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1、Targeted Sequencing of Cancer-Related Genes in Colorectal Cancer Using Next-Generation SequencingSae-Won Han1,2., Hwang-Phill Kim2., Jong-Yeon Shin3,4, Eun-Goo Jeong2, Won-Chul Lee3,5, Kyung-Hun Lee1, Jae-Kyung Won6, Tae-Yong Kim1, Do-Youn Oh1,2, Seock-Ah Im1,2, Yung-Jue Bang1,2, Seung-Yong Jeong7,
2、Kyu Joo Park7, Jae-Gahb Park7, Gyeong Hoon Kang6, Jeong-Sun Seo3,5,8,9, Jong-Il Kim3,4,5,8, Tae-You Kim1,2,5,10*1Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea, 2Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea, 3Genomic Medici
3、ne Institute, Medical Research Center, Seoul National University, Seoul, Korea, 4Psoma Therapeutics Inc., Seoul, Korea, 5Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Korea, 6Department of Pathology, Seoul National University Hospital, Seoul, Korea, 7Department
4、 of Surgery, Seoul National University Hospital, Seoul, Korea, 8Department of Biochemistry, Seoul National University College of Medicine, Seoul, Korea, 9Macrogen Inc., Seoul, Korea, 10Department of Molecular Medicine Accepted April 10, 2013; Published May 21, 2013Copyright: ? 2013 Han et al. This i
5、s an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This study was supported by a grant from the Korean Healthcare
6、 Technology R (2) the average base quality (phred Q score) for the position should be 20; and (3) the read-allele frequency at the position should be 20%. For detection of the somatic mutations (SNVs and indels) in the cancer tissues, we used the following conditions: (1) nonsynonymous SNVs or indel
7、s in the cancer tissues; (2) the SNV allele count should be zero in targeted sequence of normal tissue; (3) the wild-type allele count should be 10 or more in targeted sequence of normal tissue; and (4) the candidate positions should not be polymorphisms according to the dbSNP132. The functional con
8、sequences of the novel missense variants were predicted using Sorting Intolerant from Tolerant (SIFT) 15. Mutation in KRAS was confirmed using Sanger sequencing. Following primers were used: codon12 and 13, forward 59-CGTCTGCAGTCAACTGGAAT-39 and reverse 59- GAGAGTGAACATCATGGACC-39; codon 61, forward
9、 59- CAGACTGTGTTCTCCCTTCTCA-39andreverse59- CTCATGTACTGGTCCCTCATTG-39; and codon 146, for- ward 59-TGGACAGGTTTTGAAAGATATTTG-39 and reverse 59-ATTAAGAAGCAATGCCCTCTCAAG-39. All sequencing reactions were done in both forward and reverse directions, and all mutations were confirmed at least twice from i
10、ndependent PCR isolates.Copy number alteration We estimated the coverage of genes by using sequencing reads mapped to the targeted regions. To detect copy number alteration for a given pair of cancer and normal tissues, coverage fold ratios (tumor/normal) for 183 target genes were calculated. After
11、the normalization step considering total read bases obtained from each tissue, coverage fold ratio was adjusted by estimated tumor cell purity described below. We defined that a gene shows copy number gain when its coverage fold ratio $2.0 and loss when #0.5. To estimate tumor purity, we used read c
12、ounts of somatic SNVs identified. Given somatic SNVs for each cancer tissue, we assumed that the cancer consists of a major clone and the SNVs are derived from the clone. Moreover, we regarded the SNVs as heterozygotes whose allele frequency is 0.5. With these assumptions, tumor purity was estimated
13、 as follows. The expected numbers of wild- type reads originated from cancer clone and normal cells were calculated. Then, the proportion of wild-type plus SNP read counts for cancer among the total number of read counts was considered as the corresponding tumor purity. In 3 samples that had no tumo
14、r specific SNV, the median value of 57 samples was used in the analysis of copy number variations. Copy number alteration was confirmed using quantitative real- time PCR with the iCycler IQ detection system (Bio-Rad Laboratories, Hercules, CA) using SYBR green I (Molecular Probe, Eugene, OR) in trip
15、licate reactions. The primers used inTargeted Sequencing in Colorectal CancerPLOS ONE | www.plosone.org2May 2013 | Volume 8 | Issue 5 | e64271the PCR reaction were as following: ERBB2, forward 59- TGCTGGAGGACGATGACATG-39 and reverse 59-CTGGA- CAGAAGAAGCCCTGC-39;SRC,forward59- CGGTTACTGCTCAATGCAGA-39
16、andreverse59-CAA- GAGCGCTCGTACCTTTC-39;TP53,forward59- CCCTTCCCAGAAAACCTACC-39andreverse59-ACT- GACCGTGCAAGTCACAG-39;PTEN,forward59- TGGCACTGTTGTTTCACAAG-39andreverse59- TGTTCCAATACATGGAAGGATG-39; BRCA1, forward 59- GTTTGCCAGAAAACACCACA-39 and reverse 59-TTTATG- CAGCAGATGCAAGG-39;BRCA2,forward59- TGCCTGGCCTGATACAATTA-39andreverse59- TTGCTGCTTCCTTTTCTTCC-39; and ACTB, forward 59- AGAGCTACGAGCTGCCTGAC and reverse 59- GGATGC- CACAGGACTCCA-39. Fluorescence in situ hybridization (FISH) analysis of
