MDT1_R200
All colony fragments were incubated at 55 C for 1 hour in 450 L of digest buffer with proteinase K (QIAGEN, Hilden, Germany) and DNA was extracted using a QIAxtractor automated genomic DNA extraction instrument, according to manufacturers instructions. The V5 and V6 region of the 16S rRNA gene were amplified using the primers 784F and 1061R (Andersson et al., 2008) with added sequencing adapters (forward: 5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGGATTAGATACCCTGGTA3; reverse: 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCRRCACGAGC TGACGAC3; Illumina overhang adaptor sequences are underlined). Ten l PCRs containing 1 l of template DNA and 0.25 M of each primer were run in triplicate per sample using the Multiplex PCR Kit (QIAGEN, Hilden, Germany). PCR cycling conditions were 95 C for 15 min, followed by 30 cycles of 95 C for 30 s, 55 C for 90 s, and 72 C for 30 s, with a final extension time of 10 min at 72 C. Amplification success was verified on a 1% agarose gel, and successful triplicate reactions were pooled and cleaned using the illustra ExoProStar PCR and Sequence Reaction Clean-Up Kit (GE Healthcare Life Sciences, Pittsburgh PA, USA). Indexing adaptors were added via PCR (8 cycles) according to the Nextera XT DNA library preparation protocol using the Multiplex PCR Kit. Indexed PCR products were purified and normalized using the SequalPrep Normalization Plate Kit (Invitrogen, Carlsbad CA, USA), subsequently quantified using a Qubit dsDNA HS Kit (Invitrogen, Carlsbad CA, USA), and ran on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA, USA) to confirm amplicon length and purity. The 16S rRNA gene amplicon library was sequenced at the KAUST BioScience Core Laboratory on the Illumina HiSeq 2500 platform using the rapid-run mode with 2 x 250 bp overlapping paired-end reads with a 10% phiX control.