Short-read full-length 16S rRNA amplicon sequencing for characterisation of the respiratory bacteriome of captive and free-ranging African elephants (Loxodonta africana).

Hypervariable region sequencing of the 16S rRNA gene plays a critical role in microbial ecology, offering insights into bacterial communities within specific niches. While providing valuable genuslevel information, its reliance on limited genetic data from targeted regions of this gene limits its overall utility. Advances in sequencing have enabled characterisation of the full-length 16S rRNA gene, enhancing species-level classification. Third-generation sequencing technologies can sequence genes in single reads, yet challenges in cost and accessibility persist. Although Illumina's short-read platforms are cost-effective and precise, they lack full-length 16S rRNA amplicon sequencing capability. This study aimed to evaluate the feasibility of a modified 150 bp paired-end 16S rRNA amplicon shortread sequencing technique on the Illumina iSeq 100 by utilising a mock microbial community and subsequently performing exploratory characterisation of captive (zoo) and free-ranging African elephant (Loxodonta africana) respiratory microbiota. Our findings support the viability of the sequencing technique through taxonomic assignment that is consistent with the microbial standard’s theoretical composition. Tentative bacterial signatures, potentially representative of distinct respiratory tract compartments (trunk and lower respiratory tract) were observed, necessitating further investigation with larger sample sizes to gain deeper insights into its implication for elephant physiology and health.