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AI summary · DOI: 10.1038/s41467-026-78927-6 · Immunology and Microbiology

Phylogenomics of captured Cryptosporidium sequenced directly from patient samples identifies clade-specific genomic architecture and host-adapted lineages

Eliza V.C. Alves‐Ferreira (National Institutes of Health, National Institute of Allergy and Infectious Diseases), A. Khan (National Institutes of Health, National Institute of Allergy and Infectious Diseases), M. D. Tettey (National Institutes of Health, National Institute of Allergy and Infectious Diseases), Fumiaki Ihara (National Institutes of Health, National Institute of Allergy and Infectious Diseases), H. Vogel (National Institutes of Health, National Institute of Allergy and Infectious Diseases), S. Botchie (University of Ghana, Noguchi Memorial Institute for Medical Research), I. Ayi (University of Ghana, Noguchi Memorial Institute for Medical Research), Mattie Christine Pawlowic (University of Dundee, Wellcome Centre for Anti-Infectives Research),
and 4 more authorsGuy Robinson (Singleton Hospital, Swansea University), RM Chalmers (Singleton Hospital, Swansea University), Hernán Lorenzi (National Institutes of Health, National Institute of Allergy and Infectious Diseases), Michael E. Grigg (National Institutes of Health, National Institute of Allergy and Infectious Diseases)
Original paper Published on 24 Oct. 2026 Nature Communications License: CC BY

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The essentials

A capture-sequencing method recovers Cryptosporidium genome-wide data directly from stool and identifies three C. ubiquitum lineages with distinct host associations.

Question

Can target-enrichment sequencing recover useful whole-genome data from Cryptosporidium in clinical stool without purifying oocysts, including at low parasite abundance? Can the resulting genomic data clarify population structure, mixed infection, and host associations in C. ubiquitum?

Method

The authors designed 74,973 RNA baits covering about 92% of the C. parvum genome and tested capture sequencing on artificial mixtures and human and animal stool samples. They used a dual-reference remapping strategy for C. ubiquitum and applied SNP-based phylogenetic, admixture, and mixed-infection analyses; 21 of 26 C. ubiquitum samples yielded sufficient DNA, and 28,038 high-quality SNPs supported the population analysis.

Findings

  • Result The capture method recovered genome-wide C. parvum reads from artificial samples containing about 25 oocysts, although coverage was low.
  • Result The dual-reference mapping strategy reduced chimeric reads and improved alignment consistency for C. ubiquitum.
  • Result Genome-wide analysis identified three C. ubiquitum groups, with Group A observed only in human isolates, Group C in humans and animals, and Group B showing evidence of admixture.
    See the passage in the paper
    Notably, Group A was exclusively observed in human-derived isolates, whereas Group C included both human and animal isolates, and Group B displayed evidence of recombination between these groups, suggesting distinct transmission ecologies among lineages.
  • Authors’ interpretation Group-specific variants were regionally clustered, a pattern the authors interpret as consistent with linked haplotypes rather than independent mutations.

Limitations

  • The bait design did not target gene-poor telomeric and subtelomeric regions, limiting coverage of those genomic areas.
  • The host-adaptation interpretation requires broader sampling and functional work.
    See the passage in the paper
    Whether they are involved in host adaptation will require expanded sampling and functional analyses.
  • The study notes that the effect of storage conditions on sequencing performance remains to be investigated.
    See the passage in the paper
    The potential effects of sample storage conditions (e.g., freezing or ethanol preservation) on TES-Seq performance are variable and need to be further investigated.

Why it matters

The authors argue that direct capture sequencing enables high-resolution Cryptosporidium population studies from stool without oocyst isolation, including archived clinical material. Their findings provide a basis for investigating zoonotic transmission, host adaptation, and genome-wide associations, while highlighting the importance of controlling mapping artifacts when references are genetically distant.

Written by AI from the paper. Passages are checked word for word against the text we analysed; the rest may contain errors.

AI-generated keywords: Cryptosporidium; target-enrichment sequencing; mixed infections; C. ubiquitum; host-associated genetic structure

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  • Comprehensive Summary
  • Layman's Summary
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  • Original abstract
Cryptosporidium is a major cause of severe, persistent diarrhea in young children. Whole-genome studies have been hindered, however, by the difficulty of recovering sufficient parasite DNA from stool, particularly when parasite numbers are low. This study evaluated whether target-enrichment sequencing could generate useful genome-wide data directly from clinical samples, without first purifying oocysts, and help clarify mixed infections and host-associated genetic structure, especially in the divergent species *C. ubiquitum*. The authors developed CryptoCap_75K, a set of 74,973 RNA baits targeting gene-rich regions of the *C. parvum* genome. The baits cover approximately 92% of the reference genome, including its coding regions, and were used to enrich parasite DNA from stool extracts before Illumina sequencing. In artificial mixtures, the method achieved roughly 1,500-fold enrichment. Under the tested sequencing conditions, about 250 *C. parvum* oocysts (0.01 ng of DNA; qPCR cycle threshold [CT] near 24) produced at least 38-fold coverage. Parasite reads were also recovered from mixtures containing an estimated 25 oocysts, but coverage was only about 3-fold, making these data less suitable for high-confidence analyses. The *C. parvum*-derived baits also captured DNA from other species, including *C. hominis*, *C. meleagridis*, and *C. ubiquitum*. The team tested the method on human and animal stool samples, including archived samples that had been frozen or preserved in ethanol. Among 115 samples from Giardia-positive adults in Colombia, Ecuador, and Egypt who had no gastrointestinal symptoms, four were positive for Cryptosporidium; one low-signal sample was excluded from sequencing. Additional clinical samples came from Ghana and the United Kingdom. In total, 32 selected human and animal samples yielded high-quality sequences. Lower CT values generally predicted more mapped reads and greater genome coverage, although sample-specific factors also affected sequencing success. In comparisons involving *C. parvum*, *C. hominis*, and *C. meleagridis*, captured genomes clustered with published genomes in phylogenetic analyses, supporting the method’s ability to recover species-level relationships. In a deliberately mixed sample containing all three species, DEploid estimated proportions of 43% *C. parvum*, 50% *C. hominis*, and 7% *C. meleagridis*. In contrast, analysis of the clinical datasets identified one dominant haplotype in each sample, with no evidence of mixed infection. The genetic distance between *C. ubiquitum* and the *C. parvum* reference presented a major analytical challenge. Direct mapping produced problematic soft-clipped and chimeric reads that could distort variant calls and phylogenetic relationships. To address this, the authors used a dual-reference remapping strategy: reads that mapped poorly to *C. parvum* were aligned to a *C. ubiquitum* reference and then returned to *C. parvum* coordinates for analysis. Applying this strategy to 21 *C. ubiquitum* isolates—14 from humans and seven from sheep—identified 28,038 high-quality SNPs and three genetic groups. Groups A and B contained only human-derived isolates, whereas Group C included isolates from both humans and sheep and carried the gp60 subtype XIIa, which has previously been associated with zoonotic transmission. Group B showed evidence of admixture between the other groups. Group-specific variants were unevenly distributed across the genome, forming clusters; some Group C variants were nonsynonymous and occurred in genes without functional annotation. The authors conclude that capture sequencing can support genome-level investigation of Cryptosporidium directly from low-burden and archived stool samples.
Created on 30 Oct. 2026

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