CryptoGenotyper: A new bioinformatics tool for rapid Cryptosporidium identification

Abstract

Cryptosporidium is a protozoan parasite that is transmitted to both humans and animals through zoonotic or anthroponotic means. When a host is infected with this parasite, it causes a gastrointestinal disease known as cryptosporidiosis. To understand the transmission dynamics of Cryptosporidium , the small subunit (SSU or 18S) rRNA and gp60 genes are commonly studied through PCR analysis and conventional Sanger sequencing. However, analyzing sequence chromatograms manually is both time consuming and prone to human error, especially in the presence of poorly resolved, heterozygous peaks and the absence of a validated database. For this study, we developed a Cryptosporidium genotyping tool, called CryptoGenotyper, which has the capability to read raw Sanger sequencing data for the two common Cryptosporidium gene targets (SSU rRNA and gp60 ) and classify the sequence data into standard nomenclature. The CryptoGenotyper has the capacity to perform quality control and properly classify sequences using a high quality, manually curated reference database, saving users' time and removing bias during data analysis. The incorporated heterozygous base calling algorithms for the SSU rRNA gene target resolves double peaks, therefore recovering data previously classified as inconclusive. The CryptoGenotyper successfully genotyped 99.3% (428/431) and 95.1% (154/162) of SSU rRNA chromatograms containing single and mixed sequences, respectively, and correctly subtyped 95.6% (947/991) of gp60 chromatograms without manual intervention. This new, user-friendly tool can provide both fast and reproducible analyses of Sanger sequencing data for the two most common Cryptosporidium gene targets.

An Update on Zoonotic Cryptosporidium Species and Genotypes in Humans

“…For example, a recent study applied the bioinformatic program TIDE to deconvolute gp60 chromatograms generated using Sanger sequencing and identified previously unrecognised mixed subtype infections and has the advantage of also being able to be applied to retrospective data [ 229 ]. Similarly, another bioinformatics tool called “CryptoGenotyper” has been developed to read both 18S and gp60 Sanger sequence data, which can also resolve double peaks in mixed infections and increase the accuracy of sequence identification [ 230 ]. Amplicon next-generation sequencing (NGS), which can identify low-abundance sequences in mixed infections, has shown that it can identify additional Cryptosporidium gp60 subtypes not identified by Sanger sequencing in various hosts [ 141 , 231 ].…”

Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts

“…The comparison of glycoprotein 60 (GP60) gene fragments from this isolate with the IOWA strain showed about 87% identity. The CryptoGenotyper tool [ 17 ] classified the IOWA strain as subtype IIaA17G2R1, whereas the isolate from the Parasitology lab was subtype IId.…”

Zoonotic pathogens in wild muskoxen (Ovibos moschatus) and domestic sheep (Ovis aries) from Greenland

“…All chromatograms were manually inspected for the presence of double peaks indicating mixed species/subtypes. For Cryptosporidium , the CryptoGenotyper (Yanta et al., 2021 ), available on the web‐based platform Galaxy (Jalili et al., 2021 ), was used for initial Cryptosporidium genotyping and gp60 subtype were confirmed by manual identification of trinucleotide repeats as previously described (Chalmers et al., 2019 ).…”

“…All chromatograms were manually inspected for the presence of double peaks indicating mixed species/subtypes. For Cryptosporidium, the CryptoGenotyper(Yanta et al, 2021), available on the web-based plat-…”