Inferring Mutational Chronology by Deep-Sequencing Tumor Subpopulations
We acoustically sonicated 1ug of purified DNA to 200-300bp using the Covaris Sonicator S220. Libraries were constructed using NEBNext DNA library Prep Master Mix Set for Illumina (New England Laboratory, #F6040L) for end-repair, 3’adenylation and ligation according to manufacturer’s instructions.  MinElute PCR Purification Kit (Qiagen, #28006) was used for the purification step during library prep.  Agarose electrophoresis was run for excision at 250 to 350 bp for size selection. We then performed 8 cycles of PCR following manufacturer’s direction, using PE5/7 primers (Illumina Inc). Agencourt AMPure XP (Beckman Coulter, #A63881) was used for final purification. Final concentration was measured by quantitative PCR using KAPA Library Quantification Kit (KAPA Biosystems, KK4835) and ABI PRISM real-time machine (Applied Biosystem 7900HT) and size distribution was determined using the 2100 Bioanalyzer (Agilent).  Each library was run at 100 single-end cycles on the Illumina HiSeq2000 or GA2 systems using 4-8 flow cell lanes.  Data was processed using the CASAVA 1.8.1 pipeline (Illumina Inc.) and sequence reads were converted to FASTQ files.