Next Generation Sequencing uncovers within-host differences in the genetic diversity of Cryptosporidium gp60 subtypes

Abstract

The extent of within-host genetic diversity of parasites has implications for our understanding of the epidemiology, diseasese verity and evolution of parasite virulence. As with many other species, our understanding of the within-host diversity of the enteric parasite Cryptosporidium is changing.The present study compared Sanger and Next Generation Sequencing of glycoprotein 60 (gp60) amplicons from Cryptosporidium hominis (n = 11), Cryptosporidium parvum (n = 22) and Cryptosporidium cuniculus (n = 8) DNA samples from Australia and China. Sanger sequencing identified only one gp60 subtype in each DNA sample: one C. hominis subtype (IbA10G2) (n = 11), four C. parvum subtypes belonging to IIa (n = 3) and IId (n = 19) and one C. cuniculus subtype (VbA23) (n = 8). Next Generation Sequencing identified the same subtypes initially identified by Sanger sequencing, but also identified additional gp60 subtypes in C. parvum and C. cuniculus but not in C. hominis, DNA samples. The number of C. parvum and C. cuniculus subtypes identified by Next Generation Sequencing within individual DNA samples ranged from two to four, and both C. parvum IIa and IId subtype families were identified within the one host in two samples. The finding of the present study has important implications for Cryptosporidium transmission tracking as well as vaccine and drug studies.

Detection of classic and cryptic Strongyloides genotypes by deep amplicon sequencing: A preliminary survey of dog and human specimens collected from remote Australian communities

“…This method has great advantages over previous genotyping techniques in that it can be undertaken directly from faecal DNA extracts and does not require culture of larvae for extraction of DNA from individual worms. Furthermore, the depth of sequencing provided by NGS allows the detection of all genotypes in a single sample [31]. However, it is also worth mentioning that no information about the genotype of individual worms is obtained using this method.…”

Detection of classic and cryptic Strongyloides genotypes by deep amplicon sequencing: A preliminary survey of dog and human specimens collected from remote Australian communities

“…This method has great advantages over previous genotyping techniques in that it can be undertaken directly from faecal DNA extracts and does not require culture of larvae for extraction of DNA from individual worms. Furthermore, the depth of sequencing provided by NGS allows the detection of all genotypes in a single sample (Zahedi et al, 2017).…”

Surveillance of Giardia and Cryptosporidium in sewage from an urban area in Brazil

“…Limiting the target sequences before testing the samples by PCR-RFLP or by PCR amplification followed by sequencing, would help, however, these molecular assays require many replicates and may result yet in extremely mixed patterns or overlapping peaks in chromatograms and also lack of amplification due to random variations of the template (RUECKER et al, 2005(RUECKER et al, , 2011SPANAKOS et al, 2015). Next generation sequence is also a promising approach to better understand the diversity of Cryptosporidium species within a raw sewage sample, in Australia NGS revealed a huge diversity of species in raw sewage samples, nevertheless, this tool requires a highly technified staff and in most places is an expensive tool (ZAHEDI et al, 2017).…”

“…Considering the environment, the characterization of these pathogens in sewage may provide insight on their occurrence and prevalence in areas without a host-based surveillance (SPANAKOS et al, 2015;XIAO et al, 2001). Nevertheless, methods able to elucidate species mixtures within a sample are necessary as the standard PCR follow sequencing only diagnostic the most abundant species (ZAHEDI et al, 2017).…”