Truseq rapid pe cluster kit

WGBS libraries were prepared using the EpiGnome Methyl‐Seq Kit (Epicentre, EGMK81312), according to the manufacturer's protocol. Library quality was assessed with the Agilent 2100 Bioanalyzer using the High‐Sensitivity DNA Kit (Agilent, CA, USA). DNA was quantified using the KAPA Library Quantification Kit by quantitative PCR (KAPA 6 Biosystems). Libraries from patient specimens were analysed with 70 bp paired‐end sequencing on the Illumina HiSeq 2500 platform using TruSeq Rapid SBS Kit ‐ HS (50 cycle) and TruSeq Rapid PE Cluster Kit ‐ HS. Six samples were multiplexed across two lanes. Sequencing was performed multiple times to gain sufficient coverage.

Genomics DNA (250 ng) from each tissue was sheared in a Covaris S220 ultrasonicator (Covaris, Woburn MA, USA) and used for the construction of a library with CancerSCAN™ probes and a SureSelect XT reagent kit, HSQ (Agilent Technologies) according to the manufacturer's protocol. This panel was designed to enrich exons of 83 genes [34 (link)] covering 366.2 kb of the human genome. After enriched exome libraries were multiplexed, the libraries were sequenced using the 100-bp paired-end mode of the TruSeq Rapid PE Cluster Kit and TruSeq Rapid SBS kit on the Illumina HiSeq 2500 sequencing platform (Illumina Inc., San Diego, CA, USA). The DNA sequence data were aligned to the human genome reference (hg19) using the MEM algorithm in BWA 0.7.5 [35 (link)]. Duplicate read removal was performed using Picard v.193 and SAMTOOLS v0.1.18 [36 (link)]. Local alignment was optimized using the Genome Analysis Toolkit (GATK) v3.1-1 [37 (link)]. We also used BaseRecalibrator from GATK for base recalibration based on known single nucleotide polymorphisms (SNPs) and indels from Mills, dbSNP138, and 1000G gold standard, 1000G phase1 and Omni 2.5. Sequencing coverage is shown in Supplementary Table 5.

Genomic DNA from each sample was sheared by the Covaris S220 (Covaris, MA, USA) and used for the construction of a library using CancerSCAN probes and the SureSelectXT reagent kit (HSQ; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. CancerSCAN was designed to enrich the exons of 83 genes, covering 366.2 kb of the human genome (Supplementary Table S1)^{41 (link)}. After enriched exon libraries were multiplexed, the libraries were sequenced on a HiSeq. 2500 sequencing platform (Illumina, San Diego, CA, USA). Briefly, a paired-end DNA sequencing library was prepared through gDNA shearing, end-repair, A-tailing, paired-end adaptor ligation, and amplification. After hybridization of the library with bait sequences for 27 h, the captured library was purified and amplified with an index barcode tag, and the library quality and quantity were measured. Sequencing of the exome library was performed using the 100 bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina).

A targeted sequencing panel (CancerSCAN^™) to cover the exonic DNA sequences of 381 cancer genes and introns across 22 genes for rearrangement detection was used (S1 Table). This panel originally designed by the Samsung Genome Institute is available through company GENINUS. Tumor DNA (200 ng for FF or 300 ng for FFPE) was sheared in a Covaris S220 ultrasonicator (Covaris Inc., Woburn, MA, USA) and used for the construction of a library using CancerSCAN probes and an HSQ SureSelectXT reagent kit (Agilent Technologies Inc., Santa Clara, CA, USA) according to the manufacturers’ instructions. After the enriched exome libraries were multiplexed, the libraries were sequenced using the 100-bp paired-end mode of the TruSeq Rapid PE Cluster Kit and TruSeq Rapid SBS kit on the Illumina HiSeq 2500 sequencing platform (Illumina Inc., San Diego, CA, USA).

For RNA-Seq, sequencing libraries were prepared using TruSeq RNA Sample Preparation kit v2 (RS-122-2001 and RS-122-2002, Illumina). Sequencing of the RNA libraries was performed on an Illumina HiSeq2500 in 100-bp paired-end mode of the TruSeq Rapid PE Cluster kit and the TruSeq Rapid SBS kit. RNA-Seq was analyzed using the RSEM^{45 (link)} pipeline with hg19 as the genome reference.

Next-generation sequencing (NGS) analysis was performed on a targeted sequencing platform (CancerSCAN™) designed at Samsung Medical Center [16 (link)]. The CancerSCAN™ panel is designed to target 375 cancer-related genes. Genomic DNA (250 ng) was sheared in a Covaris S220 ultrasonicator (Covaris, Woburn, MA, USA), and target-capture was performed with the SureSelect XT reagent kit, HSQ (Agilent Technologies) according to the manufacturer’s protocol.
After enriched exome libraries were multiplexed, the libraries were sequenced on a HiSeq 2500 sequencing platform (Illumina). Briefly, a paired-end DNA sequencing library was prepared through gDNA shearing, end-repair, A-tailing, paired-end adaptor ligation and amplification. After hybridization of the library with bait sequences for 27 h, the captured library was purified and amplified with an index barcode tag, and the library quality and quantity were assessed. The exome library was sequenced via the 100-bp paired-end mode of the TruSeq Rapid PE Cluster Kit and the TruSeq Rapid SBS Kit (Illumina).
Sequence reads were mapped to the human genome (hg19) by means of Burrows-Wheeler Aligner (BWA). Duplicate read removal was performed with Picard and SAMtools. Local alignment was optimized with the Genome Analysis Toolkit (GATK). Variant calling (SNVs, small indels, CNVs and gene fusion) was done only in regions targeted in CancerSCAN™.

Whole transcriptome sequencing libraries were constructed using a TruSeq RNA sample preparation v2 kit (Illumina). Sequencing was performed using the 100-bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina). The sequencing reads were mapped to the GRCh37.75 human reference genome by using STAR version 2.4.0 26 (link). To quantify gene expression, mapped reads were processed by RSEM version 1.2.18 29 (link). The 'edgeR' method was used to identify differentially expressed genes (DEGs) and the results were filtered on the absolute value of the log 2 (fold change) to more than two 30 (link). We performed gene ontology (GO) analysis using DAVID 31 (link).