NE14V2
Bacterial libraries were prepared using the 515-Y and 926R primer pair that amplifies V4 and V5 hypervariable regions of the 16S rDNA (Parada, Needham, & Fuhrman, 2016) and contained universal 5 tail sequences from Alvarado et al. (2018). The negative controls without any potential DNA source were included in the sequencing libraries. We used specially designed mock communities of bacteria (D6305, Zymo Research, CA, USA) as a positive control. Bacterial libraries were prepared by using a two-step protocol as suggested by Berry et al. (2011). A first PCR was carried out for each root and soil sample in 25 L reaction mixture containing 1X Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific Inc, MA, USA), an additional 1.5 mM MgCl2 (3.0 mM total), 200 nM of each primer, and 8-10 ng DNA. Thermocycling conditions were: 95oC for 2 min, followed by 30 cycles of 95oC for 30 sec, 55oC for 30 sec, and then 60oC for 4 min. PCR products were checked on a 1% agarose gel for an amplicon length of 450-550 bp, and purified by bead-prep using carboxylated magnetic beads (GE Healthcare life sciences, PA, USA) as described in Rohland and Reich (2012). The 2 uL of purified PCR products from the first PCR were used as template to carry out a 25 L second PCR to add index and flowcell sequences to the amplicons. Thermocycling conditions for the second PCR were: 95oC for 2 min, followed by 15 cycles of 95oC for 30 sec, 60oC for 30 sec, and then 72oC for 1 min. The indexed amplified products from the second PCR were purified by bead-prep, and DNA concentration was quantified by using Quantiflour dsDNA System. An equal mass of 35 ng from each PCR product was combined to obtain a single pooled library. The pool was then purified with bead-prep, and subsequent quality control. Sequencing was carried out on a MiSeq Desktop Sequencer (Illumina Inc, CA, USA) running in paired end 2x300 mode at the Center for Biotechnology and Genomics at Texas Tech University.