GSM8991795: SVA-positive vesicular fluid sample, subject 7; Sus scrofa domesticus; OTHER
SVA positive vesicular fluid samples were taken from SVA positive pigs (as confirmed by RT-PCR). Collected fluid was immersed in oral fluid from the test subject. Oral fluid was collected from healthy pigs as controls. These samples were stored at -80°C until processing. RNA from VFE samples underwent the same Trizol extraction procedure with libraries generated the same day to minimize RNA degradation. Nanopore sequencing libraries were generated in a microfluidic device, which also previously performs RT-LAMP and bead purification. A brief description is as follows: 1) 1µl of sample was added to a premade mixture of 12.5µl LAMP mastermix (M1700S, New England Bio), 9µl H2O, and 2.5µl LAMP primer mix, then loaded into the microfluidic device; 2) The device is placed on a hotplate to perform isothermal amplification at 65°C for 60 min; 3) The device is then removed from the hotplate, allowed to briefly cool, then loaded with SPRI paramagnetic beads for DNA isolation (SPRIselect, Beckman Coulter) ; 4) a magnet is applied underneath the device to immobilize the beads; 5) 75% ethanol solution is loaded to wash the beads; 6) a mixture of ONT fragmentation solution is loaded into the device and DNA is eluted into the solution from the bead surface (Oxford Nanopore RBK004); 7) The device is incubated for 5 min for fragmentation and barcoding of DNA; 8) A fresh tubing is plugged into the microfluidic outlet and then the library is flushed out, ready for pooling. 4-8 samples are pooled per sequencing run, with 1ul sequencing adaptor  (Oxford Nanopore RBK004)solution added to the pooled libraries.