Generating small mammals mitogenome reference dataset for Malaysian species

Abstract

Malaysia’s biological diversity is among the richest in the world but rapidly declining due to various human activities and climate change. Despite the continuous loss in biodiversity with the most recent death of our last Sumatran Rhino in May 2019, Malaysia's biodiversity data is still poorly characterized and systematically documented. Most species data have limited visibility with very abysmal publications which are mostly restricted to morphological traits and lack genomic data. As in line with the country’s National Policy on Biological Diversity 2016- 2025 to combat biodiversity loss and global effort to sequence all life by 2028 (Earth Biogenome Project), this study has focused on generating Malaysian small mammals mitogenome reference dataset. The genomic DNA of two fresh tissue samples (Balionycteris maculata and Callosciurus notatus) were extracted, fragmented to 300 bp, and further constructed into Illumina-compatible libraries using BEST protocol. Next, about 15 amplification cycles during library indexing was used to produce maximum data output with high complexity, and low clonality suggesting the rare variants could be easily detected. Prior to sequencing using BGISEQ-500 platform, the 3-indexed libraries (including extraction blanks) with approximately 300-400 bp were pooled to equimolar DNA (<12,000 pmol/L). Approximately 5 gigabases raw sequence data per sample were generated comprising of the whole genome data (mitochondrial DNA and nuclear DNA). The new high quality mitogenomes has been successfully assembled using MITOBIM and PALEOMIX with an average size of 16-17 Kbp and an average depth of coverage of 140.27x. The detailed pipeline and challenges on mitogenome assembly for species with and without reference genome in Genbank was discussed. The mitogenomes were further annotated to its 37 designated genes via MitoZ. The robust pipeline of mitogenome sequence generation established in this work could be applied to generate more genomic data from thousands of tissue samples available from local biodiversity key players such as Perbadanan Taman Negara Johor, FRIM and PERHILITAN. The further enrichment of DNA reference database will strongly magnify species detection in invertebrate-derived DNA (iDNA) research for biodiversity assessment, wildlife forensics to monitor illegal trade of endangered species in this region, as well as population genetic studies.