AMPLICON of oral metagenome: human child
Cleft area was swabbed 3 times, using sterile FLOQSwabs (COPAN, USA), spinning the swab along the deepest area of the cleft at a perimeter of 0.5 cm around it. The samples were stored at -20 C until DNA extraction. Bacteria released from swab tips by 10s manual agitation in 1 mL of molecular grade water. DNA extracted from 200 l of the water using NucleoSpin 96 soil kit using an EpMotion 5075vt, using lysis buffer SL1 with 150 l Enhancer SX and eluted in 70 l elution buffer. The 16S rRNA gene amplification procedure was performed using a two-step procedure to amplify the hypervariable V3-V4 region of the 16S rRNA gene, using PCRBIO HiFi Polymerase (PB10.41, PCRBIOSYSTEMS, UK) in 25 L reactions (2 L template, 5 L reaction buffer, 1 L forward primer, 1 L reverse primer, 0.12 L Polymerase, 15.88 L molecular grade water (W4502, Sigma, UK). PCR settings: 1 min denaturation (95C), cycles of 15 s at 95C, 15 s at 56C and 30 s at 72C, final extension (72C) for 5 min. PCR products were purified using HighPrep PCR Clean Up System, using 0.65:1 beads to amplicon ratio (vol/vol). The first step used 30 amplification cycles and the modified broad range primers Uni341F (5-CCTAYGGGRBGCASCAG-3) and Uni806R (5-GGACTACHVGGGTWTCTAAT-3). The second step used 15 amplification cycles and primers developed in-house, which contains sequencing adaptors and unique combinations of forward and reverse indeces. A negative template-free control and a positive control (2.0 l, 1.0 ng/l; HM-782D, BEI Resources, VA, US) were included. Normalization using SequalPrep Normalization Plate (96) Kit, concentrated using DNA Clean and Concentrator-5 kit. Quantified using the Quant-iT HS-DNA Assay Kit, adjusted to 1.65 ng/l (4 nM) and sequencing on Illuimina MiSeq, in combination 16 pM. For each run, a 5.0% PhiX internal control was included. All reagents used were from the MiSeq Reagent Kits v2 . Automated cluster generation and 250 paired-end sequencing with dual-index reads were performed. Fastq-files generated directly on the MiSeq instrument. Up to 192 samples, including controls were sequenced per run.