Detection of Amphibian Species based on Cytochrome b Metabarcoding using High-Throughput Sequencing Approach

Abstract Amphibians are essential in terrestrial and freshwater ecosystems, contributing to multiple ecological functions. The presence of amphibians in a particular area usually signifies a healthy ecosystem as it indicates that the habitat meets the requirements of their life cycles. However, the amphibian population have been declining due to human activities including pollution and habitat destruction. Researchers are using new methods to monitor amphibian diversity to address this decline. Conventional sampling methods were compared with new DNA metabarcoding techniques using advanced sequencing platforms. Therefore, this study aims to analyze the early detection of amphibian species using Cytochrome b (Cyt b ) gene with metabarcoding methods and next generation sequencing techniques. Thirty-one samples were used in this study and underwent processes such as extraction, amplification using Cyt b gene, library preparation, and sequencing. The result showed that the total number of reads was 658,655, with reads in OTUs numbered 474,061. Out of 31 samples, only three species were successfully detected: Fejervarya limnocharis with the highest abundance (20.6%), followed by Polypedates leucomystax and Pulchrana baramica . The results indicate that Cyt b has low capabilities in detecting amphibian species, which might be due to its highly conserved region and the lack of databases for this gene. For future studies, an enhanced DNA metabarcoding procedure should be applied, including better choice of genetic marker and bioinformatics pipelines to minimize error and to improve the accuracy and reliability of detecting amphibian’s species using NGS and DNA metabarcoding. This would enhance efforts to protect amphibian biodiversity.