16S amplicon sequencing of Arabidopsis thaliana rhizosphere
The 16S rRNA gene amplicon libraries were constructed using the 16S metagenomic sequencing library protocol for the Illumina MiSeq system (Illumina, 2013). The protocol was adapted to incorporate 16S V3-V4 phasing primers to increase complexity and decrease the need for PhiX spiking (De Muinck et al., 2017). Moreover, the barcoded PCR primers described by Baym and co-workers (2015) were used in a total PCR reaction volume of 25 l instead of 50 l. Three l of the purified samples was run on a 1% agarose gel to check the amplicon size and the concentration of double-stranded DNA was determined with the Qubit Broad-Range kit (Thermofisher Scientific). Each sample was adjusted to 2 ng/l and pooled in a 1:1 ratio. The pooled library was sent for sequencing on a 2x300 MiSeq at the USEQ sequencing facility (Utrecht University, the Netherlands). The fungal amplicon libraries were constructed in the same way but using primers targeting the internal transcribed spacer (ITS) region 2 of the ribosome encoding genes (Ihrmark et al., 2012).