Single genome amplification and direct amplicon sequencing of Plasmodium spp. DNA from ape fecal specimens

Abstract

Conventional PCR followed by molecular cloning and sequencing of amplified products is commonly used to test clinical specimens for target sequences of interest, such as viral, bacterial or parasite nucleic acids. However, this approach has serious limitations when used to analyze mixtures of genetically divergent templates1-9. This is because Taq polymerase is prone to switch templates during the amplification process, thereby generating recombinants that do not exist in vivo 4. When amplicons are cloned prior to sequence analysis, the resulting sequences may also contain a substantial number of Tag-induced substitutions1-4. Finally, cloning of amplicons can lead to a non-proportional representation of sequences due to the re-sampling of only certain templates1-4. These confounders can be avoided by using single genome amplification (SGA) followed by direct sequencing of SGA amplicons1-5. While SGA is not required for many research applications, we have shown it to be essential for deciphering the diversification pathways of human and simian immunodeficiency viruses (HIV/SIV) in acute and chronic infection4-7, the detection of simian foamy virus (SFVCPZ) super-infection in wild-living chimpanzees8, and most recently, the molecular identification and characterization of Plasmodium spp. infections in wild-living apes9. Here, we describe SGA-direct amplicon sequencing of Plasmodium spp. DNA from ape fecal samples.