title: DNA-seq of H. sapiens: breast cancer patients
design: Genomic DNA from human peripheral blood was isolated either by the standard phenol/chloroform extraction and ethanol precipitation method [15] (n=85) or by the Kingfisher mL instrument (n=16; Thermo Scientific/Thermo Fisher Scientific, Waltham, MA, USA). All tissue samples were immediately prior to DNA extraction manually pulverized under liquid nitrogen by mortar and pestle. Genomic DNA from non-tumor tissues was isolated by the DNEasy Blood & Tissue Kit (n=4; QIAGEN, Hilden, Germany). Genomic DNA from tumors was isolated either by phenol/chloroform extraction and ethanol precipitation (n=29), AllPrep DNA/RNA/Protein Mini Kit (n=15; QIAGEN, Hilden, Germany) or by DNEasy Blood & Tissue Kit (n=56; QIAGEN). All DNA samples were stored at -20 C until use.  DNA was quantified using Quant-iT PicoGreen dsDNA Reagent and Kits (Invitrogen/Thermo Fisher Scientific) and the plate reader Infinite 200 (Tecan Group Ltd., Mnnedorf, Switzerland). DNA purity, determined by the A260/280 and A260/230 ratios, was assessed by NanoDrop Spectrophotometer 2000 (Thermo Scientific/Thermo Fisher Scientific). Only pure DNA characterized by a well-defined singular peak in absorbance spectra at 260 nm and the A260/280 ratio between 1.8 and 2.0 was used for subsequent library preparation. Where necessary, samples were purified by repeated centrifugation and dilution in the Amicon Ultra-0.5mL Centrifugal Filters  30 KDa MWCO (Millipore, Merck, Darmstadt, Germany). 200 ng of DNA was used for library preparation. The kit used for preparation was Agilent Sureselect XT Low Input with the Enzymatic Fragmentation module, according to the manufacturers protocol. Before capture, libraries were pooled in batches of 16 to 18 (1500-1600 ng total) and captured as pools. No other deviations in protocol. For sequencing, the final pool contained tumor:normal DNA in ration 5:1. Samples are paired, e.g. M068K and M068T are normal and tumor sample, resp., from the same patient. Only M215K does not have a tumor for pairing.