title: Metagenome sequencing (16S full-length) of Control : adult oral
design: The sequencing libraries is prepared according to the PacBio amplicon Template Preparation and Sequencing protocols to amplify the 27F and 1492R region. The input gDNA 2ng was PCR amplified with 10 LA PCR Buffer II (Mg2+free), 2.5mM of dNTP mix, 2.5mM MgCl2,  500nM each of the F/R PCR primer, and 5U of TaKaRa LA Taq (Takara, Kusatsu, Japan). The cycle condition for PCR was 5 min at 94C for heat activation, and 29 cycles of 30 sec at 94C, 30 sec at 53C and 90sec at 72C, followed by a 5-min final extension at 72C. The primer pair with asymmetric barcoded adapters for the amplifications were as follows: 27F-F: 5- AGRGTTYGATYMTGGCTCAG -3, 1492-R: 5- RGYTACCTTGTTACGACTT -3. The PCR product was purified with SMRTbell cleanup beads. The purified product is then quantified using Quant-IT PicoGreen (Invitrogen) and qualified using the TapeStation D5000 Screen Tape (Agilent Technologies, Waldbronn, Germany). For PacBio Sequel IIe sequencing, 500ng of pooled amplicon DNA was used for library preparation. Total 10uL library was prepared using PacBio SMRTbell prep kit 3.0. SMRTbell templates were annealed Sequel II Bind Kit 3.1 and Int Ctrl 3.1. The Sequel II Sequencing Kit 2.0 and SMRT cells 8M Tray was used for sequencing. SMRT cells (Pacific Biosciences) using 10hr movies were captured for each SMRT cell using the PacBio Sequel IIe (Pacific Biosciences) sequencing platform by Macrogen (Seoul, Korea). The subsequent steps are based on the PacBio Sample Net-Shared Protocol, which is available at https://www.pacb.com/