Dataset Information

Dual-molecular barcode sequencing detects rare variants in tumor and cell free DNA in plasma.

ABSTRACT: Conventional next generation sequencing analysis has provided important insights into cancer genetics. However, the detection of rare (low allele fraction) variants remains difficult because of the error-prone nucleotide changes derived from sequencing/PCR errors. To eliminate the false-positive variants and detect genuine rare variants, sequencing technology combined with molecular barcodes will be useful. Here, we used the newly developed dual-molecular barcode technology (Ion AmpliSeq HD) to analyze somatic mutations in 24 samples (12 tumor tissues and 12 plasma) from 12 patients with biliary-pancreatic and non-small cell lung cancers. We compared the results between next generation sequencing analysis with or without molecular barcode technologies. The variant allele fraction (VAF) between non-molecular barcode and molecular barcode sequencing was correlated in plasma DNA (R2?=?0.956) and tumor (R2?=?0.935). Both methods successfully detected high VAF mutations, however, rare variants were only identified by molecular barcode sequencing and not by non-molecular barcode sequencing. Some of these rare variants in tumors were annotated as pathogenic, and therefore subclonal driver mutations could be observed. Furthermore, the very low VAF down to 0.17% were identified in cell free DNA in plasma. These results demonstrate that the dual molecular barcode sequencing technologies can sensitively detect rare somatic mutations, and will be important in the investigation of the clonal and subclonal architectures of tumor heterogeneity.

SUBMITTER: Hirotsu Y

PROVIDER: S-EPMC7042261 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Dual-molecular barcode sequencing detects rare variants in tumor and cell free DNA in plasma.

Hirotsu Yosuke Y Otake Sotaro S Ohyama Hiroshi H Amemiya Kenji K Higuchi Rumi R Oyama Toshio T Mochizuki Hitoshi H Goto Taichiro T Omata Masao M

Scientific reports 20200225 1

Conventional next generation sequencing analysis has provided important insights into cancer genetics. However, the detection of rare (low allele fraction) variants remains difficult because of the error-prone nucleotide changes derived from sequencing/PCR errors. To eliminate the false-positive variants and detect genuine rare variants, sequencing technology combined with molecular barcodes will be useful. Here, we used the newly developed dual-molecular barcode technology (Ion AmpliSeq HD) to ...[more]

PMID: 32099048

Similar Datasets

Project description:BackgroundIn the setting of localized colon cancer (CC), circulating tumor DNA (ctDNA) monitoring in plasma has shown potential for detecting minimal residual disease (MRD) and predicting a higher risk of recurrence. With the tumor-only sequencing approach, however, germline variants may be misidentified as somatic variations, precluding the possibility of tracking in up to 11% of patients due to a lack of known somatic mutations. In this study, we assess the potential value of adding white blood cells (WBCs) to tumor tissue sequencing to enhance the accuracy of sequencing results.Patients and methodsA total of 148 patients diagnosed with localized CC were prospectively recruited at the Hospital Clínico Universitario in Valencia (Spain). Employing a custom 29-gene panel, sequencing was conducted on tumor tissue, plasma and corresponding WBCs. Droplet digital PCR and amplicon-based NGS were performed on plasma samples post-surgery to track MRD. Oncogenic somatic variants were identified by annotating with COSMIC, OncoKB and an internal repository of pathogenic mutations database. A variant prioritization analysis, mainly characterized by the match of oncogenic mutations with the evidence levels defined in OncoKB, was carried out to select specific targeted therapies.ResultsUtilizing paired tumor and WBCs sequencing, we identified somatic mutations in all patients (100%) within our cohort, compared to 89% using only tumor tissue. Consequently, the top 10 most frequently mutated genes for plasma monitoring were altered. The sequencing of WBCs identified 9% of patients with pathogenic mutations in the germline, with APC and TP53 being the most frequently mutated genes. Additionally, mutations in genes related to clonal hematopoiesis of indeterminate potential were detected in 27% of the cohort, with TP53, KRAS, and KMT2C being the most frequently altered genes. There were no observed differences in the sensitivity of monitoring MRD using ddPCR or amplicon-based NGS (p = 1). Ultimately, 41% of the patients harbored potentially targetable alterations at diagnosis.ConclusionThe germline testing method not only enhanced sequencing results and raised the proportion of patients eligible for plasma monitoring, but also uncovered the existence of pathogenic germline variations, thereby aiding in the identification of patients at a higher risk of hereditary cancer syndromes.

Project description:BACKGROUND: Towards lower latitudes the number of recognized species is not only higher, but also phylogeographic subdivision within species is more pronounced. Moreover, new genetically isolated populations are often described in recent phylogenies of Neotropical birds suggesting that the number of species in the region is underestimated. Previous COI barcoding of Argentinean bird species showed more complex patterns of regional divergence in the Neotropical than in the North American avifauna. METHODS AND FINDINGS: Here we analyzed 1,431 samples from 561 different species to extend the Neotropical bird barcode survey to lower latitudes, and detected even higher geographic structure within species than reported previously. About 93% (520) of the species were identified correctly from their DNA barcodes. The remaining 41 species were not monophyletic in their COI sequences because they shared barcode sequences with closely related species (N?=?21) or contained very divergent clusters suggestive of putative new species embedded within the gene tree (N?=?20). Deep intraspecific divergences overlapping with among-species differences were detected in 48 species, often with samples from large geographic areas and several including multiple subspecies. This strong population genetic structure often coincided with breaks between different ecoregions or areas of endemism. CONCLUSIONS: The taxonomic uncertainty associated with the high incidence of non-monophyletic species and discovery of putative species obscures studies of historical patterns of species diversification in the Neotropical region. We showed that COI barcodes are a valuable tool to indicate which taxa would benefit from more extensive taxonomic revisions with multilocus approaches. Moreover, our results support hypotheses that the megadiversity of birds in the region is associated with multiple geographic processes starting well before the Quaternary and extending to more recent geological periods.

Dataset Information

Dual-molecular barcode sequencing detects rare variants in tumor and cell free DNA in plasma.

Publications

Dual-molecular barcode sequencing detects rare variants in tumor and cell free DNA in plasma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets