Duplex PCR-Nanopore sequencing assay for Cryptosporidium species and subtype determination

The protozoan parasite Cryptosporidium is a global cause of gastrointestinal disease and cryptosporidiosis outbreaks are common. Current widely used methods for Cryptosporidium species and subtype determination, i.e., analysis of the small subunit ribosomal RNA (ssu RNA) and the 60 kDa glycoprotein (gp60) using Sanger sequencing, are complex and labor intensive. As such, developing a more rapid, accurate, automated and cost-effective method for species and subtype determination is desired. In this study, we describe the design of a new method for rapid simultaneous PCR-amplification of the ssu rRNA and gp60 loci followed by Nanopore sequencing. Species and subtypes assessed by Sanger sequencing, were successfully detected using Nanopore sequencing with 95 % concordance. Further, sequence similarity was assessed by comparing ssu rRNA and gp60 sequences attained from the two methods. Minor differences between the two methods in regards to ssu rRNA sequences were detected, however these differences did not affect species determination. In regards to the gp60 gene, all sequences were identical. Overall, duplex PCR followed by Nanopore sequencing correlated well with Sanger sequencing, which is commonly used for Cryptosporidium species and subtype determination. As such, this newly developed method will replace Sanger sequencing at the Public Health Agency of Sweden.