Citation: Çelik, I.; Keskin, E. Revealing the Microbiome of Four Different Thermal Springs in Turkey with Environmental DNA Metabarcoding. Biology 2022, 11, 998. https://doi.org/10.3390/ biology11070998 Academic Editor: Pierangelo Luporini Received: 16 May 2022 Accepted: 20 June 2022 Published: 30 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biology Article Revealing the Microbiome of Four Different Thermal Springs in Turkey with Environmental DNA Metabarcoding I¸ sılay Çelik 1,2 and Emre Keskin 2, * 1 Biotechnology Institute, Ankara University, Ankara 06135, Turkey; isilaycelik@ankara.edu.tr 2 Evolutionary Genetics Laboratory (eGL), Department of Fisheries and Aquaculture, Agricultural Faculty, Ankara University, Ankara 06135, Turkey * Correspondence: keskin@ankara.edu.tr Simple Summary: The physicochemical conditions of thermal springs are one of the most significant barriers for detecting microbial life. According to the findings of various studies, high-throughput DNA sequencing technology can be utilized to perform more precise and detailed microbiome assessments. The main goal of this paper was to determine the microbiome in a thermal spring by metabarcoding environmental DNA obtained from four different sources and revealing how temperature and chemical composition affect the microbiome. This research also aimed to gather information that will aid in determining the best gene region and bioinformatic pipeline. The findings revealed a link between four different thermal springs’ physicochemical parameters and microbial composition and we found various manipulable steps in this study. This research is also first comprehensive thermal spring metabarcoding study conducted in Turkey. Abstract: One of the most significant challenges for detecting microbial life in thermal springs by conventional techniques such as culturing is these places’ physicochemical (temperature, heavy metal content, pH, etc.) conditions. Data from several studies suggest that high-throughput DNA sequencing technologies can be used to perform more accurate and detailed microbiome analyses. The primary aim of this paper was to determine the microbiome in the thermal source by metabarcoding environmental DNA isolated from four different sources and reveal the reflection of differences caused by temperature and chemical content on the microbiome. DNA was extracted from water filtered with enclosed filters and using the Illumina high-throughput sequencing platform, V3 and V4 regions of the 16S rRNA gene were sequenced. The results showed a correlation between physicochemical conditions and microorganism composition of four different thermal springs. Springs with extremely high temperature (89–90 ◦ C) were dominated by hyperthermophiles such as Hydrogenobacter and Thermus, while a spring with a high temperature (52 ◦ C) was dominated by thermophiles such as Thermoanaerobaculum and Desulfurispora, and a spring with a low temperature (26 ◦ C) and high salinity was dominated by halophiles and sulfur-oxidizers such as Hydrogenovibrio and Sulfirimonas. With this research, we observed many manipulable steps according to the work of interest. This study sought to obtain data that will help decide the right gene region and choose the optimal bioinformatic pipeline. Keywords: microbiome; environmental DNA (eDNA); metabarcoding; thermal spring; high throughput sequencing; extremophiles 1. Introduction The entire genetic material of microorganisms in a particular niche is defined as the microbiome [1]. Microbial communities represent the world’s earliest inhabitants and shape the dynamics of environments ranging from the mammalian digestive tract to hydrothermal vents [2]. It covers a broad spectrum from nutrient cycling to the protection of human health [3,4]. For this reason, the detection, classification, and analysis of these communities that live and work together in a particular environment gain importance Biology 2022, 11, 998. https://doi.org/10.3390/biology11070998 https://www.mdpi.com/journal/biology