Untitled

Abstract

Background: Recombinase Polymerase Amplification (RPA) is a relatively new isothermal methodology for amplifying DNA. RPA is similar to traditional PCR in that it produces an amplicon that is defined by the annealing of two opposing oligonucleotide primers. However, while PCR relies on repeated heating and cooling cycles to denature and amplify DNA fragments, RPA is performed at a single moderate temperature and uses enzymatic activity to drive amplification. While RPA is commonly used in fieldbased monitoring of pathogens, it is unknown whether RPA is a viable alternative to PCR for the amplification of ancient DNA. Methods: In this study, PCR and RPA were used to amplify shotgun and mitochondrial DNA enriched libraries made from extracts from four ancient bison bone samples. Sequencing data from the amplified libraries were examined for biases in sequence content (read length and GC content), fraction of unique reads mapping to a reference sequence, and mitochondrial polymorphisms detection accuracy. Results: In comparison to PCR, RPA had a variable effect on sequence content, except in the mitochondrial DNA enriched libraries where RPA consistently reduced mean read length by approximately 30 bp. RPA increased the number of unique shotgun reads that mapped to a cattle nuclear reference by between 9% and 99% versus PCR. In contrast, RPA reduced the fraction of unique mitochondrial DNA enriched reads by > 26%, possibly due to the preferential amplification of small unmappable molecules.Both RPA and PCR data allowed the identification of similar variants in mitochondrial DNA enriched libraries, suggesting that the accuracy of the two amplification methods is comparable. Importantly, RPA was able to generate sequencing libraries at approximately a sixth of the cost of PCR. These results indicate that RPA is a viable alternative to PCR for amplification of shotgun libraries made from ancient DNA but may not be suitable for all ancient DNA applications.