NFBP will conduct in-silico validation of barcoding primers to select panels of suitable genetic markers that are able to differentiate various species based on the genetic barcodes. This will be followed by tissue dissection of fish specimens for DNA extraction of representative species. PCR amplification and sequencing of barcoding regions using validated primers will be carried out for each species.
NFBP will additionally perform eDNA metabarcoding on the environmental samples using High throughput Sequencing technology to categorize the aquatic biodiversity of the Karnali river. The collected water samples from the river will be addressed for DNA extraction following sequencing using latest Next Generation Sequencing methods.
NFBP will build a fish reference library of all representative fish species from the collected specimens from Karnali river basin using conventional Sanger sequencing. A genetic database of all the species detected from environmental samples (eDNA) using NGS technology will also be constructed, which will then be compared to the fish reference library.
NFBP is made possible with the development of fish and eDNA sample collection Standard Operating Protocols (SOP) for field sites, SOPs for fish tissue dissection, DNA extraction, Polymerase Chain Reaction (PCR) and Sequencing protocol for fish barcoding region of tissue and environmental samples. There has been development of species-specific primers for common and/or rare native fish species.
In addition, NFBP has made biosafety protocols according to the MSDS (Material Safety Data Sheet) of especially hazardous reagents used for fish molecular and morphological labs.
NFBP will organize seminars or workshops in Nepal to share research findings. Monthly disclosure of project updates will be shared amongst all collaborator parties. All novel protocols and/or discoveries made during the progress of this research are property of NFBP and collaborator parties.
At International level, the study information will be shared with experts worldwide through scientific publications and presentations.
NFBP has implemented an internship program where students from various universities around Nepal will join the project either in field sampling and/or laboratory work for a short period. This gives an opportunity for local students to become skilled at practical knowledge required for any scientific work and to learn about the environment and biodiversity of fish in Nepal.
During field sampling, they will be trained on aspects of sample collection, logistics management and chiefly to be a meticulous explorer. As a lab intern, they will be trained on all facets of general molecular biology training including DNA Extraction, conventional/real-time PCR, Gel electrophoresis, Sequencing and General bioinformatics.