Quantifying Replication Slippage Error in Cryptosporidium Metabarcoding Studies

Genetic variation in Cryptosporidium, a common protozoan gut parasite in humans, is often based on marker genes containing trinucleotide repeats, which differentiate subtypes and track outbreaks. However, repeat regions have high replication slippage rates, making it difficult to discern biological diversity from error. Here, we synthesized Cryptosporidium DNA in clonal plasmid vectors, amplified them in different mock community ratios, and sequenced them using next-generation sequencing to determine the rate of replication slippage with dada2. Our results indicate that slippage rates increase with the length of the repeat region and can contribute to error rates of up to 20%. DNA repeat regions differentiate subtypes and track Cryptosporidium outbreaks but have high replication slippage rates. Using synthesized DNA, we demonstrate that slippage rates increase with the length of repeat region and can contribute to error rates of up to 20%.