Nextera library preparation protocol

To fully identify and explore the functional capacity of potentially novel Geodermatophilaceae isolates, whole genome shotgun sequencing was performed on the gDNA of the stone isolates identified as members of Geodermatophilaceae according to the 16S rRNA sequencing described above. Sequencing libraries for the eleven Geodermatophilaceae isolates were prepared using the Illumina Nextera Library Preparation protocol according to the manufacturer's instructions (Illumina Inc., San Diego, CA). Sequencing was completed on an Illumina HISeq 2500 HiSeq2500 platform (Illumina Inc., San Diego, CA) to produce 250 bp paired-end reads at the Hubbard Center for Genome Studies (UNH, Durham, NH). Raw sequencing data was demultiplexed using bcl2convert.

To investigate the prokaryotic community profiles of different DNA extracted, sequences corresponding to the V4 hypervariable region the 16S subunit rRNA gene were amplified by PCR using the Earth Microbiome Project 515F/806R universal primers under conditions described previously (Caporaso et al., 2012 (link)). For each sample, triplicate amplifications were performed. The triplicates were pooled, and all samples were normalized to equal DNA concentrations. Paired-end sequencing of the amplification product was performed using the Illumina HiSeq 2500 platform at the Hubbard Center for Genome Studies (University of New Hampshire, Durham, NH, United States). Read lengths of 250 bp were obtained and processed as described below.
To generate metagenomes for the C. glauca root nodule samples, whole genome shotgun sequencing was performed. Sequencing libraries for the samples were prepared using the Illumina NextEra Library Preparation protocol according to the manufacturer’s instructions and were sequenced on an Illumina HiSeq 2500. The same triplicate nodule DNA extractions were used for both the metabarcoding and metagenome shotgun sequencing methods, and metagenome libraries for both methods were sequenced at 150 bp read lengths at the Hubbard Center for Genome Studies (University of New Hampshire, Durham, NH, United States).

Isolated DNA was quantified using Qubit (Thermofisher) and presented comparable optical density quality ratio, concentration 26 – 75 ng/µL and fragment size distribution. Samples were diluted to 0.5 ng/µL with Tris (10 mM), and an input of 1 ng was processed following Illumina´s Nextera library preparation protocol. DNA was tagmented for 5 min at 55°C. The resulting DNA was then amplified and indexed using 12 PCR enrichment cycles and a dual combination of barcode primers. After amplification, DNA library clean-up was performed using AMPure XP bead purification and resuspended in resuspension buffer. Resulting libraries presented the similar molarity of 11 – 25 nM were pooled based on their molarity in hybridization buffer and denatured following the Nextera XT protocol. Libraries were sequenced using the Illumina NextSeq HighOutput using 2 × 150 bp paired end sequencing, providing 34 – 41 million clusters per sample. Data were analysed with MEGAN for abundance and metabolic gene pathways estimation was performed using eggNOG and InterPro2GO.