Microsatellite Genotyping Protocol

Genotyping was carried out for a subset of samples (n = 254) by amplifying seven neutral microsatellite markers (TA1, PolyA, PfPK2, TA109, 2490, 313, and 383) and by determining length of each amplified marker. A semi-nested PCR was used to amplify five of the seven microsatellite markers (TA1, PolyA, PfPK2, TA109 and 2490), while a single round RCR was used to amplify the remaining two of the seven markers (313 and 383) using published primers and PCR conditions^{29 (link),30 (link)} . Fluorescent labelled PCR products were analysed on an ABI 3100 Genetic Analyzer sequencer (Applied Biosystems) to determine their length. Peak Scanner Software version 1.0 (Applied Biosystems, https://peak-scanner-software.software.informer.com/1.0/) was used to manually score peaks. A peak height > 300 relative fluorescence units (rfu) was considered as a positive peak^{11 (link)}. For samples producing more than one peak, the highest peak was defined as the dominant allele in the sample while minor peaks were defined as minor alleles if their peak heights were > 300 rfu and > 30% of the highest peak.

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Valdivia H.O., Anderson K., Smith D., Pasay C., Salas C.J., Braga G., Lucas C.M., Lizewski S.E., Joya C.A., Kooken J.M., Sanchez J.F, & Cheng Q. (2022). Spatiotemporal dynamics of Plasmodium falciparum histidine-rich protein 2 and 3 deletions in Peru. Scientific Reports, 12, 19845.

Publication 2022

ABI 3100 Genetic Analyzer sequencer Peak Scanner Software version 1.0

Alleles Fluorescence Genetic Microsatellite markers Nested pcr Polya Primers

Corresponding Organization :

Other organizations : Naval Medical Research Unit Six, QIMR Berghofer Medical Research Institute

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Variable analysis

independent variables

Microsatellite markers amplified (TA1, PolyA, PfPK2, TA109, 2490, 313, and 383)

dependent variables

Length of each amplified microsatellite marker

control variables

Published primers and PCR conditions
Fluorescent labeling of PCR products
Analysis on ABI 3100 Genetic Analyzer sequencer
Peak height threshold of 300 relative fluorescence units (rfu) for positive peaks
Definition of dominant allele as the highest peak and minor alleles as peaks > 300 rfu and > 30% of the highest peak

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