Metagenomic sequencing was performed with samples from SQ-amended (n = 2) and unamended control (n = 1) microcosms of rumen fluid, taken after 168 h of incubation. Libraries were prepared using the NEBNext Ultra FS II DNA Library Prep Kit (New England Biolabs, USA) for Illumina and sequenced on a NovaSeq6000 (1/2 SP flow cell, 2×100 bp reads; Illumina, USA). Quality control was conducted using a Snakemake v8.20.3 workflow to trim reads based on quality scores. Reads were quality-checked with FastQC v0.12.1, and summary statistics were compiled using MultiQC v1.21. Adapter sequences and phiX contamination were removed with BBDuk (BBMap v39.06), retaining reads of at least 50 bp. Right-end k-trimming was applied (k-mer = 21, minimum k-mer = 11, hamming distance = 2) with "tpe" and "tbo" options, and quality trimming was performed with a Q-score of 15. The quality-controlled read library was quality checked using FastQC v0.12.1 and quality statistics were merged using MultiQC v1.21 for comparison to the non-trimmed quality. BBMap’s reformat.sh was used to interleave the read library.
Reads were assembled with SPAdes v4.0.0 in "meta" mode, using k-mers from 21 to 121 (step size: 10). Contigs shorter than 1,000 bp were removed using reformat.sh of BBMap. Metabat v2.15 was used to bin putative metagenome-assembled genomes (MAGs) without coverage data (minimum length: 1,500 bp). Coverage information for the assembly was generated with BBMap (≥98% identity) using the read library, followed by sorting with SAMtools v1.20. A depth file was generated using jgi_summarize_bam_contig_depths and the assemblies were binned into putative MAGs again using the depth file in Metabat. Dereplication of putative MAGs was performed with dRep v3.5.0´, applying a minimum completeness threshold of 50% and a maximum contamination threshold of 10%. Taxonomic classification of dereplicated MAGs was conducted using GTDB-Tk v2.4.0’s classify_wf (database r220, ANI screening skipped). Barrnap v0.9 was used to identify and recover 5S, 16S, and 23S rRNA gene sequences in the genomes. Coverage and the percentage of reads mapped to each MAG were determined with BBMap with a mapping identity of 95%. Average amino acid identity (AAI) was calculated using the Enveomics Collection. Whole-genome average nucleotide identity (ANI) was calculated using FastANI (version 1.33).