Page 180 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 8. Species-specific responses of enriched thermokarst lake sediments
After purification of the amplified library using AMPure XP beads (Beckman Coulter, Indianapolis, IN), libraries were checked for quality and size distribution using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and the Qubit dsDNA HS Assay Kit. Quantification of the library was performed with the Qubit dsDNA HS Assay Kit. The libraries were pooled, denatured and sequenced with the Illumina MiSeq system (Illumina, San Diego, CA). Paired- end sequencing of 2 x 301 base pairs was performed using the MiSeq Reagent Kit v3 (Illumina, San Diego, CA) according to the manufacturer’s protocol.
Metagenome analysis. Metagenome datasets were processed as previously described (in ‘t Zandt et al. 2019). Taxonomic assignments for MAGs were based on the Genome Taxonomy Database with the GTDB-TK tool v0.2.1 (Chaumeil et al. 2019). Reads containing parts of the 16S rRNA gene were identified by performing a BLASTN search of the quality-filtered reads to the SILVA SSU Ref NR 99 release 132 database using a length and similarity fraction of 50% and 70%, respectively (Quast et al. 2012). Mapping was done in the CLC Genomics Workbench 11.0 (CLCbio, Aarhus, Denmark). Mapped reads were size filtered for a minimum length of 200 base pairs. Reads were submitted to the SILVAngs pipeline and processed with the default settings for Illumina MiSeq reads. Taxonomic frequencies were exported and used for the analysis of the taxonomic composition of the microbial samples. The bins were gene- called by Prodigal() and annotated (cut-off = E-50) using a custom HMM database and HMMER (http://hmmer.org/) (Eddy 2011). To build this database, proteins from the TrEMBL database of EMBL were selected based on their presence in the KEGG metabolic pathways and clustered using Linclust(--kmer-per-seq 160 --min-seq-id 0.5 --similarity-type 1 --sub-mat blosum80 --cluster-mode 2 --cov-mode 0 -c 0.7). The clusters were subsequently aligned with mafft() (--anysymbol) and HMM profiles were created with hmmbuild (default) for each cluster. Proteins with special interest for this study, like methyl-coenzyme M reductase (Mcr), were manually curated and complemented with HMMs of phylogenetic groups of interest.
For functional gene searches Prokka-annotated MAGs were imported in Artemis v17 (Rutherford et al. 2000; Seemann 2014). For retrieval of HdrDE sequences, reviewed Swiss- Prot HdrDE sequences from Methanosarcina acetivorans, M. barkeri, M. mazei and M. thermophila were downloaded from the UniProt Knowledgebase (UniProtKB) on 09-04-2020 (The UniProt Consortium 2014). The annotated Methanosarcina MAGs were blasted against
178