title: 5272_mix-Lib6
design: DNA isolation was performed using Epicentre MasterPure Complete DNA and RNA Purification Kit (Lucigen) following the manufacturers protocol with the following modifications: pre-lysis incubation of samples with Lysozyme (50 mg/mL) and Mutanolysin (2 mg/mL) at 37C and included an RNAase step for 30 minutes at 37C. Library preparation was performed as previously described (30, 38) with the following modifications: during the first PCR amplification step, forward primer 1 was added to a reverse primer mixture consisting of primers 2 and 3. This reverse primer reaction mixture consisted of primer 2, a truncated version of primer 3, which was added at a 10:1 volumetric ratio (primer 2:primer 3, respectively) to enhance amplification and avoid primer dimer formation initially observed in PCR reactions with only primer 1 and primer 3. The second PCR amplification step, the barcode addition, was completed using primer 4 and barcode primers (BC1-12) listed in Table S1. All PCR reactions were completed using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA) following the manufacturers protocol. For deep sequencing on Illuminas HiSeq 2500 platform, a custom sequencing primer was used.