Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

Baldwin, Don A.; Feldman, Michael; Alwine, James C.; Robertson, Erle S.

The six capture eluates (1 l) were reamplified by GenomePlex reactions (WGA3; Sigma-Aldrich), purified by Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA) using the manufacturer's protocol, and assessed for yield by Qubit double-stranded DNA (dsDNA) assays (Life Technologies, Inc.) and for size distribution by agarose gel electrophoresis. Sequencing libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA), with dual indexing and bead library normalization according to the manufacturer's protocols. After Qubit quantitation, libraries were submitted to the Washington University Genome Technology Access Center (St. Louis, MO) for quantitative PCR (qPCR) quality control measurements, library pooling, and sequencing on one flow cell of an Illumina MiSeq instrument with paired-end 250-nt reads. Approximately 400,000 reads from the six OSCC libraries generated were aligned to the PathoChip metagenome or the human genome using the Bowtie2 aligner (36) in sensitive-local mode. Reads mapping to HPV16 with MapQ scores of 20 or better were identified using Integrative Genomics Viewer 2.3.25 (37) .