Basespace platform

1 ug of RNA was used as starting material for library preparation. The kit
employed was TruSeq RNA Library Prep Kit v2 (Illumina) over polyadenylated RNA and the
manufacturer’s instructions were followed. The sequencing was performed on an
Illumina NextSeq 500 instrument. The raw reads obtained from the run were aligned against
the Syrian golden hamster genome (MesAur1.0) in the Basespace platform by Illumina, with
the tool “RNA-Seq Alignment”.

DNA was extracted from the longest core sample from each site (BPF1, 58 cm; BPF2, 30 cm) using the Qiagen DNA PowerSoil DNeasy DNA Extraction kit (Qiagen, Germany) with 0.5 g of starting material at King’s Bay AS Marine Laboratory, no more than 3 h after removal from the ground. DNA was quantified on each 2 cm soil sample using a Qubit™ 4 Fluorometer (Thermo Fisher Scientific, United States). To get a total of 10 ng/μl for each metagenome, BPF1 samples were pooled into two groups (0–30 cm and 30–58 cm), and all BPF2 samples were pooled (0–30 cm). These three samples were sequenced on an Illumina MiSeq with a V3 flow cell, using a 600 Nextera cycle kit with 275–300 bases paired-end reads. Data were downloaded from Illumina’s BaseSpace platform and analyzed with KBase (Arkin et al., 2018 (link)) with default program settings. Forward and reverse fastq reads were assembled with 98% of the two reads surviving, and adapters were trimmed with Trimmomatic v0.36 (Bolger et al., 2014); then, all three metagenomes were assembled separately with MetaSPAdes v3.14.1 (Nurk et al., 2017) and annotated with Prokka v. 1.14.6 (Seemann, 2014 (link)). Metagenome libraries of soil samples and whole genomes of isolates were compared with read mapping in terms of “reads per kilobase per read library” through Bowtie2 (Langmead and Salzberg, 2012 (link)) and in-house python scripts.

The raw reads from the 183 considered animals were analyzed by using the DRAGEN Germline App v.3.9.5 on the BaseSpace™ platform (Illumina, San Diego, CA, USA), using the default setting parameters. The DRAGEN workflow includes both alignment and variant calling algorithms: the reads were first mapped and aligned to the C. dromedarius CamDro3 reference genome (Genebank accession: GCA_000803125.3), then the variant calling was performed producing a genotype vcf.gz output file overall, including 17,679,716 variants.

Cells were collected immediately after the experiment was completed and processed for RNA-Seq on the Illumina Basespace platform. Total RNA was extracted from osteoblasts using an RNeasy Mini kit (Qiagen, Valencia, CA, USA). Raw read counts per gene were mapped to corresponding human homologs, using homology information from the Mouse Genome Informatics database (The Jackson Laboratory). The iPathwayGuide was used to score the biological pathways using impact analysis (43 (link), 44 (link)). Impact analysis uses two values to measure magnitude of pathway being examined (1) the overrepresentation of differentially expressed genes in a pathway and (2) the perturbation of that pathway computed by propagating the measured expression changes across the pathway topology. These aspects are captured independent probability values; pORA, representing the probability of obtaining a number of differentially expressed genes on the given pathway greater or equal to chance observations; pAcc, represents the p-value obtained from total perturbation accumulation. Combining pORA and pAcc is done to produce a unique global p value using Fisher’s method, which is then corrected for multiple comparisons using the false discovery rate method.

DNA samples were quantified on a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and detected under 0.8% agarose gel. A total of 5–50 ng DNA was used to generate amplicons using a MetaVx™ Library Preparation Kit (Genewiz, New Zealand, USA). Three relatively conserved variable regions (V3, V4, and V5) of 16S rRNA were amplified consistent with the previous report [18 (link)]. DNA library was verified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), quantified using real time PCR (Applied Biosystems, Carlsbad, CA, USA), and multiplexed and loaded on an Illumina MiSeq instrument following the instructions of the manufacturer (Illumina, San Diego, CA, USA). Sequencing was conducted using a 2 × 250 paired-end (PE) configuration, and image analysis and base determination were performed using the MiSeq Control Software on the MiSeq. The initial taxonomy analysis was carried out on Illumina BaseSpace platform.