Primers list.

<p>Primers list.</p

Mutation analysis using the Ion Torrent PGM.

<p>(A) A sample with a known <i>EGFR</i> exon 19 deletion mutation. (B) A sample with <i>EGFR</i> exon 21 mutation (p.L858R) is present (39% T > G transversion). (C) Sequencing result of <i>EGFR</i> exon 19 wild-type case with lack of in frame deletion. (D) A sample with wild-type <i>EGFR</i> exon 21 "hot-spot" position (100% T, no T >G transversion). Using the Integrative Genomics Viewer (IGV) software, we were able to determine the mutation and fraction of mutant copies.</p

Study workflow.

<p>Study procedure was divided to three main stages: 1) Sequential cutting of the samples using the same knife 2) De-paraffinization using the same tools 3) DNA extraction and mutation analysis.</p

Mutation characteristics of the samples.

<p>Mutation characteristics of the samples.</p

Distribution of the different substitution allele frequencies in the non-”hot-spot” positions.

<p>As shown, allele frequencies higher than 0.3% were present in less than 5% of possible positions and we therefore set 0.3% allele frequency as the detection limit of our method.</p

Comparison of EGFR exon 21 "hotspot" locus (c.2573T>G) mutation fraction and sequencing noise.

<p>There was no significant difference in the fraction of <i>EGFR</i> exon 21 (cDNA position 2573) "hotspot" mutation (T>G) from the same base alteration fraction in the other T residues in the sequence (noise). Statistical significance was assessed by Wilcoxon test.</p