Miseq 2x250 v2 kit

PCR was performed using universal primers flanking the variable 4 (V4) region of the bacterial 16S rRNA gene [76 (link)]. Genomic DNA samples were amplified in duplicate. Each reaction contained 10–30 ng genomic DNA, 10 μM each primer, 12.5 μl 2x HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA), and water to a final reaction volume of 25 μl. PCR was carried out under the following conditions: initial denaturation for 3 min at 95°C, followed by 25 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 55°C and elongation for 30 s at 72°C, and a final elongation step for 5 min at 72°C. PCR products were purified with the QIAquick 96-well PCR Purification Kit (Qiagen, Germantown, MD, USA) and quantified using Qubit dsDNA HS Assay kit (Invitrogen, Oregon, USA). Samples were equimolar pooled and sequenced by the University of Wisconsin–Madison Biotechnology Center with the MiSeq 2x250 v2 kit (Illumina, San Diego, CA, USA) using custom sequencing primers.

PCR was performed using universal primers flanking the variable 4 (V4) region of the bacterial 16S rRNA gene [16 (link)]. PCR products were purified with the QIAquick 96-well PCR Purification Kit (Qiagen, Germantown, MD, USA). Samples were quantified by Qubit Fluorometer (Invitrogen, Carlsbad, CA) and equimolar pooled. The pool was sequenced at the University of Wisconsin-Madison Biotechnology Center with the MiSeq 2x250 v2 kit (Illumina, San Diego, CA, USA). DNA extraction blanks, PCR blanks, and technical duplications for both extractions and PCRs were employed to ensure proper sample handling throughout the library preparation process.

PCR was performed using primers 515F and 806R for the variable 4 (V4) region of the bacterial 16S rRNA gene [21 (link)]. PCR reactions contained 1 ng μl^-1 DNA, 10 μM each primer, 12.5 μl 2X HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA), and water to 25 μl. PCR program was 95°C for 3 min, then 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, the final step was 72°C for 5 min. PCR products were purified by gel extraction from a 1.5% low-melt agarose gel using a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). Samples were quantified using the Qubit dsDNA HS assay (Invitrogen, Carlsbad, CA, USA) and equimolar concentrations pooled. The pool was sequenced with the MiSeq 2x250 v2 kit (Illumina, San Diego, CA, USA). All DNA sequences generated in this study are deposited in the NCBI Short Read Archive under BioProject Accession Number: PRJNA595821 and PRJNA596889.