Shallow Whole-Genome Sequencing Pipeline

Fifteen LGGs (van Thuijl et al. 2014 (link)), two SCCs (Bhattacharya et al. 2011 (link)), and the breast cancer cell line BT474 were used to develop and illustrate the shallow WGS pipeline presented. All material used from LGG and SCC tumors was derived from FFPE archival samples. Patient consent was obtained for SCCs as published previously (Bhattacharya et al. 2011 (link)). LGG samples were collected from five Dutch hospitals (VU University Medical Center in Amsterdam, Academic Medical Center in Amsterdam, Radboud University Medical Center in Nijmegen, St. Elisabeth Hospital in Tilburg, and Isala Klinieken in Zwolle). Sample collection was approved by the Medical Ethics Committees of all five hospitals. Areas containing > 60% tumor cells were outlined on hematoxylin and eosin-stained slides, and 10 subsequent 10-μm sections were used for DNA isolations.
DNA from the LGG samples was isolated as previously described (van Essen and Ylstra 2012 (link)). DNA concentrations were measured with the Nanodrop 2000 (Fisher Scientific), and 500 ng was used as input for Shallow WGS laboratory preparation. DNA from the SCC samples was isolated as previously described (Bhattacharya et al. 2011 (link)), DNA concentrations were measured with the Qubit 2.0 fluorometer dsDNA BR Assay (Life Technologies), and 250 ng DNA used as input for shallow WGS laboratory preparation. The BT474 breast tumor cell line was cultured and DNA isolated as previously described (Krijgsman et al. 2013 ). DNA concentration was measured with the Qubit fluorometer and 250 ng used as input.