Nimblegen

A CGH was performed using oligonucleotide microarrays (Roche NimbleGen, Inc., Reykjavik, Iceland) in order to identify copy number alterations (CNA) for both age-of-onset subgroups and has been described before [10 (link)]. The degrees of genomic instability were also described in that same study. Both datasets were included in the gene expression omnibus (GEO): LOCRC (GSE108166) and EOCRC (GSE108220).

Enriched and input DNA was UDG fragmented and terminally labeled with Cy5 and Cy3, respectively, and competitively hybridized to custom tiling arrays (Roche NimbleGen) as previously described [21] (link). Arrays contain 50-mer probes overlapping by an average of 42 bp, covering both strands of chromosomes III, VI, and XII, representing 14% of the yeast genome. Nimblescan software was used to obtain Tukey by-weight mean-adjusted log2 Cy5/Cy3 ratio files, and a 50 base-pair pseudomedian sliding window was applied to all files to eliminate outliers [22] (link), [23] (link). To allow for direct peak-height comparison between BrdU chromatin immuno-precipitation on microarrays (ChIP-Chip) samples, each data set was normalized by subtracting the average signal of known unreplicated regions in 200 mM hydroxyurea (HU) within each data set [24] (link). Known unreplicated regions included ChrIII 184–192 kb, ChrVI 80–86 kb, and ChrXII 845–870 kb. Accession number of the replication profile is GSE61743.

Sequencing libraries were prepared from ctDNA using KAPA DNA Library preparation kits (Kapa Biosystems, Wilmington, MA, USA), and genomic DNA sequencing libraries were prepared using Illumina’s TruSeq DNA Library preparation kits (Illumina, San Diego, CA, USA). Libraries were hybridized to custom-designed biotinylated oligonucleotide probes (Roche NimbleGen, Madison, WI, USA) targeting cancer-related genes ranging from 16 to 1,021, including but not limited to all driver mutations in lung cancer (EGFR, ALK, ROS1, RET, KRAS, NRAS, TP53, BRAF, ERBB2, and MET).

The cfDNA libraries were constructed by using the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA) according to the manufacturer's protocol. Libraries were hybridized to custom-designed biotinylated oligonucleotide probes (Roche NimbleGen, Madison, WI, USA) covering ~1 Mbp of genomic regions in the genes and exons most frequently mutated in solid tumors. DNA sequencing was performed using the HiSeq 3000 Sequencing System (Illumina, San Diego, CA) with 2 × 151 bp paired-end reads.

A customized capture array (NimbleGen, Roche) was designed to enrich the genes on chromosome 15q11-q13 (including NIPA1, NIPA2, CYFIP1, SNRPN, SNURF, UBE3A, ATP10A, GABRB3, GABRA5, and GABRG3) according to the Build GRCh 37 assembly genome annotation of NCBI. The sequencing regions included all exons, 1 Kb upstream of the transcription start site, and 3′UTR. To avoid the nonspecific binding of genomic elements to capture arrays, RepeatMasker (http://ftp.genome.washington.edu/RM/RepeatMasker.html) was used to exclude highly repetitive elements from the probe. The method similar to the WindowMasker program was used to identify these regions. Genomic DNA was captured on a NimbleGen’ array following the manufacturer’s protocols. Briefly, the genomic DNA of participants was fragmented to a size of 200 bp by ultrasonoscope. The DNA was sheared by sonication and adaptors were ligated to the resulting fragments. Subsequently, the extracted DNA was amplified by ligation-mediated polymerase chain reaction (PCR), purified, and hybridized to the capture array at 42 °C using the manufacturer’s buffer. The resulting fragments were purified and subjected to DNA sequencing on the Illumina HiSeq2500 Analyzers platform according to the manufacturer’s protocol (Fig. S1).