Selective Enrichment and Analysis of Microbial DNA from Contaminating Vertebrate host DNA
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).  Illumina library construction and sequencing (MiSeq, GAIIx): genomic DNA (500 ng) was sheared using a Covaris S2 device to obtain an average fragment size of ~350 bp. Illumina paired-end sequencing libraries were constructed using the NEBNext® DNA library prep reagent set for Illumina (NEB #E6040) following the standard Illumina sample preparation protocol. PCR library amplifications were performed with an MJ Research Thermo Cycler PTC-225 (City, State). Illumina PE 1.0 and 2.0 primers, or PE 1.0 and 2.0-derived indexing primers, were used to amplify adapter-ligated library fragments by PCR. Libraries were amplified using optimized PCR conditions described in [33].