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High-throughput sequencing of kDNA amplicons for the analysis of Leishmania minicircles and identification of Neotropical species

Published online by Cambridge University Press:  16 November 2017

Laboratoire Evolution et Diversité Biologique, CNRS, Université Toulouse III Paul Sabatier, ENSFEA, IRD, UMR5174 EDB, Toulouse, France UMR MIVEGEC, IRD 224 – CNRS 5290 – Université de Montpellier, F34394 Montpellier, France
GeT–PlaGe, Genotoul, INRA Auzeville, 31326 Castanet-Tolosan, France
UMR MIVEGEC, IRD 224 – CNRS 5290 – Université de Montpellier, F34394 Montpellier, France
Laboratoire Evolution et Diversité Biologique, CNRS, Université Toulouse III Paul Sabatier, ENSFEA, IRD, UMR5174 EDB, Toulouse, France
*Corresponding author. Laboratoire EDB, 118 route de Narbonne, 31062 Toulouse CEDEX 9, France. E-mail:


Leishmania kinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitive Leishmania detection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization of Leishmania minicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18 Leishmania strains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis of Leishmania relationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications of Leishmania spp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications in Leishmania research.

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Copyright © Cambridge University Press 2017 

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High-throughput sequencing of kDNA amplicons for the analysis of Leishmania minicircles and identification of Neotropical species
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