ASV of microbiome : sputum
DNA was extracted using the DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Extracted DNA was quantified using Quant-IT PicoGreen (Invitrogen). Sequencing libraries were prepared following the PacBio Amplicon Template Preparation and Sequencing protocols, targeting the 27F and 1492R regions. Input gDNA (2 ng) was polymerase chain reaction (PCR)-amplified with 10 LA PCR Buffer II (Mg2+-free), 2.5 mM dNTP mix, 2.5 mM MgCl2, 500 nM each of forward and reverse PCR primers, and 5 U of TaKaRa LA Taq (Takara, Kusatsu, Japan). The PCR conditions were initial heat activation at 94C for 5 min, followed by 25 cycles of 30 s at 94C, 30 s at 53C, and 90 s at 72C, with a final extension at 72C for 5 min. The primer pairs with asymmetric barcoded adapters used for the amplifications were: 27F-F: 5- AGRGTTYGATYMTGGCTCAG -3 and 1492-R: 5- RGYTACCTTGTTACGACTT -3. PCR products were purified using SMRTbell cleanup beads, quantified using Quant-IT PicoGreen (Invitrogen), and qualified using the TapeStation D5000 Screen Tape (Agilent Technologies, Waldbronn, Germany). For PacBio Sequel IIe sequencing, 500 ng of pooled amplicon DNA was used for library preparation. A total of 10 L of the library was prepared using the PacBio SMRTbell prep kit 3.0. SMRTbell templates were annealed using the Sequel II Bind Kit 3.1 and Int Ctrl 3.1. Sequencing was performed using the Sequel II Sequencing Kit 2.0 and SMRT cells 8M Tray. Data acquisition involved 10-hour movies captured for each SMRT cell using the PacBio Sequel IIe (Pacific Biosciences) sequencing platform, conducted by Macrogen (Seoul, Korea). Subsequent steps followed the PacBio Sample Net-Shared Protocol, which is available on the PacBio website (https://www.pacb.com/).