Genomic Characterization of Cryptosporidium spp. via iNextEra Library Preparation and Hybridization Bait Capture.

Next-generation DNA sequencing (NGS) is used to study the genome sequences of Cryptosporidium spp., but NGS is challenging when pure Cryptosporidium oocysts are limited in number or not available. Varying levels of parasites present in fecal samples, combined with the abundance of host cells, bacterial and other microbial cells, and undigested food particles, often result in fecal DNA samples with ~0.1% Cryptosporidium DNA, making genome-scale sequencing of Cryptosporidium from such samples cost-prohibitive. DNA extractions from fecal samples are, however, widely available and commonly used for polymerase chain reaction (PCR)-based diagnostics which can detect fg levels of Cryptosporidium DNA in complex DNA mixtures. Here, we describe an Illumina NGS sample preparation protocol (iNextEra) that can generate libraries from a wide range of DNA input (<1 ng to >60 ng). We then use those libraries within a modified myBaits capture hybridization protocol using CryptoCap_100K baits to enrich Cryptosporidium genomic DNA from a complex DNA background to increase the percentage of generated sequence reads that map to target Cryptosporidium reference genome sequences. Thus, iNextEra libraries and capture hybridization facilitate genome-level sequencing of this critical pathogen from widely available samples with less cost, thereby opening new opportunities to understand the complex biology of this important pathogen.