Whole genome sequencing of Cryptosporidium from low DNA inputs without whole genome amplification

Authors: HSHIEH, KATHERINE - Oak Ridge Institute For Science And Education (ORISE); Molokin, Aleksey; Maloney, Jenny

Submitted to: Molecular Biology Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2025
Publication Date: 12/29/2025
Citation: Hshieh, K., Molokin, A., Maloney, J.G. 2025. Whole genome sequencing of Cryptosporidium from low DNA inputs without whole genome amplification. Molecular Biology Reports. 53. Article e225. https://doi.org/10.1007/s11033-025-11395-1.
DOI: https://doi.org/10.1007/s11033-025-11395-1

Interpretive Summary: Cryptosporidium is a single-celled parasite that causes diarrheal illness in humans and other animals and can be spread through food and water. Genomes can be used to develop new tools to understand how this parasite makes its way into food and water, which will help to prevent contamination of fresh produce. However, the amount of DNA needed for genome sequencing is difficult to obtain from most Cryptosporidium isolates. We tested three sequencing kits for genome sequencing from very small quantities of DNA. Two of the kits worked well under these conditions and could produce high quality genomes of Cryptosporidium even when DNA was undetectable. This research provides methods for sequencing high quality Cryptosporidium genomes from as few as 1,000 cells. These methods will benefit researchers by creating avenues for sequencing genomes from more isolates to produce the data needed to better study and control this important foodborne pathogen.

Technical Abstract: Cryptosporidium is a protozoan parasite that causes diarrheal illness in humans and other animals and has a low infectious dose. The development of new tools to study and mitigate Cryptosporidium infection is aided by access to a diverse library of genomic material. However, culture methods to amplify the quantity of cells available from an individual isolate are not available, so Cryptosporidium DNA often falls below the input threshold for many genome sequencing methods. Here, we assess the suitability of whole genome amplification (WGA) free methods of obtaining high quality whole genome sequences from low inputs of DNA from Cryptosporidium. Three commercially available library kits from Illumina, New England Biolabs (NEB), and Qiagen were compared. Testing was conducted on extractions from Cryptosporidium oocyst counts ranging from 1 million (n=1) to 1 oocyst (n=4 for 100,000 and n=8 for all other cell levels), representing DNA inputs ranging from approximately 5 ng to 0.0005 pg. Similar performance was observed for the Illumina and NEB library prep kits for which 99% coverage of the reference genome was obtained from isolates containing between 1 million and 1,000 oocysts. Because only 36% of the Qiagen preps produced successful libraries, sequencing was not performed on this group. This research indicates that high quality Cryptosporidium whole genome sequences can be generated from DNA extracted from as few as 1,000 oocysts, representing an estimated 5 pg of input DNA, without the need for WGA, creating new avenues for producing genomes from samples with limited DNA content.