Metagenome Sequencing on Illumina HiSeq 2500

Metagenome libraries were constructed with size-selected, sonicated DNA fragments of 500 to 700 bp with the NEBnext Ultra DNA II library kit for Illumina (E7645S), as previously described (94 (link)). Paired-end sequencing (2 × 125 bp) of metagenomic libraries was conducted at the University of Utah High-Throughput Genomics Core Facility at the Huntsman Cancer Institute with an Illumina HiSeq 2500 platform. Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 paired end flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq PE Cluster Kit v4-cBot. Following transfer of the flowcell to an Illumina HiSeq 2500 instrument (HCS v2.2.38 and RTA v1.18.61), a 125 cycle paired-end sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC-401-4003).