2.3 Metagenomic sequencing
We sent contents of the dissected sections of the gastrointestinal tract to Azenta Life Sciences (South Plainfield, NJ, USA) for metagenomic analysis. Genomic DNA was isolated using the NucleoMag DNA Microbiome Kit (Takara Bio, Shiga, Japan) and was quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, US). NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) was used for library preparation following manufacturer’s recommendations. Briefly, genomic DNA was fragmented by acoustic shearing with a Covaris S220 instrument, followed by end-repair. Adapters were ligated after adenylation of the 3’ends followed by enrichment by a limited cycle PCR. DNA libraries were quantified using Qubit 2.0 Fluorometer and by real time PCR (Applied Biosystems, Carlsbad, CA, USA). Sequencing libraries were sequenced on an Illumina HiSeq instrument using a 2 x 150bp Paired End (PE) configuration. Image analysis and base calling were conducted using the HiSeq Control Software (HCS). Raw BCL files were converted to FastQ files and de-multiplexed using bcl2fastq v.2.1.9 (Illumina), keeping only >Q30 reads with 150 bp in length. A de novo approach was followed for assembling reads using Spades v3.10 (Bankevich et al., 2012), with a minimum contig length of 1,000 bp and using the newly assembled genome of Phyllotis vaccarum (Storz et al., 2023) as a reference genome to discard reads aligned to the host. QUAST (Gurevich et al., 2013) was used to generate statistics and EMBOSS tools getorf was used to find the open reading frames within the de novo assembled genome. BLAST+ (v.2.6.0) (Altschul et al., 1990) was used to query assembled contigs in the nucleotide database of Genbank.