title: shotgun DNA sequencing of human stool from melanoma patients
design: Stool samples underwent shotgun metagenome sequencing at the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory. DNA was extracted as above, and quantified using a fluorometer (Qubit, Invitrogen). DNA was then mechanically sheared to the desired insert size of the final library using the Covaris S-series system, and products brought to 15 ul using Agencourt AMPure XP beads (Beckman Coulter). The Apollo 324 system (Takara Bio) was then used for end-repair, A-tailing, Illumina adaptor and barcode ligation, and size-selection to generate the libraries. Libraries are run through 10-15 cycles of PCR with Kapa Biosystems Library Amplification kits, followed by further size selection with Blue Pippin Prep (Sage Science). Final library quantification is achieved using the Qubit Fluorometer (for concentration) and the Agilent 2100 Bioanalyzer (for library insert size and length). Libraries were sequenced on the Illumina HiSeq 2500 on a 2X101bp paired end run.