18s rRNA NGS
Library for 16S and 18S marker gene were prepared following Illumina Protocol. For the 16S, the hypervariable V3-V4 region was amplified using the 341F and 805R primers (Herlemann et al., 2011) while for 18S, the V9 region was amplified using the 1389F and 1510R primers (Piredda et al., 2017) both modified with the required Illumina sequencing adaptors. 16S and 18S PCR amplification was performed on a total volume of 25 l: 12.5 l of appTaq RedMix (Appleton Wood LTD, UK), 1 l of forward and 1 l of reversed primers modified with Illumina over-hanger (10 uM) (IDT, Belgium), 2.5 l of extracted DNA and 8 l of PCR grade water (Merck, Germany). Thermal protocol for 16S gene was 95C for 3 mins followed by 30 cycles at 95C for 15s, 57C for 15s and 72C for 30s, with a final extension step at 72C for 7 mins. For 18S marker gene the thermal protocol was 98C for 3 mins, followed by 30 cycles at 98c for 10s, 56C for 30s and 72C for 15s with a final extension step at 72C for 7 mins. PCR products were cleaned using Agencourt AMPure XP PCR Purification beads (Beckman Coulter), following the manufacturers instructions. 2.5 l of purified PCR product was used in a short secondary PCR, to attach Nextera XT indices, in the presence of 2.5 l of Nextera i5 and i7 index, 12.5 l Appletonwood Taq and 5 l of PCR water. Thermal cycling conditions consisted of an initial denaturation step of 3 min at 95C followed by 8 cycles each of 30 s at 95C, 30s at 55C and 30 s at 72C followed by a final extension step of 5 min at 72C. PCR products were purified using Agencourt AMPure XP PCR Purification beads as described previously. PCR products were quantified using PicoGreen dsDNA quantification assays (Thermo Fisher Scientific), on a POLAR star Omega (BMG Labtech) plate reader. Nextera XT amplicons were then pooled in equimolar concentration. The length of amplicons was verified with Agilent bioanalyzer DNA kit (Agilent, USA). Final quantification of the pooled amplicon library was determined with the NEBNext Library Quant Kit for Illumina (New England BioLabs) prior to sequencing on the Illumina MiSeq (2  300 bp) at University of Essex (UK).