16S profiling of human lung microbiota:Bronchoalveolar lavage fluid
PCRs were commenced running a pre-amplification for 20 cycles using GM3 and 1061R. One µl of this PCR was transferred to the second 20µl PCR which was done using the standard primers and was run for another 20 cycles. Subsequent PCRs included 5 ng of DNA extract, 15 pmol of each forward primer 341F 5Õ-NNNNNNNNNNTCCTACGGGNGGCWGCAG and reverse primer 785R 5Õ- NNNNNNNNNNTGACTACHVGGGTATCTAAKCC in 20 µL volume of 1 x MyTaq buffer containing 1.5 units MyTaq DNA polymerase (Bioline) and 2 µl of BioStabII PCR Enhancer (Sigma). This provided coverage of the V3-V4 region for microbiome analysis. For each sample, the forward and reverse primers had the same 10-nt barcode sequence. PCRs were carried out for 30 cycles using the following parameters: 2 min 96¡C pre-denaturation; 96¡C for 15 s, 50¡C for 30 s, 70¡C for 90 s. DNA concentration of amplicons of interest was determined 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 AMPure XP beads (Agencourt) to remove primer dimer and other small mis-priming products, followed by an additional purification on MinElute columns (Qiagen). Purified amplicon pools of DNA (100 ng) were used to construct Illumina libraries using the Ovation Rapid DR Multiplex System 1-96 (NuGEN). Illumina libraries were pooled, size selected, and sequenced on an Illumina MiSeq using V3 Chemistry (Illumina).