Soil and Root Microbiome DNA Extraction and Sequencing

Total DNA was extracted from 250 mg of bulk or rhizosphere soil with MoBio’s PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc, Carlsbad, CA). Three grams of root material were pulverized with the Geno Grinder (HORIBA Canada Inc., London, Ontario) and 50 mg were extracted using the PowerPlant Pro DNA Isolation Kit (MO BIO Laboratories, Inc.). DNA samples were quantified by fluorescence detection using the Life Technologies Qubit^® dsDNA HS quantitation kit (Invitrogen, Waltham, MA). Libraries for sequencing were prepared according to Illumina’s “16 S Metagenomic Sequencing Library Preparation” guide (Part#15044223Rev.B). Amplicon libraries for the bacterial 16 S rRNA gene were prepared using primers F343 and R803 [42 (link)] whereas for the fungal ITS1 region the primers ITS1F and 58A2R [43 (link)] were used. We also used the gene coding for the group I chaperonins (CPN60, also known as GroEL or hsp60) to have an independent confirmation of the 16 S rRNA gene results. The cpn60 UT was amplified using the type I chaperonin universal primer cocktail containing a 1:3 ratio of H279/H280:H1612/H1613 as previously described [44 (link), 45 ]. The three pools were then loaded on an Illumina MiSeq sequencer and sequenced in-house using a 600-cycles MiSeq Reagent Kit v3.