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.