Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

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

Magnetic bead capture was used to create libraries of targeted sequences for deep sequencing. Selected PathoChip probes with high signals for candidate organisms were synthesized as 5=-biotinylated DNA oligomers (Integrated DNA Technologies, Coralville, IA, USA), mixed as a 36-probe panel, including six probes for HPV16 (Fig. 6A) , and hybridized to pools of tumor targets. Targets were captured by pooling the TransPlex products used for PathoChip screening (100 tumors over six pools) and then adding a probe panel aliquot containing 2.5 pmol of each probe to 150 ng of each target pool in 100-l reaction mixtures with 1ϫ TMAC buffer (3 M tetramethylammonium chloride, 0.1% Sarkosyl, 50 mM Tris-HCl, 4 mM EDTA, pH 8.0). The reaction mixtures were denatured at 100°C for 10 min followed by hybridization at 60°C for 3 h. M-280 streptavidin Dynabeads (Life Technologies, Carlsbad, CA, USA) (1,530 g) were then added with continuous mixing at room temperature for 3 h, followed by three washes of the magnetically captured bead-probe-target complexes with 1 ml 2ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and three washes with 1 ml 0.1ϫ SSC. Captured single-stranded target DNA was eluted in 50 l Tris-EDTA (TE) at 100°C for 10 min.