A mitochondrial DNA metabarcoding system based on tNGS enables multiplex identification of vertebrate species in samples containing degraded and mixed DNA.

Next-generation sequencing (NGS) has facilitated the widespread use of DNA metabarcoding in fields such as biodiversity, population research, and food adulteration. To improve the identification of animal-derived DNA in samples containing degraded and mixed DNA, a DNA metabarcoding system targeting the mitochondrial Cytb, COI, 16 S rRNA, and 12 S rRNA genes using targeted NGS (tNGS) was developed for vertebrate species. In line with the International Society for Forensic Genetics (ISFG) recommendations for non-human DNA analysis, the forensic performance of the novel assay was systematically evaluated, including assessments of sensitivity, mixtures, inhibitors, degraded DNA, processed meat products, and case sample studies. Sensitivity studies showed that 10 pg of genomic DNA per reaction from a single species was sufficient to detect at least one marker for all tested species, whereas 100 pg enabled reliable species identification using all four markers. For mixed samples with a total DNA input of 1 ng, all ten species were accurately identified in an equimolar mixture. Minor components were also detected in binary mixtures at a 99:1 ratio (1% minor contributors). Notably, the assay recovered fragmented DNA from experimentally degraded samples and highly processed food matrices. Analysis of casework samples further demonstrated that the tNGS-based multiplex assay enables broad-spectrum vertebrate identification and holds great practical value for curbing wildlife trafficking, mitigating food fraud, and facilitating biodiversity monitoring.