title: 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.affiliation: Department of Molecular and Medical Genetics, Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan.
authors.forename: Yoji
authors.initials: Y
authors.lastname: Kukita
authors.affiliation: DNA Chip Research Inc., Yokohama 230-0045, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Matoba
authors.affiliation: Department of Thoracic Oncology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Uchida
authors.affiliation: Department of Molecular and Medical Genetics, Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Hamakawa
authors.affiliation: Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Doki
authors.affiliation: Department of Thoracic Oncology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Imamura
authors.affiliation: Department of Molecular and Medical Genetics, Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan katou-ki@mc.pref.osaka.jp.
authors.forename: Kikuya
authors.initials: K
authors.lastname: Kato
mesh_terms: D058893:DNA Barcoding, Taxonomic; D004273:DNA, Neoplasm; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D008297:Male; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D010950:Plasma Cells / Q000378:metabolism*; D015203:Reproducibility of Results; D013274:Stomach Neoplasms / Q000235:genetics
chemical_list: D004273:DNA, Neoplasm
keywords: barcode sequences; circulating tumour DNA; massively parallel DNA sequencer