Field-deployable genetic methods for subspecies identification of long-tailed macaques (Macaca fascicularis) based on Oxford Nanopore technologies

Abstract

Background: Long-tailed macaque (Macaca fascicularis) is an ecologically and biomedically important primate widely distributed throughout Southeast Asia. Accurate subspecies identification is essential for conservation, ecological monitoring, and biomedical research. Morphological classification is often unreliable in hybrids.

Objective: To develop and evaluate a field-deployable genetic workflow for subspecies identification of M. fascicularis using fecal swab samples and Oxford Nanopore Technologies (ONT).

Methods: Fecal swab samples were collected from 30 individuals representing M. fascicularis fascicularis (n = 10), M. fascicularis aurea (n = 10), and hybrids (Mff×Mfa; n = 10). DNA from NAPSeq-preserved swabs was PCR-amplified for the mitochondrial hypervariable segment I (HVSI) and the sex-determining region Y (SRY) gene, followed by ONT sequencing and phylogenetic analysis.

Results: All samples yielded adequate DNA concentration (37.6 ± 17.7 ng/µL) and purity (A260/A280 = 1.90 ± 0.12). HVSI (881 bp) and SRY (846 bp) target genes were successfully amplified. ONT sequencing produced 560,171 raw reads (HVSI) and 220,740 raw reads (SRY). Phylogenetic analysis based on HVSI and SRY determined maternal and paternal lineages, respectively, enabling identification of subspecies and hybrid ancestry. Subspecies classification was concordant between morphology-based and ONT-based analyses for all samples, demonstrating the reliability and accuracy of the genetic workflow.

Conclusion: ONT-based sequencing of DNA extracted from fecal samples provides a practical and reliable approach for field-deployable subspecies identification in M. fascicularis, which would be applicable for ecological surveillance and quality control of non-human primate models in biomedical research.