Experimenet 203
Quantitative detection of mutation alleles derived from lung cancer in plasma cell-free DNA by use of anomaly detection with deep sequencing data
To amplify target regions of EGFR gene, PCR primer pairs were designed with Primer3 (http://frodo.wi.mit.edu/). One of a primer pair has a 5-nt index (to descriminate individuals) and adaptor sequences for semicomdactor-sequencing. PCR amplification was done in a 50 ?l reaction mixture containing plasma DNA obtained from 300 ?l of plasma (about 10 ng), 1x KOD -Plus- Buffer (Toyobo), 1 mM MgSO4, 0.2 mM dNTPs, 20 pmol each of primers and 1 unit of KOD -Plus- DNA polymerase (Toyobo). For analysis of sequencing read error, we used genomic DNA from plasma or leukocyte of healthy individuals as PCR templates. The PCR cycling profile was 2 min at 94?C for the initial heating, followed by 40 cycles of 15 sec at 94?C for denaturation, 30 sec at 55?C for annealing and 50 sec at 68 ?C for extension. The PCR products were purified with a QIAquick 96 PCR Purification Kit (Qiagen) or a MinElute PCR Purification Kit (Qiagen), and concentration was determined using a Quant-iT? PicoGreen? dsDNA Assay Kit (Life Technologies) or ND-1000 Spectrophotometer (NanoDrop Technologies). Then, we mixed equal molar amounts of purified PCR products, and diluted to create sequencing libraryies with dH2O (final DNA concentration: 23 pM).