Ampure xp magnetic bead assay

Picogreen dsDNA assay (Thermofisher, MA, USA) will be used for sample DNA concentration measurement. For sequencing, the V3-V4 region of the 16S rRNA gene region will be amplified using the individually distinguishable dual index primer sets. The rDNA amplification primers will be the universal primer set 319F/806R and they will be altered to also encode the Illumina sequencing primer and barcode labelling sequences. PCR will include 30 s at 98 °C, 30 cycles of 10 s at 98 °C, 15 s at 58 °C, and 15 s at 72 °C and three minutes at 72 °C. The AMPure XP magnetic bead assay (BeckmanCoulter Genomics, Danvers, MA, USA) will be used for purification of the amplified DNA. The following formula will be used for recalculation into nM and equalized to 12 nM: $$\left[\mathrm{nM}\mathrm{DNA}\right]=\frac{\left(\mathrm{DNA}\text{concentration}\left(\frac{\mathrm{ng}}{\mathrm{\mu l}}\right)x1e6\left(\frac{\mathrm{\mu l}}{\mathrm{L}}\right)\right)}{(\text{sample fragment size in}\mathrm{bp}x\mathrm{656,4}\left(\mathrm{g}/\text{mole}\right)}$$
If DNA concentrations fell below 12 nM, pooled DNA will be concentrated by vacuum evaporation.

Sample DNA concentration was measured with the Picogreen dsDNA assay (Thermofisher, MA, USA). A PCR amplifying the V3/V4 region of the 16S rRNA gene region was performed with individually distinguishable dual index primer sets, which were developed to distinguish low diversity microbiomes on each sample as has previously been described by Fadrosh et al. [15 (link)]. The universal primer set 319F/806R, altered to also encode the Illumina sequencing primer and barcode labelling sequences, was used during the PCR. PCR conditions were as follows: 30 s at 98 °C, then 30 cycles of 10 s at 98 °C, 15 s at 58 °C, and 15 s at 72 °C and a final step of 3 min at 72 °C.
The amplified DNA was purified with the AMPure XP magnetic bead assay (BeckmanCoulter Genomics, Danvers, MA, USA) quantified as above, recalculated into nM with the formula: “[nM DNA] = DNA concentration (ng/μl) x 1e6 (μl/L) / (Sample fragment size in bp x 656,4 (g/mole))” and equalized to 12 nM. To ensure quality, pooled DNA that did not reach at least 8 nM was not used for 16S rRNA gene sequencing analysis.