BIODIVERSITAS ISSN: 1412-033X Volume 24, Number 4, April 2023 E-ISSN: 2085-4722 Pages: 2251-2260 DOI: 10.13057/biodiv/d240438 The importance of DNA barcode reference libraries and selection primer pair in monitoring fish diversity using environmental DNA metabarcoding DEWI IMELDA ROESMA  , DJONG HON TJONG, SYAIFULLAH, NOFRITA, MUHAMMAD NAZRI JANRA, FURQAN DWIKI LINTANG PRAWIRA, VIOLA MUTIARA SALIS, DYTA RABBANI AIDIL Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Andalas. Jl. Limau Manis, Padang 25175, West Sumatra, Indonesia. Tel./fax.: +62-751-71671,  email: dewiroesma@sci.unand.ac.id Manuscript received: 13 December 2022. Revision accepted: 25 April 2023. Abstract. Roesma DI, Tjong DH, Syaifullah, Nofrita, Janra MN, Prawira FDL, Salis VM, Aidil DR. 2023. The importance of DNA barcode reference libraries and selection primer pair in monitoring fish diversity using environmental DNA metabarcoding. Biodiversitas 24: 2251-2260. Environmental DNA (eDNA) metabarcoding has become an alternative method used for biodiversity monitoring of an ecosystem. The eDNA metabarcoding has advantages compared to the conventional method because it is non-invasive, quick, and requires less cost. However, the effectiveness of the eDNA method is highly dependent on the coverage of the DNA barcode reference and primer pair. A study using the eDNA method was conducted for fish biodiversity monitoring in Singkarak Lake. Two-liter water samples were collected using sterile bottle samples at each sampling site (five sites). The universal primers (Fish FI and Fish R1) used for Next-generation sequencing (GRIDION, Nanopore, Oxford Technologies). The study detected 152 fish species using eDNA metabarcoding. Ten species out of the 30 originally reported in Singkarak Lake were detected using eDNA metabarcoding. The low percentage of fish detected is thought to be due to several factors; incomplete/unavailability of freshwater fish DNA barcodes in Indonesia registered in the database repository, inappropriate primer pair selection, low DNA quality, and the absence of target species DNA in collected water samples. The results demonstrated the significance of correctly registering DNA barcodes to the database and appropriate primer pair selection to identify eDNA metabarcoding. This study provides recommendations using eDNA metabarcoding for monitoring in future work. Keywords: DNA barcode, eDNA metabarcoding, next-generation sequencing, primer pair, Singkarak Lake INTRODUCTION Indonesia has the second-highest mega biodiversity in the world (von Rintelen et al. 2017). Unfortunately, not all biodiversity data is known related to the number and types of species, especially freshwater fish groups. Data from various databases (library repositories, official websites, articles, and books) are still separate and uncollected well (Widjaja et al. 2014). These data are essential for managing and conserving fishery resources (Menning et al. 2018). Collecting data and information to assess the biodiversity of freshwater fish has been carried out using conventional methods, which are the direct collection and morphological identification with the help of taxonomists and the existing literature (Hogg et al. 2018). However, there are big challenges related to using a morphological approach, which is the lack of taxonomists; the identification can be subjective; the lack of literature; the identification doubts of cryptic species, and it takes time, effort, and great expense to estimate all communities (Bean et al. 2017; Ellingsen et al. 2017). Therefore, a more reliable approach, environmental DNA (eDNA) metabarcoding, is needed to collect biodiversity data and information (Ficetola et al. 2008). The eDNA metabarcoding is a promising approach using DNA barcodes as identification references combined with High Throughput Sequencing (HTS) namely Next- Generation Sequencing (NGS) (Shokralla et al. 2012). The eDNA metabarcoding approach is non-invasive because it only takes DNA from living or dead organisms in the environment from water, soil, or sediment (Ficetola et al. 2008). The eDNA metabarcoding has been used in various monitoring studies to assess the community diversity of multiple taxa groups (Aylagas et al. 2014; Pawlowski et al. 2014; Thomsen and Willerslev 2015; Valentini et al. 2016; Yamamoto et al. 2017; Andriyono et al. 2019). Despite being a promising approach, there are some limitations in using eDNA metabarcoding, including incomplete DNA barcode databases and inappropriate primer pairs. Unregistered DNA barcodes in the databases will impact the limitation and occurrence of species identification errors (Hebert et al. 2016). Freshwater ecosystems are currently facing a lot of anthropogenic pressure from various community activities (Carew et al. 2013), one of which is Singkarak Lake, the largest lake in West Sumatra. The Singkarak Lake, used for various activities, has experienced a decline in fish biodiversity, based on survey data using conventional methods from 1913 to 2011 (Weber and de Beaufort 1913, 1916; Syandri 2008; Roesma 2011). The results indicate differences in the type of species found from time to time. The lack of studies and unavailable species database