Shotgun sequencing of Brachypodium distachyon rhizosphere
Libraries were prepared using Illumina DNA Prep (M) Tagmentation library preparation kit (Illumina Inc., San Diego, CA, USA) following the manufacturer's user guide. Initial DNA concentration was evaluated using the Qubit dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA). Eukaryotic DNA was depleted in leaf tissue samples using NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, Ipswich, MA, USA) following the manufacturer's user guide to decrease the probability of recovery of host genomic, chloroplast, and mitochondrial DNA sequences (Feehery et al., 2013). DNA (500 ng) was used for depletion of the eukaryotic DNA as recommended by Molecular Research LP (MR DNA; Shallowater Texas USA). The enriched microbial DNA was quantified using the Qubit dsDNA HS Assay Kit (Life Technologies). Subsequently, 50 ng of DNA was used to prepare the libraries. The samples underwent simultaneous fragmentation and addition of adapter sequences, which were utilized during a limited-cycle PCR in which unique indices were added to the sample. Following library preparation, library concentration and mean library size was determined using the Qubit dsDNA HS Assay Kit (Life Technologies) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. Libraries were pooled in equimolar ratios (0.6 nM), and sequencing was performed on a NovaSeq 6000 platform (Illumina Inc.) to a depth of 10 million 2 x 150 bp reads.