Phusion Hot Start II DNA Polymerase

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Publication protocol

16S rRNA gene amplification

For each pooled extraction, the V4 region of the 16 S ribosomal RNA (16 S rRNA) gene was amplified in triplets using the universal primers 515 F and 806 R adapted with linker regions and barcoded sequences used for dual-indexing. Platinum SuperFi DNA Polymerase (Thermo Fisher Scientific) and the Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) were both tested for amplification. Each PCR reaction consisted of 12.5 μl of 2x PCR master mix, 6 μl of Microbial DNA-Free water (Qiagen GmbH), 1.25 μl of each primer (0.5 mM each, Metabion, Martinsried, Germany) and 4 μl of template in a total reaction volume of 25 μl. PCR cycling conditions comprised of a pre-denaturation step of 30 s at 98 °C, followed by 30 cycles of 98 °C for 10 s, 55 °C for 15 s and 72 °C for 60 s, and a final 10 min extension step at 72 °C. For a selection of four samples, five additional cycles were added to the amplification procedure to examine if additional cycles may be favorable for samples with low concentrations. The amplicon triplets were pooled, purified using 0.7x AMPure XP beads (Beckman Coulter, Brea, USA) and quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Amplicon integrity was verified for a representative number of 11 samples using a BioAnalyzer 2000 (Agilent, Palo Alto, USA) prior to pooling equimolar amounts (10 nM) of each amplicon for sequencing. For the blank samples, the maximum volume (5 μl) of sample was added to the library, as the concentrations prior to sequencing were below 10 nM. Illumina MiSeq. 2 × 250 bp paired-end sequencing (Illumina V2 chemistry) was performed in the Transcriptome and Genome Analysis Laboratory at the University of Göttingen. All generated read files analyzed in this study were uploaded to the NCBI Sequence Read Archive (SRA) (SRP125723).


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