Identification and subtyping of Cryptosporidium spp. using Nanopore sequencing

Cryptosporidium is a parasite that causes gastrointestinal issues such as diarrhoea and stomach pain. The main transmission routes are through contaminated water or food, between humans and from animal to humans. Cryptosporidium infects through oocysts which contain four sporozoites that releases when entering a host and can continue to breed inside the body. Cryptosporidium can cause massive outbreaks if established in a water source used for drinking water. To prevent and detect an outbreak it´s important to trace the transmission back to the source. The GP60-gene is used to identify and subtype Cryptosporidium and is a very useful tool during contact tracing. The purpose of this study was to identify species and subtype of Cryptosporidium using nanopore sequencing. In this study the GP60-gene was amplified using a Nested PCR protocol and then sequenced using nanopore sequencing. The sequences acquired where then used to make a search in BLAST to identify the species. The GP60 subtyping method uses the hypervariable region on the GP60-gene. A series of tandem repeats are used to identify the subtype. In this study seven positive Cryptosporidium faeces samples were amplified and sequenced. Nanopore sequencing was possible for five of the seven samples with C. parvum identified in four of these samples. Targeting the GP60-gene to determine species and subtype works well for the most common human pathogen species of Cryptosporidium. Further optimization is required before the method can be implemented för diagnostic use.