title: Soil nemtaode metagenome from Natural Pasture
design: The recovered nematodes were transferred to an Eppendorf tube after being centrifuged for 10 min at 15,000 rpm to reduce the remaining water. The pellets were used for DNA extraction by using Kit PureLink Genomic DNA Mini. The eluted DNA was stored at -20C until used as a polymerase-chain- reaction (PCR) template. The 3' part of SSU rDNA was amplified by PCR using Integrated DNA Technologies forward primer sequence NF1: 5'-GGT GGT GCA TGG CCG TTC TTA GTT - 3';, and reverse primer sequence 18Sr2b: 5'- TAC AAA GGG CAG GGA CGT AAT  3';. Reactions were carried out at a 25 l final volume including 16.25 l ultra-pure water, 5 l buffer Phusion HF X5 (Biolabs), 0.5 l dNTPs, 1 l F primer, 1 l R primer, 0.25 l Phusion High Fidelity DNA Polimeras Biolabs Phusion HF DNA Polymerase and 1 l of DNA from each sample.  96-Well Thermal Cycler (Applied Biosystems). The PCR program involved a five- stage procedure: 1) denaturation step of 30 s at 98C; 2) a denaturation step of 10 sec at 98C; 3) an annealing step of 30 s at 56C; 4) an extension step of 1.5 min at 72C and 5) a final extension step of 10 min. at 72C.The step 2-4 X20 cycles. All the PCR products were diluted with ultra-pure water, and undergo a second PCR amplification using F primer, with adaptors for nematodes. Negative controls were included for each amplification. For Ilumina sequencing, NEM ILL Reverse: 5-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GTA CAA AGG GCA GGG ACG TAA -3'; and NEM ILL Forward: 5'- TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG GGT GGT GCA TGG CCG - 3'. The PCR program involved (1) a denaturation step of 30 s at 98C; (2) a denaturation step of 10 s at 98C; (3) an annealing step of 30 s at 56C; (4) an extension step of 1 min and 30s at 72C; and (5) a final extension step of 10 min at 72C. An amount of five l of all PCR products, along with 1 kb DNA ladder (Thermo Scientific), was electrophoresed in 1% agarose gel (SeaKem) and stained with 1 l GelStar. No PCR products were observed in the negative controls, and the correct size of the positive band was observed. The remaining 20 l DNA of each sample was electrophoresed in 0.5% low-melt agarose (CSL- AG100) and stained with 3 l gel.