I d na agarose gel

ITS sequencing followed methods described more fully in Cornman et al. [27 (link)], in which an approximately 900-bp fragment is subjected to 300-bp paired-end sequencing, recovering non-overlapping fragments of the ITS1 and ITS2 spacer regions. Briefly, amplicons were produced in two steps, first using ‘standard’ primers to generate a high concentration of input template, followed by less efficient ‘fusion’ primers that incorporate exogenous sequencing adapters. The first amplification reaction used primers ITS5a [28 (link)] (5’—ACC TTA TCA TTT AGA GGA AGK ARA ART CGT AAC AAG GT—3’) and ITS4 [29 ] (5’—TTC CTC CGC TTA TTG ATA TGC TTA ARY TCA GC—3’). The thermocycler program consisted of an initial denaturation step of 95°C for 5 min, followed by 40 cycles of 30 s at 95°C, 35 s at 47°C, and 1.5 min at 72°C, and a final extension of 72°C for 10 minutes. An appropriately sized amplification product was confirmed for each reaction by electrophoresis of 5 μL of the reaction product through a 1.5% I.D.NA agarose gel (Cambrex Corporation, East Rutherford, NJ) at 100 V for 45 min. Polymerase chain reaction (PCR) products were cleaned with the Qiagen PCR Purification Kit (Valencia, CA) and quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, Grand Island, NY). Samples were diluted in 10 mM Tris buffer (pH 8.5) to a final concentration of 5-ng/μL.

Bacteria were characterized using PCR that targeted a portion of the 16S rRNA gene. Amplicons were produced in two steps, first using standard primers to generate a high concentration of input template. This was followed by less efficient fusion primers that incorporate exogenous sequencing adapters that were used for the creation of full-length Illumina MiSeq sequencing libraries. Sequencing of 16S rRNA used primer pair sequences for the V3 and V4 region that create a single amplicon of ~460 base pairs (bp). The primers for the first amplification reaction were 16S For (5’–CCTACGGGNGGCWGCAG– 3’) and 16S Rev (5’–GACTACHVGGGTATCTAATCC– 3’) [19 (link)]. The thermocycler program consisted of an initial denaturation step of 95°C for 3 min, followed by 35 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C, for 7 minutes. An appropriately sized amplification product was confirmed for each reaction by electrophoresis of 5 μL of the reaction product through a 1.2% I.D.NA™ agarose gel (Cambrex Corporation, East Rutherford, NJ) at 100 V for 45 min. PCR products were cleaned with a QIAquick PCR Purification Kit (Qiagen CT # 28104, Valencia, CA) and quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, Grand Island, NY). Samples were diluted in 10 mM Tris buffer (pH 8.5) to a final concentration of 5 ng/μL.