V3V4 sequencing of sputum
Libraries for V3-V4 amplicon sequencing were prepared using NEBNext Ultra DNA Library preparation kit. The amplicons were subjected to a sequence of enzymatic steps for repairing the ends and tailing with deoxyadenosine (dA) followed by ligation of adapter sequences. These adapter ligated fragments were then cleaned up using solid-phase reversible immobilization (SPRI) beads. The clean fragments were indexed during limited cycle Polymerase Chain Reaction (PCR) to generate final libraries for paired-end sequencing. The resulting libraries were quantified before loading on the cBot for cluster generation and sequenced on Illumina MiSeq system platform.