Dr Jia Jin Marc Chang1, Ms Aubane Carré de Malberg1, Ms Wan Wen Rochelle Chan2, Dr Yin Cheong Aden Ip3, Dr Zeehan Jaafar1,2, Dr Danwei Huang1,2
1Lee Kong Chian Natural History Museum, National University Of Singapore, Singapore, 2Department of Biological Sciences, National University of Singapore, Singapore, 3School of Marine and Environmental Affairs, University of Washington, Seattle, United States of America
Biography:
Marc is a Research Fellow from the Lee Kong Chian Natural History Museum, National University of Singapore. He current research work involves the use of Autonomous Reef Monitoring Structures to study unseen marine biodiversity in Singapore's coral reefs. Previously, he has worked on projects ranging from DNA barcoding, in situ sequencing, environmental DNA and even genome assembly. He is keenly interested in further operationalising long read sequencing for more DNA sequencing projects.
Abstract:
Environmental DNA (eDNA) metabarcoding is a powerful approach for species detection utilizing short, standardized DNA markers from samples collected from the environment. While the mitochondrial cytochrome c oxidase subunit I is the preferred DNA barcode for eukaryotes, especially invertebrates, alternative genes are sometimes used depending on the study taxa. In fish, commonly-sequenced mitochondrial genes for eDNA include cytochrome b, 12S, or 16S. One challenge is the unequal representation of these barcodes in gene databases, potentially hindering downstream species identification. While there are economical methods to scale-up DNA barcode generation, doing so for multiple genes per species can be labour-intensive. A common workaround is to sequence complete mitogenomes to capture all target mitochondrial genes. Genome skimming based on short-read sequencing is the most common approach; but it is inefficient and challenging to scale up due to the substantial amount of off-target reads generated. Instead, we propose the use of long-range polymerase chain reaction (LRPCR) to amplify fish mitogenome products that can be easily multiplexed and sequenced readily using long-read technology like Oxford Nanopore. We demonstrate the utility of this workflow through the generation of ~100 fish mitogenomes on a MinION flow cell. Nanopore-sequenced mitogenomes sequenced were found to be ~98% accurate when benchmarked against Illumina sequencing. Our proposed workflow greatly improves scalability of sequencing mitogenomes, not only for improving sequence databases for species identification, but also benefiting research such as mitogenome-based phylogenomics.