title: full-length 16S rRNA gene sequencing of the microbes on cigarette butt surface
design: the bacterial 16S rRNA genes were amplified using the universal bacterial primers 27F (5-AGRGTTYGATYMTGGCTCAG-3) and 1492R (5-RGYTACCTTGTTACGACTT-3). Primers were tailed with PacBio barcode sequences to distinguish each sample. Amplification reactions (20 L volume) consisted of 5 FastPfu buffer 4 L, 2.5 mM dNTPs 2 L , forward primer (5 M) 0.8 L, reverse primer (5 M) 0.8 L, FastPfu DNA Polymerase 0.4 L, template DNA 10 ng and DNase-free water. The PCR amplification was performed as follows: initial denaturation at 95  for 3 min, followed by 27 cycles of denaturing at 95  for 30 s, annealing at 60  for 30 s and extension at 72 for 45 s, and single extension at 72  for 10 min, and end at 4  (T100 Thermal Cycler PCR thermocycler, BIO-RAD, USA). After electrophoresis, The PCR products were purified using the AMPure PB beads (Pacifc Biosciences, CA, USA) and quantified with Qubit 4.18 (Thermo Fisher Scientific, USA).