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High-throughput resequencing of target-captured cDNAs in cancer cells.
Cancer Sci. 2011 Sep 20;
Authors: Ueno T, Yama****a Y, Soda M, Fukumura K, Ando M, Yamato A, Kawazu M, Choi YL, Mano H
Abstract
The recent advent of whole exon (exome)-capture technology coupled with second-generation sequencers has made it possible to readily detect genomic alterations that affect encoded proteins in cancer cells. Such target resequencing of the cancer genome, however, fails to detect most clinically relevant gene fusions, given that such oncogenic fusion genes are often generated through intron-to-intron ligation. To develop a resequencing platform that simultaneously captures point mutations, insertions-deletions (indels), and gene fusions in the cancer genome, we chose cDNAs as the input for target-capture and extensive resequencing, and we now describe the versatility of such a cDNA-capture system. As a test case, we constructed a custom target-capture system for 913 cancer-related genes, and we purified cDNA fragments for the target gene set from five cell lines of chronic myeloid leukemia. Whereas our target gene set included ABL1, it did not include BCR; however, the sequence output faithfully detected reads spanning the fusion points of these two genes in all cell lines, confirming the ability of cDNA-capture to detect gene fusions. Furthermore, computational analysis of the sequence data set successfully identified nonsynonymous mutations and indels, including those of TP53. Our data may thus support the feasibility of a cDNA-capture system coupled with massively parallel sequencing as a simple platform for the detection of a variety of anomalies in protein-coding genes among hundreds of cancer specimens.
PMID: 21929543 [PubMed - as supplied by publisher]
More...
High-throughput resequencing of target-captured cDNAs in cancer cells.
Cancer Sci. 2011 Sep 20;
Authors: Ueno T, Yama****a Y, Soda M, Fukumura K, Ando M, Yamato A, Kawazu M, Choi YL, Mano H
Abstract
The recent advent of whole exon (exome)-capture technology coupled with second-generation sequencers has made it possible to readily detect genomic alterations that affect encoded proteins in cancer cells. Such target resequencing of the cancer genome, however, fails to detect most clinically relevant gene fusions, given that such oncogenic fusion genes are often generated through intron-to-intron ligation. To develop a resequencing platform that simultaneously captures point mutations, insertions-deletions (indels), and gene fusions in the cancer genome, we chose cDNAs as the input for target-capture and extensive resequencing, and we now describe the versatility of such a cDNA-capture system. As a test case, we constructed a custom target-capture system for 913 cancer-related genes, and we purified cDNA fragments for the target gene set from five cell lines of chronic myeloid leukemia. Whereas our target gene set included ABL1, it did not include BCR; however, the sequence output faithfully detected reads spanning the fusion points of these two genes in all cell lines, confirming the ability of cDNA-capture to detect gene fusions. Furthermore, computational analysis of the sequence data set successfully identified nonsynonymous mutations and indels, including those of TP53. Our data may thus support the feasibility of a cDNA-capture system coupled with massively parallel sequencing as a simple platform for the detection of a variety of anomalies in protein-coding genes among hundreds of cancer specimens.
PMID: 21929543 [PubMed - as supplied by publisher]
More...