Qubit fluorometric quantitation

Prior to DNA extraction, the aliquots were homogenised a second time, to avoid variability caused by changes in the microenvironments after 48 hours of storage. For the first five samples, three different commercially available stool DNA extraction kits were compared: PSP® Spin Stool DNA Kit (Stratec Biomedical, Germany), ZymoBIOMICS DNA Miniprep Kit (Zymo Research, USA) and QIAamp PowerFecal DNA Isolation Kit (Qiagen, formerly moBio, Germany). The workflow is shown in Fig 1. Briefly, for each kit, DNA was extracted from 200 mg aliquots of stool from each of the four storage conditions, according to the manufacturer’s instructions. Aliquots frozen at -80°C were not thawed prior to extraction. DNA purity (A260/A280 and A260/A230), and yield (ng/μL) were determined using the BioDrop μLite spectrophotometer (BioDrop, UK) and by Qubit Fluorometric Quantitation (Invitrogen, USA) at the Central Analytical Facility (CAF) of Stellenbosch University. We used the Kruskal-Wallis test [13 ] for non-normally distributed data to determine whether the different extraction kits or storage methods influenced the DNA yield significantly (p < 0.05). DNA degradation was assessed by gel electrophoresis. The remaining five samples were subjected to the same storage condition comparisons as described above and DNA was extracted with the kit that showed the best performance.

DNA was quantified (Qubit Fluorometric Quantitation, Invitrogen, Life Technologies) and sheared into fragments of 400–600 bp. Illumina libraries were generated using a) the PCR free protocol (NoPCR) [15 (link)] or b) the standard library preparation using the KAPA enzyme [16 (link)] with eight PCR cycles. NoPCR libraries were sequenced on the Illumina HiSeq 2000 platform for 100 paired-end cycles and standard PCR libraries were sequenced on Illumina MiSeq for 150 paired ends cycles using V4 or V5 SBS sequencing kits and proprietary reagents according to manufacturer's recommended protocol (https://icom.illumina.com/). Data were analysed from the Illumina sequencing machines using RTA1.6, RTA1.8 or GA v0.3 analysis pipelines.

Total endophytic community DNA was extracted from surface disinfected root samples using the PowerPlant^®Pro DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA, USA) and the rhizosphere soil community DNA was extracted using the MoBio PowerSoil^® extraction kit (MoBio Laboratories Inc., Carlsbad, CA, USA). DNA exactions were conducted following the manufacture’s protocols. The DNA yield was quantified using Qubit^®Fluorometric Quantitation (Invitrogen) and in a SYBR Safe (Invitrogen) 1% agarose gel by comparison with a high DNA mass ladder (Invitrogen) using a Bio-Rad Gel Doc XR System (Bio-Rad Laboratories, Mississauga, ON, Canada).

Genomic DNA was extracted from the fin clips with the DNeasy 96 Blood and Tissue Kit (Qiagen) according to the manufacturer’s protocol. PCR was carried out using Colorless GoTaq Flexi Reaction Buffer (Promega) with PCR dnd primers^{19 (link)}. The PCR product was purified with QIAquick PCR purification Kit (Qiagen) and measured via Qubit Fluorometric Quantitation (Invitrogen). Purified and quantified PCR products were cloned into PCR4-TOPO using the TOPO TA cloning kit for sequencing (Invitrogen). Cloned fragments were sequenced with M13 forward and reversed primers using BigDye 3.1.

16S Metagenomic Sequencing kit (Illumina, San Diego, CA, USA) was used for library preparation. V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using the primers (5′-CCTACGGGNGGCWGCAG-3′ and 5′ GACTACHVGGGTATCTAATCC-3′) provided by the manufacturer and following the recommended protocol as described before (29 (link)). Library concentration was assessed by Qubit Fluorometric Quantitation (Invitrogen, Waltham, MA, USA). Short-read paired-end amplicon sequencing was performed using Illumina^® MiSeq Instrument for 600 cycles.