Selective Enrichment and Analysis of Microbial DNA from Contaminating Vertebrate host DNA
Genomic DNA Preparation  Bacterial cells (E. coli MGL 1655), IMR90 cells (ATCC CCL-186) were suspended in 50 mls of lysis buffer, 50 mM Tris (pH 7.5), 20 mM EDTA, 1% SDS, 200 µl/ml Proteinase K (NEB #P8102S), and incubated at 50oC for 3 hours with frequent mixing. The samples were extracted with Tris-EDTA equilibrated phenol, once with methylene chloride, and then two volumes of ethanol were added. The product was then spun and the pellet air dried and suspended in 200 µl of 10 mM Tris (pH 7.5), 1 mM EDTA (TE). 20 µl of 0.4 mg/ml RNase A was added and the sample incubated at 37oC for one hour. The samples were phenol/ methylene chloride extracted as before, ethanol precipitated, and the dried pellets brought up in TE to a concentration of 100 µg/ml. DNA quality and quantity were assayed by agarose gel electrophoresis of the samples alongside a DNA marker (2-log DNA ladder, NEB #N3200S).   Metagenomic DNA Preparation  Human buffy coats/leukocytes blood (Innovative Research) was suspended in 50 mls of lysis buffer and treated as above.   SOLiD 4, Ion Torrent PGM, and Illumina Library Construction and Sequencing  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).