TY - JOUR
T1 - Efficient genotyping of Schistosoma mansoni miracidia following whole genome amplification
AU - Valentim, Claudia L.L.
AU - LoVerde, Philip T.
AU - Anderson, Timothy JC
AU - Criscione, Charles D.
N1 - Funding Information:
Supported by NIH R21 AI072704 (TJCA), SFBR Forum Grant (CDC), NIH Training Grant D43TW006580 (PTL), and NIH schistosome supply grant AI30026. The molecular work at the Southwest Foundation for Biomedical Research was conducted in facilities constructed with support from Research Facilities Improvement Program Grant C06 RR013556 from the National Center for Research Resources, National Institutes of Health. We thank the following: the Wellcome Trust Sanger Institute for providing the genome sequence and repeat masked sequence; Matt Berriman and Najib El-Sayed for genome support; and Guilherme Oliveira for supplying the LE line.
PY - 2009/7
Y1 - 2009/7
N2 - Small parasites and larval stages pose a problem for molecular analyses because limited amounts of DNA template are available. Isothermal methods for faithfully copying DNA have the potential to revolutionize studies of such organisms. We evaluated the fidelity of multiple displacement amplification (MDA) for amplifying DNA extracted from a single miracidium of Schistosoma mansoni. To do this we genotyped DNA extracted from 28 F1 miracidia following MDA using 56 microsatellite markers. Because these miracidia were obtained from a cross between a male and female worm of known genotypes, we were able to predict the alleles present in the progeny and quantify the genotyping error rate. We found just 8/1568 genotypes deviated from Mendelian expectations. Furthermore, because 1 of these resulted from a genuine mutation, the error rate due to MDA is 7/1568 (0.45%). We conclude that many hundreds of microsatellites or other genetic markers can be accurately genotyped from a single miracidium using this method, greatly expanding the scope of population genetic, epidemiological and evolutionary studies on this parasite.
AB - Small parasites and larval stages pose a problem for molecular analyses because limited amounts of DNA template are available. Isothermal methods for faithfully copying DNA have the potential to revolutionize studies of such organisms. We evaluated the fidelity of multiple displacement amplification (MDA) for amplifying DNA extracted from a single miracidium of Schistosoma mansoni. To do this we genotyped DNA extracted from 28 F1 miracidia following MDA using 56 microsatellite markers. Because these miracidia were obtained from a cross between a male and female worm of known genotypes, we were able to predict the alleles present in the progeny and quantify the genotyping error rate. We found just 8/1568 genotypes deviated from Mendelian expectations. Furthermore, because 1 of these resulted from a genuine mutation, the error rate due to MDA is 7/1568 (0.45%). We conclude that many hundreds of microsatellites or other genetic markers can be accurately genotyped from a single miracidium using this method, greatly expanding the scope of population genetic, epidemiological and evolutionary studies on this parasite.
KW - Mendelian
KW - Microsatellites
KW - Miracidia
KW - Multiple displacement amplification
KW - Schistosoma
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U2 - 10.1016/j.molbiopara.2009.02.010
DO - 10.1016/j.molbiopara.2009.02.010
M3 - Article
C2 - 19428677
AN - SCOPUS:64549116720
SN - 0166-6851
VL - 166
SP - 81
EP - 84
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -