Hiseq 3000 sequencing system

Fresh specimens were fixed and cross-linked with 37% formaldehyde according to known methods, followed by cell lysis. Then, the quality of the samples was examined, and those deemed to be good quality were utilized for Hi-C fragment preparation. Chromatin digestion was performed using restriction endonuclease (HindIII/MboI) and the digestion effect was examined. Next, on completion of biotin labeling, blunt-end ligation, and DNA purification, Hi-C samples were prepared and subsequently taken for DNA quality testing. The samples that passed the DNA quality test were utilized for standard library preparation. On completion of removal of biotin, sonication, end repair, and “A” tailing, the fragments containing biotin were captured and bound to ligated adapters, and eventually Hi-C libraries were obtained after polymerase chain reaction (PCR) amplification and a final quality control. The next sequencing step was completed utilizing a HiSeq 3000 sequencing system (Illumina, San Diego, CA, USA). The specimens were further analyzed for the overall understanding of intrachromosomal (cis-acting) and interchromosomal (trans-acting) and NPC specific chromatin interaction sites. Sample information is shown in Table 1.

Both cfDNA and gDNA libraries were constructed with the KAPA DNA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA) using the manufacturer's protocol. Capture probes were designed to cover coding sequences and hot exons of 1021 genes that are frequently mutated in solid tumors. A detailed description of the capture experiments has been reported previously [28 (link)]. Libraries were hybridized to custom-designed biotinylated oligonucleotide probes (Integrated DNA Technologies, Iowa, IA, USA). DNA sequencing was performed using the HiSeq 3000 Sequencing System (Illumina, San Diego, CA) with 2 × 101-bp paired-end reads. In Table S2, all genes included in our panel are listed. Clonal hematopoietic mutations were filtered as previously described [29 ], including those in DNMT3A, IDH1, and IDH2 and specific alterations within ATM, GNAS, and JAK2.

Targeted region capture combined NGS was performed for the 41 NSCLC patients. Genomic DNA sequencing libraries were prepared using the protocols recommended by the TruSeq DNA Library Preparation Kit (Illumina, San Diego, CA, USA). For samples close to the minimum input requirement, additional pre-capture polymerase chain reaction cycles were performed to generate sufficient product for hybridization. The libraries were hybridized to custom-designed probes (Integrated DNA Technology, Coralville, IA, USA) including all exons of 170 genes and selected introns of anaplastic lymphoma kinase, RET, and ROS1 for the detection of genomic rearrangements. DNA sequencing was performed on a HiSeq3000 sequencing system (Illumina) with 2×75 bp paired-end reads. The reads were aligned to the human genome build GRCh37 using BWA (a Burrows-Wheeler aligner). Somatic single nucleotide variant and indel calls were generated using MuTect and GATK, respectively. Somatic copy number alterations were identified with CONTRA. Genomic rearrangements were identified by software developed in-house to analyze chimeric read pairs.