Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

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

Whole-genome amplifications (WGAs) of genomic DNA and/or cDNA from random-primed, reverse-transcribed total RNA were performed with the Illustra GenomiPhi v2 kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), the Ovation WGA system (NuGEN, San Carlos, CA, USA), and GenomePlex or TransPlex kits (WGA2 and WTA2; Sigma-Aldrich, St. Louis, MO) using manufacturer-recommended protocols and input amounts. Amplification products were purified with the QIAquick PCR purification kit (Qiagen), and 2 g was used for Cy3 dye labeling by the SureTag labeling kit (Agilent). Cy5 dye labeling was performed on 2 g of human reference DNA from the Agilent SureTag kit, without prior WGA (experiment 1, Table 1 ) or after WGA (other experiments), as a control to report probe cross-hybridization to human DNA. Labeled DNA was purified with SureTag kit spin columns, and specific activities were calculated for use in hybridization reactions.