Truseq library construction

DNA was extracted from cell lines using a QIAamp DNA Blood Mini QIAcube Kit (QIAGEN, Valencia, CA). In brief, the samples were incubated in proteinase K for 16 hr before DNA extraction. DNA purification was performed using the QIAamp DNA Blood Mini QIAcube Kit following the manufacturer’s instructions (QIAGEN, Valencia, CA). Genomic DNA from tumor samples were used for Illumina TruSeq library construction (Illumina, San Diego, CA) according to the manufacturer’s instructions. Paired-end sequencing resulting in 100 bases from each end of the fragments was performed using Illumina HiSeq2000 instrumentation.

Whole exome sequencing of matched tumor-normal samples and identification of candidate neoantigens was performed as previously described using the VariantDx and ImmunoSelect-R pipelines (Personal Genome Diagnostics, Baltimore, MD) (17 (link),18 (link)). Briefly, the pre-treatment tumor sample underwent pathological review for confirmation of lung cancer diagnosis and assessment of tumor purity. Slides from the FFPE block were macrodissected to remove contaminating normal tissue and peripheral blood was used as matched normal. DNA was extracted from tumor and matched peripheral blood using the Qiagen DNA FFPE and Qiagen DNA blood mini kit respectively (Qiagen, CA). Fragmented genomic DNA from tumor and normal samples was used for Illumina TruSeq library construction (Illumina, San Diego, CA) and exonic regions were captured in solution using the Agilent SureSelect v.4 kit (Agilent, Santa Clara, CA) according to the manufacturers’ instructions as previously described (17 (link)–20 (link)). Paired-end sequencing, resulting in 100 bases from each end of the fragments for the exome libraries was performed using Illumina HiSeq 2000/2500 instrumentation (Illumina, San Diego, CA). Depth of coverage was 209× and 80× for the tumor and matched normal respectively.

Whole exome sequencing was performed on the pre-treatment and post-progression tumor and matched normal samples. Tumor samples underwent pathological review for confirmation of lung cancer diagnosis and assessment of tumor purity. Slides from each FFPE block were macrodissected to remove contaminating normal tissue. Matched normal samples were provided as peripheral blood. DNA was extracted from patients’ tumors and matched peripheral blood using the Qiagen DNA FFPE and Qiagen DNA blood mini kit respectively (Qiagen, CA). Fragmented genomic DNA from tumor and normal samples was used for Illumina TruSeq library construction (Illumina, San Diego, CA) and exonic regions were captured in solution using the Agilent SureSelect v.4 kit (Agilent, Santa Clara, CA) according to the manufacturers’ instructions as previously described (16 (link), 23 (link), 35 (link)). Paired-end sequencing, resulting in 100 bases from each end of the fragments for the exome libraries was performed using Illumina HiSeq 2000/2500 instrumentation (Illumina, San Diego, CA). The mean depth of coverage for the pre-treatment and resistant tumors was 214x and 217x respectively, allowing us to identify sequence alterations and copy number changes in >20,000 genes (Supplementary Table S2).

Genomic purification, library construction, exome capture, next-generation sequencing, and bioinformatic analyses of tumor and normal samples were performed at Personal Genome Diagnostics (PGDx). In brief, genomic DNA from tumor and normal samples were fragmented and used for Illumina TruSeq library construction (Illumina). The exomic regions were captured in solution using the Agilent Canine All Exon kit according to the manufacturer’s instructions (Agilent). Paired-end sequencing, resulting in 100 bases from each end of the fragments, was performed using a HiSeq 2000 Genome Analyzer (Illumina). The tags were aligned to the canine reference sequence (CanFam2.0) using the ELAND algorithm of CASAVA 1.7 software (Illumina). The chastity filter of the BaseCall software of Illumina was used to select sequence reads for subsequent analysis. The ELAND algorithm of CASAVA 1.7 software (Illumina) was then applied to identify point mutations and small insertions and deletions. Known polymorphisms recorded in dbSNP131 (Single Nucleotide Polymorphism database, build 131) (CanFam2.0) were removed from the analysis. Potential somatic mutations were filtered and visually inspected as described previously (38 (link)).

Sample preparation, library construction, targeted capture, next-generation sequencing, and bioinformatic analyses of tumor and normal samples were performed as previously described (23 , 37 (link)). Briefly, DNA was extracted from matched FFPE or frozen tumor tissue and buffy coat samples using the Qiagen DNA FFPE Tissue Kit or Qiagen DNA Blood Mini Kit (Qiagen GmbH). Genomic DNA from tumor and normal samples was fragmented and used for Illumina TruSeq library construction (Illumina) as previously described (23 , 37 (link)). Targeted regions of interest were captured using Agilent SureSelect in-solution capture reagents and a custom targeted panel for genes of interest according to the manufacturer’s instructions (Agilent). Paired-end sequencing, resulting in 150 bases from each end of the fragment for targeted libraries, was performed using the Illumina MiSeq (Illumina).