Urine NGS DNA Extraction Protocol

The amount of urine required for NGS has not yet been established; however, many studies have reported that at least 30–50 mL has proven to be successful for studies of the bladder and genitourinary microbiota [14 (link),23 (link),24 (link),25 (link)]. Therefore, in this study, we used 50 mL urine samples for NGS. Upon collection, samples were immediately sent to the lab to be stored at −20 °C [26 (link)]. Within 1–2 days, samples were sent frozen with dry ice in the genetic lab. DNA was extracted with the Chemagic DNA Stool Kit (PerkinElmer Inc., Waltham, MA, USA) according to the manufacturer’s recommendations with pretreatment and modification. The urine specimens (50 mL) were centrifuged at 3000 rpm for 15 min, and the urinary pellets were washed twice with a 10-fold volume of phosphate-buffered saline (PBS). The washed pellets were resuspended in 700 uL of lysis buffer, and this suspension was then added to a silica bead tube. For cell lysis, the bead-suspension mix was vortexed at maximum speed for 5 min. After centrifugation, the supernatant was further subjected to thermal disruption, proteinase K digestion, and bead-binding and elution according to the manufacturer’s instruction. The DNA concentration was determined fluorometrically on the Qubit^® 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) using the Qubit^TM dsDNA HS Assay Kit.