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

Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

doi:10.1093/dnares/dsv010

Cited by 75 publications

(84 citation statements)

References 36 publications

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“…Because insertion/deletion errors are dominant in the Ion Torrent sequencers, errors in the barcode tags can be monitored by the size of the tags. As revealed in the previous experiment , the erroneous barcode tags were accumulated in the fraction of low numbers of reads. Thus, the fraction whose error‐free tags occupied <90% was removed (Fig.…”

Section: Resultsmentioning

confidence: 83%

Characterization of the T‐cell receptor beta chain repertoire in tumor‐infiltrating lymphocytes

et al. 2016

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Tumor‐infiltrating lymphocytes (TILs) are direct effectors of tumor immunity, and their characterization is important for further development of immunotherapy. Recent advances in high‐throughput sequencing technologies have enabled a comprehensive analysis of T‐cell receptor (TCR) complementarity‐determining region 3 (CDR3) sequences, which may provide information of therapeutic importance. We developed a high‐fidelity target sequencing method with the ability for absolute quantitation, and performed large‐scale sequencing of TCR beta chain (TCRB) CDR3 regions in TILs and peripheral blood lymphocytes (PBLs). The estimated TCRB repertoire sizes of PBLs from four healthy individuals and TILs from four colorectal cancer tissue samples were 608,664–1,003,098 and 90,228–223,757, respectively. The usage of J‐ and V‐regions was similar in PBLs and TILs. Proportions of CDR3 amino acid (aa) sequences occupying more than 0.01% of the total molecular population were 0.33–0.43% in PBLs and 1.3–3.6% in TILs. Additional low coverage sequencing of 15 samples identified five CDR3 aa sequences that were shared by nine patients, one sequence shared by 10 patients, and one sequence shared by 12 patients. The estimated size of the TCRB repertoire in TILs was significantly smaller than that in PBLs. The proportion of abundant species (>0.01%) in TILs was larger than that in PBLs. Shared CDR3 aa sequences represent a response to common antigens, and the identification of such CDR3 sequences may be beneficial in developing clinical biomarkers.

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Section: Resultsmentioning

confidence: 83%

Characterization of the T‐cell receptor beta chain repertoire in tumor‐infiltrating lymphocytes

et al. 2016

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“…One cannot simply remove EA-derived variants based on VAF, as there exist clinically actionable variants at VAFs comparable to the frequency of EAs. UMI consensus calling can help resolve down to 0.01 VAF with confidence by polishing out sequencing error, 20 but EAs originate from a physical molecule prior to the ligation of UMIs and thus this strategy is also unhelpful in the elimination of EAs. INDEL-aware variant calls may produce extraneous variant calls by considering the soft-clipped sequences as true INDEL variants.…”

Section: Discussionmentioning

confidence: 99%

Characterization and Mitigation of Fragmentation Enzyme-Induced Dual Stranded Artifacts

Gregory

Ngankeu

Orwick

et al. 2020

Preprint

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words)High-throughput short-read sequencing relies on fragmented DNA for optimal sampling of input nucleic acid. Several vendors now offer proprietary enzyme cocktails as a cheaper and more streamlined method of fragmentation when compared to acoustic shearing. We have discovered that these enzymes induce the formation of library molecules containing regions of nearby DNA from opposite strands. Sequencing reads derived from these molecules can lead to artifactderived variant calls appearing at variant allele frequencies less than 5%. We present Fragmentation Artifact Detection and Elimination (FADE), software to remove these artifacts from mapped reads and mitigate artifact-related effects on downstream analysis. We find that the artifacts principally affect downstream analyses that are sensitive to a 1-3% artifact bias in the sequencing reads, such as targeted resequencing and rare variant discovery.

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“…Deep sequencing with molecular barcodes was performed using the non-overlapping integrated read sequencing system26.…”

Section: Methodsmentioning

confidence: 99%

Transient appearance of circulating tumor DNA associated with de novo treatment

Kato¹,

Uchida²,

Kukita³

et al. 2016

Sci Rep

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The limitation of circulating tumor DNA (ctDNA) is its inability to detect cancer cell subpopulations with few or no dying cells. Lung cancer patients subjected to the EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment were prospectively collected, and ctDNA levels represented by the activating and T790M mutations were measured. The first data set (21 patients) consisting of samples collected in the period from before initiation of EGFR-TKI to at least 2 weeks after initiation: the ctDNA dynamics generally exhibited a rapid decrease and/or a transient increase. In 4 patients, we detected a transient increase of ctDNA bearing activating mutations not identified in biopsy samples. ctDNA with the same genotypical pattern was identified in 7 out of the 39 patients of the second data set intended to include samples until the onset of disease progression. In 6 of the 7 patients, this unique ctDNA appeared in the early period after treatment initiation, and did not reappear even after disease progression or chemotherapy. In another patient, similar ctDNA appeared upon radiation therapy. The identification of ctDNA with a unique genotype indicates the presence of cancer cell subpopulations that normally contain few or no dying cells, but generate dead cells because of the treatment.

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

Cited by 75 publications

References 36 publications

Characterization of the T‐cell receptor beta chain repertoire in tumor‐infiltrating lymphocytes

Characterization of the T‐cell receptor beta chain repertoire in tumor‐infiltrating lymphocytes

Characterization and Mitigation of Fragmentation Enzyme-Induced Dual Stranded Artifacts

Transient appearance of circulating tumor DNA associated with de novo treatment

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