Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

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

Independently, low-complexity regions in the metagenome were (36) . Criteria for unique regions were 250 to 300 bp and Ͻ50 contiguous bp with Ͼ70% identity to a sequence in any other metagenome accession. Conserved viral regions were similarly identified using criteria of 70 to 300 bp and Ͼ70% identity to at least one other virus but not to human sequences. Agilent-designed probes that mapped to unique or conserved regions of the pathogen genomes, or any prokaryotic or eukaryotic pathogen accession, were added to the microarray design by default if fewer than 10 probes were available for the source accession. Otherwise, the probes were filtered for minimum interprobe spacing of 100 bp and distribution that roughly covers the full length of each accession while limiting the number of probes to 10 to 20 per accession. The number of probes was not restricted for known oncogenic viral agents, creating a saturation tiling set covering these accessions. Entire genome sequences were covered to the extent possible with all available Agilent-designed probes. The microarray was supplemented with an additional number of predesigned aCGH probes for 660 genes and 602 intergenic regions from the human genome and Saccharomyces cerevisiae. Probes and accession annotations are available in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/ geo/). Sample preparation. Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA). Plasmid minipreps were prepared from pBR322 subclones carrying JC or BK polyomavirus genomes (J. C. Alwine, University of Pennsylvania, Philadelphia, PA) and from pUC19 carrying the human papillomavirus 16 (HPV16) genome (obtained from Peter Howley, Harvard Medical School, Boston, MA).