4HaTTeR is a modular assay. Samples can be processed with either or both of the two modules: Diversity or Resistance+.
3. Ease of use and standout aspects versus other library-prep kits
Ease of use: SOP-driven and modular, with adjustable mPCR cycles by parasitaemia and clear QC decision thresholds (bead clean ups, gel purification guidance). The modular design (Resistance vs Diversity modules) and explicit SOP thresholds stood out compared with more monolithic kits.
4. Biggest challenges or limitations before using this library-prep kit
Prior challenges: The only challenge noted originated from technical point. We lacked metadata to link the results to the original samples. For the reagents and machines were good performing.
5. How the kit improved research outcomes
Improvements: produced high coverage across targets, robust read depths at canonical resistance positions, and standardized, reproducible outputs (VCFs, codon tables, resistance summaries) that promises to support routine surveillance and reanalysis.
6. Panel performance on the MiSeq i100
Performance: libraries sequenced on the Illumina MiSeq i100 yielded robust per-locus read depths (from hundreds to tens of thousands), giving confidence in calls at canonical resistance codons when QC thresholds were met.
Final pooled libraries were normalised by molarity and sequenced on an Illumina MiSeq i100 using paired-end amplicon chemistry.
Read depths supporting these Alternative calls ranged from the low hundreds to tens of thousands, indicating robust coverage for most loci.
The integrated laboratory and computational workflow… produced high coverage, reproducible amplicon data suitable for routine molecular surveillance.
The dataset also demonstrates that an Illumina MiSeq i100, MAD4HaTTeR-based amplicon approach, combined with rigorous library QC and reproducible bioinformatics (Malaria Profiler on Terra), yields high-coverage, actionable variant calls at key antimalarial resistance loci.
7. Plans to continue using the Paragon CleanPlex Malaria NGS Panel and goals
Yes. Planned uses include routine molecular surveillance, periodic resequencing to monitor allele-frequency trends, and follow-up work to add haplotype phasing and phenotype correlation so resistance markers can be linked to outcomes.
Recommended next steps include haplotype phasing and linkage analysis to determine co-occurrence of resistance markers, phenotypic correlation with susceptibility or clinical outcomes where available, and periodic resequencing to monitor allele frequency trends. Another future goal is to use the malaria surveillance as surveillance as a tool to monitor transmission across borders of Malawi and the introduction of the new mutation.
8. Recommendation to other researchers
Recommendation: Yes. The panel and workflow deliver high-coverage, reproducible, actionable variant calls at key antimalarial resistance loci and integrate with containerised bioinformatics for reproducibility, making it suitable for surveillance programs.
Objectives
The project focused on the following objectives:
- Validation and verification of the Illumina MiSeq i100 Plus platform
- Training researchers on targeted malaria sequencing workflows
- Identification of resistance-associated nucleotide variants
- Generation of reproducible genomic surveillance outputs
- Establishment of scalable workflows for future surveillance programs
According to the team, the project combined practical and theoretical training sessions over five days while integrating laboratory and bioinformatics workflows into a unified surveillance pipeline. The training was delivered by Separations, Paragon Genomics’ regional distributor, whose hands-on guidance was instrumental in enabling the team to implement the full workflow from library preparation through bioinformatics analysis.
Interested in Learning More?
Explore our infectious disease sequencing solutions or contact us to discuss custom panel development for your surveillance program.
Why the CleanPlex® Malaria NGS Panel?
The team highlighted many advantages of using the CleanPlex® Malaria NGS Panel.
One major benefit was the modular MAD4HaTTeR assay design, which allowed users to separately process resistance and diversity targets depending on project needs. Rather than forcing every target into a single workflow, researchers had more control over how they approached different sequencing goals.
The researchers also noted that the workflow was highly user-friendly with relatively short hands-on time compared to other library preparation methods.
Standout Features Included:
- Modular assay architecture
- Flexible target selection
- Streamlined workflow
- High sequencing coverage
- Reproducible outputs
- Easy integration into surveillance pipelines
Performance on the MiSeq™ i100 Plus
Normalized pooled libraries were sequenced using paired-end amplicon chemistry on the MiSeq i100 Plus platform. The team reported:
- Excellent sequencing QC metrics
- Strong Q30 performance
- Robust per-locus read depth
- High coverage across resistance loci
- Sequencing runtimes of approximately 7 hours
The researchers also highlighted the ability to generate standardized and reproducible outputs, including:
This integration of laboratory and computational workflows supported routine malaria surveillance applications.
Challenges Associated with Malaria Genomic Surveillance
Before implementing the workflow, there were challenges associated with malaria genomic surveillance:
- Long and error-prone library preparation workflows
- High consumable costs
- Complex sample processing
- Limited sequencing standardization
- Difficulty generating reproducible surveillance outputs
While whole genome sequencing (WGS) provides broader genomic diversity analysis, targeted sequencing workflows can provide a much more focused and scalable solution for routine surveillance applications.
Future Applications and Surveillance Goals
Following the success of the training and validation effort, the group plans to continue using the CleanPlex® Malaria NGS Panel for future surveillance and research initiatives.
Planned applications include:
- Monitoring allele frequency trends
- Tracking resistance-associated mutations
- Cross-border transmission surveillance
- Haplotype phasing studies
- Correlation of resistance markers with clinical outcomes
- Expanded pathogen surveillance programs
The team also indicated plans to pursue additional funding for larger-scale surveillance and advanced downstream analyses.
Supporting Scalable Pathogen Genomics
The researchers strongly recommended the combination of the Illumina MiSeq i100 Plus and the CleanPlex® Malaria NGS Panel for malaria and broader pathogen surveillance applications.
According to the team, the workflow delivers:
- High-coverage sequencing
- Reproducible variant calls
- Actionable resistance profiling
- Scalable targeted sequencing workflows suitable for research and surveillance environments
CleanPlex® Infectious Disease Panels
Paragon Genomics continues to support infectious disease surveillance through highly multiplexed targeted sequencing workflows optimized for:
- Malaria surveillance
- Pathogen genomics
- Drug resistance monitoring
- Public health sequencing
- Custom infectious disease panels
- Research and translational genomics