Selective Enrichment and Analysis of Microbial DNA from Contaminating Vertebrate host DNA
Human/fish DNA was extracted from the magnetic bead pellet by first washing the pellet 2 x with 1 ml bind/wash buffer while leaving the tube on the magnetic rack. The wash buffer was carefully removed with a pipette without disturbing the beads. The sample pellet was then suspended in 150 µl of TE and 15 µl of Proteinase K by gentle vortexing (1200 rpm) or by flicking the tube. (Note: The pellet maybe difficult to suspend initially due to the high concentration of genomic DNA bound to the beads.) The resulting slurry was incubated in a heat block or thermomixer at 75oC for 20 minutes, with frequent mixing. After a brief centrifugation of the sample at 13,000 rpm in a microcentrifuge, tubes were placed on the magnetic rack for 2–5 minutes to concentrate the beads on the inner wall of the tube. The resulting supernatant, containing methylated host DNA, was then transferred to a fresh microcentrifuge tube. Samples were either used directly for downstream analysis or stored at –20oC. Libraries for SOLiD 4 and Ion Torrent PGM sequencing were prepared using NEBNext® Fast DNA Library Prep Set (NEB #E6270) with adaptors and primers compatible with their corresponding platform.  Briefly, genomic DNA was sheared to an average size of 200 bp using the Covaris S2.  Sheared DNA was end repaired, ligated to adaptors, and size selected to an average size of 220-240 bp using E-Gels® (Invitrogen) or Ampure XP beads (Agencourt Bioscience) prior to nick translation and amplification.  Sequencing templates were prepared on the SOLiD EZ Bead (Life Technologies).  50 bp sequencing was performed on the SOLiD 4 Platform (Life Technologies). Similarly, sequencing templates were prepared on the Ion One Touch using the Ion One Touch System Template Kit (Life Technologies).  100 bp sequencing was performed on the Ion Torrent PGM using 316 chips (Life Technologies).