title: Sequencing the V3-V4 region of the 16S rRNA gene: BAL sample from an adult female
design: Amplicon library preparation was performed using an Illumina MiSeq System. For each sample, the V3 and V4 region of the bacterial 16S rRNA gene was amplified, using the primers recommended for the Illumina [Part#15044223F/Part#15044223R], which amplifies bacterial and archaeal 16s rRNA genes. Each amplicon was generated of 25 ?l reactions with the following reaction conditions: 5 ?l PCR grade water, 12.5 ?l  2x KAPA HiFi HotStart ReadyMix, forward and reverse primers (5 ?l ) and 2.5 ?l template DNA. Reactions were run on a Veriti Thermal Cycler (Applied Biosystems) with the following cycling conditions: initial denaturing at 95C for 3 min followed by 25 cycles of denaturation at 95C for 30 seconds, annealing at 55C for 30 seconds, and extension at 72C for 30 seconds, with a final extension of 5 minutes at 72C. Purification was then performed using  Ampure XT as per the manufacturer instructions.