Related Experiment Video
Updated: May 29, 2026
Microfluidics-based High-throughput Circulating Tumor Cell Sorting and Single-cell Sequencing Technology
Published on: November 14, 2025
High-throughput resequencing of target-captured cDNA in cancer cells
Toshihide Ueno1, Yoshihiro Yamashita, Manabu Soda
1Division of Functional Genomics, Jichi Medical University, Tochigi, Japan.
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 cDNA as the input for target capture and extensive resequencing, and we 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 CML. Our target gene set included Abelson murine leukemia viral oncogene homolog 1 (ABL1), but it did not include breakpoint cluster region (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 dataset successfully identified non-synonymous mutations and indels, including those of tumor protein p53 (TP53). Our data might 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.
Insights
A novel cDNA-capture sequencing method effectively detects cancer-related genomic anomalies, including point mutations, indels, and gene fusions. This versatile platform offers a simplified approach for comprehensive cancer genome analysis.
Area of Science:
- Genomics and Cancer Research
- Molecular Biology
- Bioinformatics
Background:
- Exome-capture technology enables detection of genomic alterations affecting cancer proteins.
- Current methods often miss clinically relevant gene fusions caused by intron-to-intron ligation.
- A unified platform for detecting point mutations, indels, and gene fusions is needed.
Purpose of the Study:
- To develop and validate a cDNA-capture resequencing platform for simultaneous detection of diverse cancer genomic alterations.
- To assess the versatility of a cDNA-capture system for identifying point mutations, insertions-deletions (indels), and gene fusions.
Main Methods:
- Utilized cDNA as input for target capture and extensive resequencing.
- Constructed a custom target-capture system for 913 cancer-related genes.
- Applied the system to five chronic myeloid leukemia (CML) cell lines and performed computational analysis.
Main Results:
- The cDNA-capture system successfully detected gene fusions, exemplified by Abelson murine leukemia viral oncogene homolog 1 (ABL1)-breakpoint cluster region (BCR) fusions in CML cell lines.
- Computational analysis identified non-synonymous mutations and indels, including in tumor protein p53 (TP53).
- Demonstrated the system's ability to capture reads spanning fusion points, confirming its utility for gene fusion detection.
Conclusions:
- A cDNA-capture system coupled with massively parallel sequencing is feasible for detecting a wide range of anomalies in protein-coding genes.
- This approach offers a simplified platform for comprehensive genomic profiling across numerous cancer specimens.
- The method's versatility supports its application in clinical cancer genomics and research.
