*3.2. Targeted Sequencing Identified Somatic Mutations in the Lung Cancers*

Targeted sequencing was performed on 76 surgically resected tumors and 8 lymph nodes obtained from 37 patients, with their blood cell samples utilized as normal controls. The mean coverage depth was 1411-fold for cancer samples (range, 106- to 5096-fold) and 1387-fold for blood cell samples (range, 76- to 6960-fold). Sequence analyses detected 314 somatic mutations with an allele fraction ≥1% from 84 cancer lesions (1–54 mutations per tumor) (Supplementary Table S3). Among these mutations, 137 mutations (44%) were present at an allele fraction ≥20% (Supplementary Table S3).

In 29 patients, the gene, amino-acid substitution and nucleotide changes that were caused by these somatic mutations within individual tumors composing the multiple lung cancers lacked consistency (Figure 1, Supplementary Table S3). Thus, there were no shared or overlapping mutations among the individual lung cancers detected in these patients. This finding demonstrated that the multiple lung cancers in these cases were independently developed primary lung cancers (Figure 1). Meanwhile, in 8 patients, the gene mutation profile was consistent among the individual tumors, suggesting the presence of intrapulmonary metastasis (Figure 2). Importantly, in these cases, nucleotide position and mutation variance were entirely consistent across the tumors (Supplementary Table S3).

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**Figure 1.** Heatmap of gene mutations in patients with double or triple primary lung cancers. These maps visualize the gene mutations in each cancer. Two or three lung cancers in each patient were characterized by different mutation profiles and all patients were diagnosed with double or triple primary lung cancers. Case 21, 22 and 28 were metachronous cancers, while the other cases in this figure were synchronous cancers. The remaining 5 cases of double primary lung cancers (cases 12, 18, 24, 26 and 34 in Table 1 and Table S2) that are not shown in this figure are described in detail in the Case presentation section. Black, red and blue indicate tumor 1 (T1), T2 and T3, respectively. r, right; S, segment; AF, allele fraction; MIA, microinvasive adenocarcinoma; AIS, adenocarcinoma in situ.

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**Figure 2.** Heatmap of gene mutations in patients with metastatic lung cancers. The mutation profiles were consistent between the individual tumors in each case and the tumors were identified as

intrapulmonary metastasis. Case 33 was synchronous cancers, while the other cases in this figure were metachronous cancers. The remaining 2 cases of metastatic lung cancers (cases 10 and 30 in Table 1 and Table S2) that are not shown in this figure are described in detail in the Case presentation section. r, right; S, segment; AF, allele fraction.
