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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

ARTHUR KOCHER ,
SOPHIE VALIÈRE ,
ANNE-LAURE BAÑULS  and
JÉRÔME MURIENNE
ARTHUR KOCHER*
Affiliation:
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
SOPHIE VALIÈRE
Affiliation:
GeT–PlaGe, Genotoul, INRA Auzeville, 31326 Castanet-Tolosan, France
ANNE-LAURE BAÑULS
Affiliation:
UMR MIVEGEC, IRD 224 – CNRS 5290 – Université de Montpellier, F34394 Montpellier, France
JÉRÔME MURIENNE
Affiliation:
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: arthur.kocher@gmail.com
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Summary

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.

Keywords

DetectiondiagnosisIlluminaLeishmania Viannialeishmini primersNew WorldPCRsand fly
Type
Special Issue Article
Information
Parasitology , Volume 145 , Special Issue 5: Advances and challenges in barcoding of microbes, parasites, and their vectors and reservoirs , April 2018 , pp. 585 - 594
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Copyright © Cambridge University Press 2017 

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References

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