ERP134512 PRJEB49966 a53e37c1-3dea-4d35-a6d5-0f92f2351d55 Deep kinetoplast genome analyses results in a novel molecular assay for detecting Trypanosoma brucei gambiense-specific minicircles The World Health Organization targeted Trypanosoma brucei gambiense (Tbg) Human African Trypanosomiasis for elimination of transmission by 2030. This is a challenging goal, partly because of the lack of sensitive molecular tools to reliably detect Tbg type 1 (Tbg1) infections in asymptomatic human carriers and/or a possible animal reservoir. Here, we aim at improving molecular diagnosis of Tbg1 infections by targeting the abundant mitochondrial minicircles within the kinetoplast of Trypanosoma brucei parasites. Using Next-Generation Sequencing of total cellular DNA extracts, we assembled and annotated the kinetoplast genome and investigated minicircle sequence diversity in 38 animal- and human-infective trypanosome strains. Computational analyses recognized a total of 241 Minicircle Sequence Classes as Tbg1-specific, of which three were shared by the 18 studied Tbg1 strains. We then developed a novel multiplex quantitative PCR assay (g-qPCR3) targeting one Tbg1-specific minicircle and three previously published Tbg1-specific or Trypanozoon-specific markers. Molecular analyses revealed that the minicircle-based assay is applicable on animals and is as specific as the TgsGP-based assay, the current golden standard for molecular detection of Tbg1. The median copy number of the targeted minicircle was equal to eight, suggesting that our minicircle-based assay may be used for the sensitive detection of Tbg1 parasites. Finally, annotation of the targeted minicircle sequence indicated that it encodes genes essential for the survival of the parasite, and will thus likely be preserved in natural Tbg1 populations. These results demonstrated that our minicircle-based assay is a promising new tool for reliable and sensitive detection of Tbg1 infections in humans and animals. Nuclear and mitochondrial genomics of Trypanosoma brucei parasites The World Health Organization targeted Trypanosoma brucei gambiense (Tbg) Human African Trypanosomiasis for elimination of transmission by 2030. This is a challenging goal, partly because of the lack of sensitive molecular tools to reliably detect Tbg type 1 (Tbg1) infections in asymptomatic human carriers and/or a possible animal reservoir. Here, we aim at improving molecular diagnosis of Tbg1 infections by targeting the abundant mitochondrial minicircles within the kinetoplast of Trypanosoma brucei parasites. Using Next-Generation Sequencing of total cellular DNA extracts, we assembled and annotated the kinetoplast genome and investigated minicircle sequence diversity in 38 animal- and human-infective trypanosome strains. Computational analyses recognized a total of 241 Minicircle Sequence Classes as Tbg1-specific, of which three were shared by the 18 studied Tbg1 strains. We then developed a novel multiplex quantitative PCR assay (g-qPCR3) targeting one Tbg1-specific minicircle and three previously published Tbg1-specific or Trypanozoon-specific markers. Molecular analyses revealed that the minicircle-based assay is applicable on animals and is as specific as the TgsGP-based assay, the current golden standard for molecular detection of Tbg1. The median copy number of the targeted minicircle was equal to eight, suggesting that our minicircle-based assay may be used for the sensitive detection of Tbg1 parasites. Finally, annotation of the targeted minicircle sequence indicated that it encodes genes essential for the survival of the parasite, and will thus likely be preserved in natural Tbg1 populations. These results demonstrated that our minicircle-based assay is a promising new tool for reliable and sensitive detection of Tbg1 infections in humans and animals. ENA-FIRST-PUBLIC 2022-01-12 ENA-LAST-UPDATE 2022-01-12