16S rRNA gene sequencing of tonsillar tissue samples
Bacterial genomic DNA was extracted and purified using QIAamp DNA Stool Mini Kit  (Qiagen) with minor modifications. Lysing Matrix D tubes (MPBio) were used for tissue disruption and an additional extraction step was introduced after incubation of bacteria with a lysis buffer (20 mg/ml lysozyme  Sigma 62970-5G-F, 2 mM EDTA, 1.2% Triton, 20 mM Tris-HCL) for an optimal yield of bacterial DNA. PCR amplification of the V4 region of bacterial 16S rRNA gene was performed with barcoded primers following a dual-index paired-end sequencing approach [46]. PCR was performed using KAPA HiFi polymerase (Roche) under the following cycling conditions: initial denaturation at 98C (2 min), 25  40 cycles of  98C (30 sec), 55C (30 sec), 72C (20 sec) and final elongation at 72C (7 min). PCR products including a negative control were electrophoresed on a 1% agarose gel to ensure high-fidelity amplification. NucleoMag NGS (Macherey-Nagel) was used for PCR clean up and size selection. Individual libraries were normalized with SequalPrepTM Normalization Plate Kit (ThermoFisher) according to the manufacturers instruction and combined into a pooled library. The combined library was qualitatively and quantitatively assessed with a High Sensitivity D1000 ScreenTape device (Agilent) and on a Qubit fluorometer (ThermoFisher). 16S rRNA V4 amplicon sequencing was performed on the Illumina MiSeq v2 platform.
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