146 Chapter 5 2022). Additional genes of interest were searched 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 and InterPro (Blum et al., 2021) web browers” search option. To obtain a read-based “Ca. Methanoperedens” granular and free-living relative abundance, an additional shallow metagenome was generated. Library preparation of the metagenome was done using the Nextera XT kit (Illumina, San Diego, CA, USA) 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, 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). The libraries were pooled, denatured, and sequenced with the MiSeq (Illumina) sequencer (San Diego, CA, USA). Paired end sequencing of 2x300 bp was performed using the MiSeq Reagent Kit v3 (San Diego, CA, USA) according to the manufacturer’s protocol yielding 1.6-1.7 Gbp for the granular fraction and 0.3 or, 1.1 Gbp for the T3 and T3 planktonic fractions, respectively. Metatranscriptomics Metatranscriptomic sequencing was performed using a TruSeq stranded with NEB rRNA depletion kit (bacteria) (Illumina, San Diego, CA, USA) on a NovaSeqX 10B (Illumina) platform, generating 150-bp paired-end reads with ~ 15 Gb throughput/ sample. Raw sequences were quality trimmed using sickle v1.33 (https://github. com/najoshi/sickle) and ribosomal RNA contaminant-filtered, mapped against the DRAM-generated scaffolds and Transcripts per Million (TPM) values generated using trancriptm v0.4 (https://github.com/sternp/transcriptm). Differential expression (log2 fold chain and p-adjusted) was evaluated using the DESeq2 library in R (Love et al., 2014).
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