Metagenomic Sequencing and Bioinformatics

Sample DNA concentrations from TCE/Cr(VI) contaminated biocathode were measured using the Qubit dsDNA HS kit. The DNA quality and concentrations were evaluated using TapeStation with the Genomic ScreenTape (Agilent Technologies). The final concentration of 1.5ng/μl has been used for the library preparation. Sequencing libraries were prepared using the NEB Next Ultra II DNA library prep kit for Illumina (New England Biolabs, United States) following the manufacturer’s protocol. Library concentrations were measured in triplicate using the Qubit dsDNA HS kit and library size estimated using TapeStation with D1000 HS ScreenTape. The sequencing libraries were pooled in equimolar concentrations and diluted to 4nM. The samples were paired-end sequenced (2x301bp) on a MiSeq (Illumina, United States) using a MiSeq Reagent kit v3, 600cycles (Illumina, United States) following the standard guidelines for preparing and loading samples on the MiSeq. Raw Illumina reads were filtered for PhiX using Usearch11 (Edgar, 2010) subsequently trimmed using Cutadapt v. 2.10 (Martin, 2011). Forward and reverse reads were used to perform de novo assembly in megahit v. 1.2.9. The total assembly length of the metagenomes, the length of the longest contig, and the shortest contig length needed to cover 50% of the genome were calculated. Individual genomes (genome bins) were subsequently extracted from each sample metagenome in mmgenome2 v. 2.1.3. Bins were quality assessed with CheckM v. 1.1.3 (Parks et al., 2015). Classification of bacterial bins was conducted with the Genome Taxonomy Database toolkit (GTDB-TK) v. 1.3.0 (Chaumeil et al., 2019). Average nucleotide identities (ANI) were calculated using FastANI v. 1.32 (Goris et al., 2007; Jain et al., 2018). Genome annotations of bacterial and archaeal genomes were firstly conducted with Prokka v. 1.14.6. Further, NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was also performed for the deepest annotation. The Whole Genome Shotgun project was deposited at DDBJ/ENA/GenBank under the multiple accession {"type":"entrez-nucleotide","attrs":{"text":"JADIIK000000000","term_id":"1930685013","term_text":"JADIIK000000000"}}JADIIK000000000-{"type":"entrez-nucleotide","attrs":{"text":"JADIIN000000000","term_id":"1930684824","term_text":"JADIIN000000000"}}JADIIN000000000. Raw data are deposited under the SRA accession SRR12879946 within the BioProject PRJNA670625.