Identification of novel SNPs by next-generation sequencing of the genomic region containing the APC gene in colorectal cancer patients in China.
We described an approach of identifying single nucleotide polymorphisms (SNPs) in complete genomic regions of key genes including promoters, exons, introns, and downstream sequences by combining long-range polymerase chain reaction (PCR) or NimbleGen sequence capture with next-generation sequencing. Using the adenomatous polyposis coli (APC) gene as an example, we identified 210 highly reliable SNPs by next-generation sequencing analysis program MAQ and Samtools, of which 69 were novel ones, in the 123-kb APC genomic region in 27 pair of colorectal cancers and normal adjacent tissues. We confirmed all of the eight randomly selected high-quality SNPs by allele-specific PCR, suggesting that our false discovery rate is negligible. We identified 11 SNPs in the exonic region, including one novel SNP that was not previously reported. Although 10 of them are synonymous, they were predicted to affect splicing by creating or removing exonic splicing enhancers or exonic splicing silencers. We also identified seven SNPs in the upstream region of the APC gene, three of which were only identified in the cancer tissues. Six of these upstream SNPs were predicted to affect transcription factor binding. We also observed that long-range PCR was better in capturing GC-rich regions than the NimbleGen sequence capture technique.
Cheng
Yin
Y
Systems Biology Division, Zhejiang-California International Nanosystems Institute (ZCNI), Zhejiang University, Hangzhou, People's Republic of China.
Wang
Jun
J
Shao
Jiaofang
J
Chen
Qiyun
Q
Mo
Fan
F
Ma
Liang
L
Han
Xu
X
Zhang
Jing
J
Chen
Chen
C
Zhang
Cixiong
C
Lin
Shuyong
S
Yu
Jiekai
J
Zheng
Shu
S
Lin
Sheng-Cai
SC
Lin
Biaoyang
B
D001483:Base Sequence; D002681:China; D015179:Colorectal Neoplasms / Q000235:genetics*; D017491:Genes, APC; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software