title: 16S rRNA gene sequencing of stool samples in melanoma patients
design: Stool samples underwent 16S rRNA gene sequencing at the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory. DNA was extracted using the Mo Bio PowerSoil DNA isolation kit, following the manufacturers protocol. The V4 region of the 16S rRNA gene was PCR amplified with the 515F/806R primer pair, which included sequencer adapter sequences used in the Illumina flowcell and sample-specific barcodes (13, 14). Each 25 L PCR reaction contained 9.5 L of Mo Bio PCR Water (Certified DNA-Free), 12.5 L of QuantaBios AccuStart II PCR ToughMix (2x concentration, 1x final), 1 L Golay barcode tagged Forward Primer (5 M concentration, 200 pM final), 1 L Reverse Primer (5 M concentration, 200 pM final), and 1 L of template DNA. The conditions for PCR were as follows: 94 C for 3 minutes to denature the DNA, with 35 cycles at 94 C for 45 s, 50 C for 60 s, and 72 C for 90 s; with a final extension of 10 min at 72 C. PCR products were quantified using PicoGreen (Invitrogen) and a plate reader (Infinite 200 PRO, Tecan). Sample PCR products were then pooled in equimolar amounts, purified using AMPure XP Beads (Beckman Coulter), and then quantified using a fluorometer (Qubit, Invitrogen). Molarity was then diluted to 2 nM, denatured, and then diluted to a final concentration of 6.75 pM with a 10% PhiX spike for sequencing on the Illumina MiSeq.  Amplicons were sequenced on a 151bp x 12bp x 151bp MiSeq run (14).