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Bulk Segregant Analysis (BSA)

Diagram outlining a genetic cross experiment. Gametocytes are mosquito-fed to an Anopheline mosquito. Sporozoites are then injected into a FRG huHep mouse. A graph shows the drug concentration response in a resistant parent. Below, recombinant progeny pools are displayed as blue and gold bars, separated into control and drug-treated groups. Panel D depicts pooled sequencing and QTL identification before and after treatment, showing changes in the proportion of the P1 allele across genome locations.

Cloning and phenotyping hundreds of recombinant progeny in order to obtain reliable quantitative trait loci (QTL) can be incredibly laborious. The MCC was the first to perform bulk segregant analysis (BSA) in P. falciparum. Through this process, uncloned bulk recombinant pools of parasites can be exposed to any condition of interest (drug exposure, nutrient limitation, etc.) and then whole genome sequenced. Since this sequence is a readout of hundreds of different recombinant parasites, we can determine the average allele inheritance at any given position in the genome. When the average inheritance patterns are compared between treated and untreated pools, we can identify significant loci associated with our phenotype of interest. We have not only identified previously validated drug resistance markers, pfcrt and kelch13, but also uncovered minor loci which further drive our phenotype of interest. We have already successfully used BSA to identify loci which contribute to CQ resistance, ART resistance, nutritional requirements, and allele selection at various stages in the parasite lifecycle across all 3 hosts.

Publications

Li X, Kumar S, McDew-White M, Haile M, Cheeseman IH, et al. (2019) Genetic mapping of fitness determinants across the malaria parasite Plasmodium falciparum life cycle. PLOS Genetics 15(10): e1008453. https://doi.org/10.1371/journal.pgen.1008453

Amambua-Ngwa A, Button-Simons KA, Li X, Kumar S, Brenneman KV, Ferrari M, Checkley LA, Haile MT, Shoue DA, McDew-White M, Tindall SM, Reyes A, Delgado E, Dalhoff H, Larbalestier JK, Amato R, Pearson RD, Taylor AB, Nosten FH, D'Alessandro U, Kwiatkowski D, Cheeseman IH, Kappe SHI, Avery SV, Conway DJ, Vaughan AM, Ferdig MT, Anderson TJC. Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1. Nat Microbiol. 2023 Jul;8(7):1213-1226. doi: 10.1038/s41564-023-01377-z. Epub 2023 May 11. PMID: 37169919; PMCID: PMC10322710.

Button-Simons KA, Kumar S, Carmago N, Haile MT, Jett C, Checkley LA, Kennedy SY, Pinapati RS, Shoue DA, McDew-White M, Li X, Nosten FH, Kappe SH, Anderson TJC, Romero-Severson J, Ferdig MT, Emrich SJ, Vaughan AM, Cheeseman IH. The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice. Commun Biol. 2021 Jun 14;4(1):734. doi: 10.1038/s42003-021-02210-1. PMID: 34127785; PMCID: PMC8203791.

Kumar S, Li X, McDew-White M, Reyes A, Delgado E, Sayeed A, Haile MT, Abatiyow BA, Kennedy SY, Camargo N, Checkley LA, Brenneman KV, Button-Simons KA, Duraisingh MT, Cheeseman IH, Kappe SHI, Nosten F, Ferdig MT, Vaughan AM, Anderson TJC. A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media. Front Cell Infect Microbiol. 2022 May 30;12:878496. doi: 10.3389/fcimb.2022.878496. PMID: 35711667; PMCID: PMC9197316.

Brenneman KV, Li X, Kumar S, Delgado E, Checkley LA, Shoue DA, Reyes A, Abatiyow BA, Haile MT, Tripura R, Peto T, Lek D, Button-Simons KA, Kappe SHI, Dhorda M, Nosten F, Nkhoma SC, Cheeseman IH, Vaughan AM, Ferdig MT, Anderson TJC. Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice. iScience. 2022 Mar 16;25(4):104095. doi: 10.1016/j.isci.2022.104095. PMID: 35372813; PMCID: PMC8971943.