PRJNA422575
Homo sapiens Genome sequencing and assembly
DeTiN estimates tumor in normal (TiN) based on tumor and matched normal sequencing data. The estimate is based on both candidate SSNVs and aSCNAs. DeTiN then applies the joint TiN estimate to reclassify SSNVs and InDels as somatic or germline.To evaluate the performance of deTiN on experimentally derived sequencing data we mixed tumor and normal cell lines in various ratios. For the tumor sample we selected the cell line CRL-2321D and for the normal CRL-2362D. DNA from these samples was mixed in equal amounts to generate a 0.5 TiN pool with total mass of 500ng. We then mixed pure tumor and pure normal with this pool to generate the other mixtures. Samples were volume checked using nanodrop to ensure we achieved the desired mixtures.We then performed library preparation. Briefly, dsDNA was quantified by Picogreen fluorescence assay using providedDNAstandards, 100ng of DNA were fragmented to obtain 150bp pieces by sanitation using a Covaris E210 instrument. Solid phase reversible immobilization purification and library construction were performed using AMPure XP Beads, KAPA Library Preparation and KAPA Library Amplification Kits. Library preparation was performed in 96-well plates on an Agilent Bravo Liquid Handler.Finally we performed hybrid selection, capture and sequencing. DNA was processed through two hybridization events using the Illumina Content Exome Rapid Capture Kit. Samples were normalized to 2ng/uL and pooled. Quantitative PCR (qPCR) was then performed on the pool in order to normalize it to 2nM, before using 0.1M NaOH to denature. Samples were sequenced on Illumina HiSeq2500 machines in Rapid Run mode using 76 base-pair, paired-end reads.