174 Chapter 6 taxonomical classification of trimmed reads, which also included the sample from 3% salinity (Menzel et al., 2016). Trimmed reads were co-assembled de novo using metaSPAdes v3.14.1 (Nurk et al., 2017) and mapped to assembled contigs using BBMap (BBTools v38.75) (Bushnell, 2014). Contigs at least 1,000-bp long were used as template for read mapping of metatranscriptomic and metaproteomic sequences, as well as binning. Sequence mapping files were handled and converted using Samtools v1.10., later used for binning with CONCOCT v2.1 (Alneberg et al., 2014), MaxBin2 v2.2.7 (Wu et al., 2016), and MetaBAT2 v2.12.1(Kang et al., 2019). Resulting metagenome-assembled genomes (MAGs) were dereplicated with DAS Tool v1.1.1 (Sieber et al., 2018) and taxonomically classified with the Genome Taxonomy Database Toolkit GTDB-Tk v2.1.0 (Chaumeil et al., 2019). For a metagenomic binning read-recruitment assessment together with a read-based “Ca. Methanoperedens” strains differentiation, SingleM v2 (https://github.com/wwood/singlem) was employed. MAG completeness and contamination was estimated with CheckM v1.1.3 (Parks et al., 2015). Metagenome-assembled genomes were annotated with DRAM v1.0 (Shaffer et al., 2020) with default options, except min_contig_size at 1,000 bp, and genes of interest were searched in annotation files as well as via BLASTp and HMM analyses. To corroborate poorly annotated genes/proteins, we opted to validate manual curations with the NCBI Batch Entrez Conserved Domains search option. See below for in-depth explanation for kamA and ablB gene mining and amino acid tree generation together with “Ca. Methanoperedens Strain Vercelli 1” biogeography study and additional metagenomic analysis, including: operon retrieval and sialic acid gene coverage calculations. To obtain a read-based “Ca. Methanoperedens” presence proxi at 3% salinities an additional metagenome was generated. Library preparation of the metagenome was done using the Nextera XT kit (Illumina, San Diego, California U.S.A.) according to the manufacturer’s instructions. Enzymatical 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, USA), 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 Inc Waltham USA).
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