High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

Abstract

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA.

• Authors: Yoji Kukita, Ryo Matoba, Junji Uchida, Takuya Hamakawa, Yuichiro Doki, Fumio Imamura, Kikuya Kato
• Journal: DNA research : an international journal for rapid publication of reports on genes and genomes (DNA Res) Vol. 22 Issue 4 Pg. 269-77 (Aug 2015) ISSN: 1756-1663 [Electronic] England
• PMID: 26126624 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Chemical References

• DNA, Neoplasm

Topics

• DNA Barcoding, Taxonomic
• DNA, Neoplasm
• High-Throughput Nucleotide Sequencing (methods)
• Humans
• Lung Neoplasms (genetics)
• Male
• Mutation
• Neoplasms (genetics)
• Plasma Cells (metabolism)
• Reproducibility of Results
• Stomach Neoplasms (genetics)