Nanopore 16S amplicon sequencing
The bacterial full-length 16S rRNA gene was amplified via PCR using the primer pair S-D-Bact-0008-a-S-16 (5-AGRGTTYGATYMTGGCTCAG-3) and S-D-Bact-1492-a-A-16 (5-TACCTTGTTAYGACTT-3) (Klindworth et al., 2013). The following reagents and concentration were used for the first PCR reaction: 200 M dNTPs, 200 nM of each primer, 1 U of VWR Taq DNA Polymerase (VWR, WR International bvba/sprl, Belbium), 1 X key Buffer supplemented with MgCl2 (1.5 mM), and 10 ng of DNA template (final volume: 50 L). PCR started with an initial denaturation step at 94C for 1 min, followed by 35 cycles of amplification (denaturing, 1 min at 95C; annealing, 1 min at 49C; extension, 2 min at 72C), and a final extension at 72C for 10 min. A negative control without DNA template was included. Agencourt AMPure XP beads (Beckman Coulter) at 0.5X concentration were used to remove primer-dimers and non-specific amplicons. DNA concentration was measured using the QubitTM dsDNA HS Assay kit (Qubit 2.0 Fluorometer, Thermofisher, Waltham, USA). Then, the Ligation Sequencing Kit 1D (SQK-LSK108) was used to prepare the amplicon library to load into the MinION. The flow cell (R9.4, FLO-MIN106) was primed and then loaded as indicated in the ONTTM protocols.