Highly multiplex molecular inversion probe panel in Plasmodium falciparum targeting common SNPs approximates whole genome sequencing assessments for selection and relatedness

Karamoko Niaré^1,2*Rebecca Crudale¹Abebe Fola^1,2Neeva Wernsman Young²Victor Asua^3,4Melissa Conrad⁵Pierre Gashema⁶Anita Ghansah⁷Stan Hangi⁸Deus S Ishengoma⁹Jean-Baptiste Mazarati⁶Ayalew Jejaw Zeleke¹⁰Philip J Rosenthal⁵Abdoulaye A Djimdé^11,12Jonathan J Juliano^13,14,15Jeffrey A Bailey^1,6*

• ¹Department of Pathology and Laboratory Medicine, Division of Biology and Medicine, Brown University, Providence, United States
• ²Center for Computational Molecular Biology, Brown University, Providence, Rhode Island, United States
• ³Institute for Tropical Medicine, Travel Medicine, Human Parasitology, University Hospital and Faculty of Medicine, University of Tübingen, Tübingen, Baden-Württemberg, Germany
• ⁴Infectious Diseases Research Collaboration, Kampala, Uganda
• ⁵Department of Medicine, University of California, San Francisco, San Francisco, California, United States
• ⁶Center for Genomic Biology, Institut d’Enseignement Supérieur de Ruhengeri, Ruhengeri, Rwanda
• ⁷Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana
• ⁸Department of Pediatrics, HEAL Africa, Goma, Democratic Republic of Congo
• ⁹National Institute for Medical Research, Dar es Salaam, Tanzania; Department of Biochemistry, Kampala International University in Tanzania, Dar es Salaam, Tanzania
• ¹⁰Department of Medical Parasitology, School of Biomedical and Laboratory sciences, University of Gondar, Gondar, Amhara Region, Ethiopia
• ¹¹Pathogens genomics Diversity Network Africa, Imm. Gwancoura,, Sotuba, Bamako, Mali
• ¹²Malaria Research and Training Centre, University of Bamako, Bamako, Mali
• ¹³Institute for Global Health and Infectious Diseases, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
• ¹⁴Division of Infectious Diseases, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
• ¹⁵Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States

Introduction. The use of next-generation sequencing technologies (NGS) to study parasite populations and their response and evolution to interventions is important to support malaria control and elimination efforts. While whole genome sequencing (WGS) is optimal in terms of assessing the entire genome, it is costly for numerous samples.Targeted approaches selectively enriching for sequence of interest are more affordable and higher throughput but sometimes lack adequate information content for key analyses.. We have developed a highly-multiplexed molecular inversion probe (MIP) panel (IBC2FULL) targeting 4,264 single nucleotide polymorphisms (SNPs) with ≥ 5% minor allele frequency (MAF) in sub-Saharan African regions from publicly available Plasmodium falciparum WGS (n=3,693). We optimized the panel alone and in combination with antimalarial drug resistance MIPs in laboratory P. falciparum strains at different parasitemias, and validated it by sequencing field isolates from Democratic Republic of Congo, Ethiopia, Ghana, Mali, Rwanda, Tanzania and Uganda and evaluating population structure, identity-by-descent (IBD), signals of selection, and complexity of infection (COI) Results. The new panel IBC2FULL consisted of 2,128 MIPs (containing 4,264 common SNPs) spaced by 5.1 -18.4 kb across the entire genome. While these microhaplotypes were developed based on variation from sub-Saharan African WGS, 59.3% (2,529) of SNPs were also common in South-East Asia. The MIPs were balanced to produce more uniform and higher depth coverage at low parasitemia (100 parasites/μL) along with MIPs targeting antimalarial drug resistance genes. Comparing targeted regions extracted from public WGS, IBC2FULL provided higher resolution of local population structure in sub-Saharan Africa than current PCR-based targeted sequencing panels.Sequencing field samples (n=140), IBC2FULL approximated WGS measures of relatedness, population structure, and COI. Interestingly, genome-wide analysis of extended haplotype homozygosity detected the same major peaks of selection as WGS.We also chose a subset of 305 high performing MIPs to create a core panel (IBC2CORE) that produced high-quality data for basic population genomic analysis and accurate estimation of COI.Discussion. IBC2FULL and IBC2CORE provide an improved platform for malaria genomic epidemiology and biology that can approximate WGS for many applications and is deployable for malaria molecular surveillance in resource-limited settings.