Polymorphisms and ambiguous sites present in DNA sequences of Leishmania clones: looking closer.
In genetic studies of Leishmania parasites, co-dominant markers are chosen for their ability to detect heterozygous polymorphisms, to infer the occurrence of inbreeding and to resolve genetic variability. The majority of DNA sequence based reports perform conventional dye terminator cycle sequencing where perfectly ambiguous sites or double peaks in the chromatogram are interpreted as heterozygous strains. However, molecular peculiarities of the parasite such as aneuploidy, mixed populations and homologous recombination advise that data from regular DNA sequence analysis should be carefully evaluated. We report here a closer look at ambiguous sites observed in 6pgd DNA sequences obtained for a multilocus sequence analysis project on Leishmania (Viannia) strains. After comparing 286 DNA sequences from biological and molecular clones of six L. (Viannia) strains we could distinguish events that contribute to genetic variation in Leishmania (recombination, mutation, chromosomal mosaics). Also, the results suggest how diversity might not be completely revealed through regular DNA sequence analysis and demonstrate the importance for molecular epidemiology research to be aware of such possibilities while choosing samples for studies.
Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. Electronic address: boitemc@ioc.fiocruz.br.
Mariana Côrtes
MC
Boité
Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil.
Taíse Salgado
TS
de Oliveira
Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil.
Gabriel Eduardo Melim
GE
Ferreira
Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil.
Marcos
M
Trannin
Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil.
Barbara Neves
BN
dos Santos
Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil.
Renato
R
Porrozzi
Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil.
Elisa
E
Cupolillo
D016054:DNA, Protozoan / Q000235:genetics*; D019143:Evolution, Molecular; D014644:Genetic Variation; D007891:Leishmania / Q000145:classification / Q000235:genetics*; D058885:Multilocus Sequence Typing; D010802:Phylogeny; D011110:Polymorphism, Genetic; D015800:Protozoan Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA
D016054:DNA, Protozoan; D015800:Protozoan Proteins
Aneuploidy; Heterozygosis; Leishmania; Polyclonal