Abstract
In this stujdy, we try to describe a geothermal microbial community by means of metagenomics and genomics of isolated microorganisms.
Sampling location and DNA isolation
Samples were collected at Favara Grande, Pantelleria, Italy 2017 (FAV1, 36° 50′ 80″ N; 11° 57′ 170″ E) and (FAV2, 36° 50′ 77″ N; 11°57′ 160″ E) during a field campaign in June 2017 (Picone et al. 2020). Soil samples (1–10, 10–15 and 12–20 cm depth) were taken using a core sampler (diameter 1.5 cm), stored in sterile 50 mL tubes and kept at 4 °C until DNA was extracted. In situ pH values were 4–4.5 with temperatures from 60 to 67 °C. Two different DNA extraction methods were used, namely Fast DNA Spin kit for soil (MP Biomedicals, Santa Ana, California), according to manufacturer’s instructions, and the CTAB method (Allen et al. 2006). DNA extraction was only successful from the FAV2 sampling site and the reads from the different depths and different extraction methods were combined for assembly and binning. For more detail see Picone et al. (2020).
Genome sequencing, assembly and binning
The metagenome was sequenced on the Illumina sequencing platform. For library preparation the Nextera XT kit (Illumina, San Diego, California) was used according to the manufacturer’s instructions. Enzymatic tagmentation was performed starting with 1 ng of DNA, followed by incorporation of the indexed adapters and amplification of the library. After purification of the amplified library using AMPure XP beads (Beckman Coulter, Indianapolis), libraries were checked for quality and size distribution using the Agilent 2100 Bioanalyzer and the High sensitivity DNA kit. Quantitation of the library was performed by Qubit using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts). The libraries were pooled, denatured and sequenced with the Illumina Miseq sequence machine (San Diego, California). Paired end sequencing of 2 × 300 base pairs was performed using the MiSeq Reagent Kit v3 (Illumina, San Diego, California) according the manufacturers protocol.
Reads were trimmed using BBDuk (BBMap), assembled by MEGAHIT v1.0.3 (Li et al. 2015) and binned using an in-house pipeline, using different binning algorithms, including BinSanity (Graham et al. 2017), COCACOLA (Lu et al. 2017), CONCOCT (Alneberg et al. 2014), MaxBin 2.0 (Wu et al. 2016), and MetaBAT 2 (Kang et al. 2019). DAS Tool 1.0 was used for consensus binning (Sieber et al. 2018) and CheckM was used to assess the MAG quality (Parks et al. 2015). The average nucleotide identity using BLAST (ANIb) is calculated using JSpeciesWS software with standard settings (Richter et al. 2016). An up-to-date Bacterial Core Gene (UBCG) phylogenetic tree was constructed using RAxML (Stamatakis 2014) on CIPRES Science Gateway V. 3.3 platform (Miller et al. 2012). PROKKA and the MicroScope platform were used to automatically annotate the draft genome (Seemann 2014; Vallenet et al. 2013) and genomic features were manually checked.
