Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

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

The PathoChip design goals were to cover all public NCBI viral genomes and genomic sequences from a broad selection of microorganisms (bacteria, fungi, and parasites) that are pathogenic to humans, using multiple probes to independent target sites in the genome of each species (Fig. 1A) . The resulting collection of pathogen sequences was assembled into a metagenome containing 58 chromosomes of 448.9 million bp and 5,206 accessions for over 4,200 viruses, bacteria, fungi, and parasites. Agilent custom probe design algorithms built for comparative genomic hybridization applications were used to identify 5.5 million probes from the metagenome. Over 3 million of these probes were predicted to have low risk of cross-hybridization with a hu-man genome sequence. Importantly, a subset of these probes that map to unique target regions of the selected pathogens was synthesized on PathoChip v2a microarrays, and a separate subset that covers regions of sequence conservation between at least two or more viruses was synthesized on PathoChip v2b arrays (Fig. 1B ). An enhanced feature of the PathoChip v2b was the inclusion of 2,085 probes tiled throughout the lengths of 22 accessions for agents known to be tightly associated with human cancers.