16S rRNA metagenomic sequencing of Homo Sapiens: induced sputum COPD patients
DNA was extracted from samples using sputum DNA collection, preservation and isolation kit (Norgen Biotek Corporation, Canada, Catalogue no. RU46100). DNA purity and concentration were determined using both spectrophotometer and a Qubit fluorescent based kit.  The V3-V4 hypervariable region of the 16S rRNA gene was PCR amplified using 16S rRNA specific 341F (5 NNNNNNNNNNCCTACGGGNGGCWGCAG) and 785R (5 NNNNNNNNNNGACTACHVGGGTATCTAAKCC) primers.  All samples were tagged with unique 10 nucleotide-sequences (barcodes) that were incorporated into the forward primer. The PCRs included about 1-10 ng of DNA extract (total volume 1l), 15 pmol of each forward primer and reverse primer in 20 L volume of 1x MyTaq buffer containing 1.5 units MyTaq DNA polymerase (Bioline GmbH, Luckenwalde, Germany) and 2 l of BioStabII PCR Enhancer (Sigma-Aldrich Co). PCRs were carried out for 30 cycles. DNA concentration of amplicons of interest was assessed by gel electrophoresis. About 20 ng amplicon DNA of each sample were pooled for up to 48 samples carrying different barcodes. The amplicon pools were purified with one volume Agencourt AMPure XP beads (Beckman Coulter, Inc., IN, USA) to remove primer dimer and other small mispriming products, followed by an additional purification on MiniElute columns (QIAGEN GmbH, Hilden, Germany). About 100 ng of each purified amplicon pool DNA was used to construct Illumina libraries using the Ovation Rapid DR Multiplex System 1-96 (NuGEN Technologies, Inc., CA, USA). Illumina libraries (Illumina, Inc., CA, USA) were pooled and size-selected by preparative gel electrophoresis.  Bacterial 16S rRNA gene amplicons were sequenced targeting the V3 (300 bp read length, paired-end protocol) region using the Illumina Miseq platform.