Lung-EGFR-Run8
Sequence analysis of EGFR exon 18, 19, 20 and 21 was performed with the 454 GS-Junior Next Generation sequencer (Roche Diagnostics, Mannheim, Germany). The protocol include the following steps: PCR amplification of the target sequence, purification of the amplified fragments, emulsion PCR, and recovery of the emulsion PCR products that are then loaded on the Titanium PicoTiterPlate (Roche Diagnostics) of the 454 GS Junior instrument for massively parallel pyrosequencing. Primers for the initial amplification run are modified with universal tail sequences and multiple identifiers (MID) nucleotides (Integrated DNA Technologies Inc, www.idtdna.com). A specific couple of MID sequences identifies univocally each sample (target sequence). Approximately 10 ng of genomic DNA were amplified for each exon. In each sequencing run up to 100 - 120 different target sequences were analyzed for parallel pyrosequencing.