16S rRNA gene amplicon sequencing of Populus associated soil metagenome
Bacterial and archaeal V3-V4 regions of the 16S rRNA gene were amplified using the primer set 515F (5- GTGCCAGCMGCCGCGGTAA-3)  806R (5- GGACTACHVGGGTWTCTAAT-3). The fungal internal transcribed spacer region 1 (ITS1) was amplified using the primers ITS1F (5- CTTGGTCATTTAGAGGAAGTAA-3)  58A2R (5- CTGCGTTCTTCATCGAT-3). The PAH-RHD genes cluster corresponding to GN bacteria (nahAc, nahA3, nagAc, ndoB, ndoC2, pahAc, pahA3, phnAc, phnA1, bphAc, bphA1, dntAc and arhA1) was amplified using the primer set 610F (5- GAG ATG CATACC ACG TKG GTT GGA-3) and 916R (5- AGC TGT TGT TCG GGA AGAYWG TGC MGT T-3) and the genes cluster corresponding to GP bacteria (narAa, phdA/pdoA2, nidA/pdoA1, nidA3/fadA1) was amplified using the primer set 641F (5- CGG CGC CGA CAA YTT YGT NGG-3) and 933R (5- GGG GAA CAC GGTGCC RTG DAT RAA-3). All microbial regions were amplified in 25 L volumes using 19.125 L of sterile water, 50 nM BSA, 20mM Taq reaction buffer (20mM Tris HCl pH 8.4), 400 nM (0.5 L at 20 M) of each primer, 500 nM of MgCl2, 200 nM dNTP, 0.25 U Taq DNA polymerase and 1 L of DNA template (5-10 ng/L) on a T100 TM Thermal Cycler (Bio-Rad). Thermal cycling conditions for bacteria and archaea were as follows: initial denaturation at 95 C for 5 min; 35 cycles at 95 C for 30 s, 55 C for 30 s, 72 C for 1 min, and a final elongation phase at 72 C for 5 minutes. For fungi, a similar protocol was used except that the annealing temperature was at 59 C and 30 cycles were performed. PAH-RHD genes also had different annealing temperatures set at 57 C and 54 C for GN and GP primer sets, respectively for a total of 30 cycles. Following amplification, PCR products were cleaned using 16 L of magnetic beads (Agencourt AMPure XP, Beckman Coulter Life Science) following the Illuminas protocol 16S Metagenomic Sequencing Library preparation guide (Part #15044223 Rev. B). Then, 400 nM of each Nextera XT unique index primers (2 L at 5 M) were added to 5 L of our PCR product, in a PCR mix containing 50 nM BSA, 20 mM Taq reaction buffer ( 20mM Tris HCl pH 8.4), 500 nM of MgCl2, 200 nM dNTP, 0.25 U Taq DNA polymerase and 12.175 L of sterile water. The PCR product was tagged under the following thermal cycling conditions: 95 C initial denaturation phase for 5 min, followed by 8 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, elongation at 68 C for 30 s, and a final elongation phase at 68 C during 5 min. The indexed amplicons were purified using magnetic beads as described above, and quantified the DNA using the Quant-iTTM Picogreen dsDNA Assay kit (Invitrogen). The libraries were combined in an equimolar ratio and sent them for 2 x 250 bp pair-end sequencing on an Illumina Miseq at The Genome Quebec Service Center (Montral, QC, Canada).