The phylum Apicomplexa represents a diverse group of obligate parasitic protists, comprising over 6000 described species with countless others awaiting characterization, many of which hold significant medical and veterinary importance (Morrison, 2009, Votýpka et al., 2016, Oberstaller et al., 2021). Notable pathogens include malaria-causing Plasmodium spp. (Haemospororida: Plasmodiidae), responsible for substantial annual mortality (Venkatesan, 2025); Toxoplasma gondii (Eucoccidiorida: Sarcocystidae), infecting approximately one-third of the global human population (Dubey, 2021); and Eimeria spp. (Eucoccidiorida: Eimeriidae), which cause economically devastating poultry and livestock coccidiosis (Bangoura and Bardsley, 2020, Blake et al., 2020, Mathis et al., 2025). The mitochondrial genomes (mitogenomes) of these parasites have garnered attention not only as potential drug targets (Mather et al., 2007, Goodman et al., 2017, Collier et al., 2025) due to their prokaryotic origins and essential functions but also as critical loci for studying speciation, evolutionary relationships, and ecological diversity (Karadjian et al., 2016, Hikosaka et al., 2010, Hikosaka et al., 2011b, Hikosaka et al., 2012, Hayakawa et al., 2012, Morgan and Godwin, 2017, Pacheco et al., 2018b, Pacheco et al., 2022, Léveillé et al., 2019, Kruth et al., 2020).

Apicomplexan mitogenomes display remarkable structural diversity. They typically encode three protein-coding genes (PCGs): cytochrome c oxidase subunits I and III (COI and COIII) and cytochrome b (COB), along with highly fragmented ribosomal RNA genes (rRNAs) dispersed throughout the genome, while lacking transfer RNA genes entirely (Pino et al., 2010, Hikosaka et al., 2013, Feagin et al., 2012, Berná et al., 2021, Collier et al., 2025, Kruth et al., 2025). Some taxa, such as Cryptosporidium spp., have undergone extreme reduction to form mitosomes devoid of genomic content (Mathur et al., 2021, Collier et al., 2025), whereas others like T. gondii exhibit complex, multi-molecule architectures with 21 repetitive sequence blocks (Namasivayam et al., 2021, Tetzlaff et al., 2024). Among characterized forms, monomeric linear mitogenomes (ranging from ∼6.6 kb to ∼11.1 kb) dominate in Piroplasmida (Hikosaka et al., 2010, Hikosaka et al., 2012, Hikosaka et al., 2013, Schreeg et al., 2016), whereas circular-mapping concatemers, termed the “6.2 kb element” in Eimeriidae and “6 kb element” in Haemosporida are highly conserved and widely studied (Hikosaka et al., 2011a, Hikosaka et al., 2011b, Hikosaka et al., 2013, Berná et al., 2021, Kruth et al., 2025).

Among these, the Eimeriidae and Haemosporida parasites are particularly well-characterized due to their biodiversity and global distribution. Eimeriidae, exemplified by the genus Eimeria with over 1800 species (Duszynski, 2011, Votýpka et al., 2016), and Haemosporida, documented to include more than 5100 avian haemosporidian genetic lineages in MalAvi database (accession date: 18 October 2024) (Bensch et al., 2009), exhibit striking diversification that frequently results in multi-species infections within individual hosts (Votýpka et al., 2016, Valkiūnas, 2004). For instance, domestic chickens can harbor up to 10 Eimeria species (Blake et al., 2021) alongside diverse haemosporidian lineages (Bensch et al., 2009, Hong et al., 2025), complicating accurate pathogen detection. Conventional molecular diagnostics, including mitochondrial DNA barcoding (e.g., COI for Eimeriidae; COB for Haemosporida) (Hellgren et al., 2004, Ogedengbe et al., 2011), often fail to resolve mixed infections and co-infections due to ambiguous chromatograms and undetectable minor genotypes that may be misinterpreted as single nucleotide polymorphisms (Valkiūnas et al., 2006, Martínez et al., 2009, Yeo et al., 2022, Jackwood, 2023, Hong et al., 2025). Although auxiliary methods like multiplex PCR (Fernandez et al., 2003, Kumar et al., 2014, Ciloglu et al., 2019, Musa et al., 2024) or fragment cloning (Pérez-Tris and Bensch, 2005, Megía-Palma et al., 2023, de Vieira et al., 2023) can address some limitations, they are constrained by amplification biases toward dominant lineages and primer mismatches limit their accuracy for detecting conspecific mixed infections or novel lineages, thereby obscuring true infection complexity. It is worth noting that sequences of the cloned PCR products may contain polymerase errors, thereby requiring analysis of multiple clones to verify the correct sequence (Bensch and Hellgren, 2020).

While next-generation sequencing has facilitated mitogenome assembly in Eimeriidae and Haemosporida parasites (Tang et al., 2015, Karadjian et al., 2016, Ciloglu et al., 2020, Zhou et al., 2023a), its reliance on short reads may increases the risk of chimeras in mixed infections due to high sequences conservation and lack of reference genomes (Pacheco and Escalante, 2023). Long-read sequencing platforms like Oxford Nanopore Technologies (ONT) have been developed to address the limitations, offer a robust alternative using amplicon-based method in polyparasitized samples across diverse taxa (Jackwood, 2023, Huggins et al., 2024a, Huggins et al., 2024b, Jiménez et al., 2024, Matoute et al., 2024, Cruz-Saavedra et al., 2024), and also enabling nearly complete mitogenome reconstruction (Pacheco et al., 2024, Pacheco et al., 2025, Hong et al., 2025). However, ONT remains unexplored for full-length mitogenome assembly in Eimeriidae and Haemosporida—a critical gap given mitogenomes' centrality to phylogenetic inference and species delimitation. This study aims to address these challenges by establishing an integrated novel workflow for: (1) one-step PCR amplification of complete mitogenomes; (2) ONT-based sequencing and assembly to resolve mixed/co-infections; (3) comparative evaluation against conventional DNA barcoding; and (4) construction of a baseline apicomplexan mitogenome reference database to bridge regional knowledge gaps in Taiwan.