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

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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

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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.

Project description:Tumor genetic sequencing identifies potentially targetable genetic alterations with therapeutic implications. Analysis has concentrated on detecting tumor-specific variants, but recognition of germline variants may prove valuable as well.To estimate the burden of germline variants identified through routine clinical tumor sequencing.Patients with advanced cancer diagnoses eligible for studies of targeted agents at Memorial Sloan Kettering Cancer Center are offered tumor-normal sequencing with MSK-IMPACT, a 341-gene panel. We surveyed the germline variants seen in 187 overlapping genes with Mendelian disease associations in 1566 patients who had undergone tumor profiling between March and October 2014.The number of presumed pathogenic germline variants (PPGVs) and variants of uncertain significance per person in 187 genes associated with single-gene disorders and the proportions of individuals with PPGVs in clinically relevant gene subsets, in genes consistent with known tumor phenotypes, and in genes with evidence of second somatic hits in their tumors.The mean age of the 1566 patients was 58 years, and 54% were women. Presumed pathogenic germline variants in known Mendelian disease-associated genes were identified in 246 of 1566 patients (15.7%; 95% CI, 14.0%-17.6%), including 198 individuals with mutations in genes associated with cancer susceptibility. Germline findings in cancer susceptibility genes were concordant with the individual's cancer type in only 81 of 198 cases (40.9%; 95% CI, 34.3%-47.9%). In individuals with PPGVs retained in the tumor, somatic alteration of the other allele was seen in 39 of 182 cases (21.4%; 95% CI, 16.1%-28.0%), of which 13 cases did not show a known correlation of the germline mutation and a known syndrome. Mutations in non-cancer-related Mendelian disease genes were seen in 55 of 1566 cases (3.5%; 95% CI, 27.1%-45.4%). Almost every individual had more than 1 variant of uncertain significance (1565 of 1566 patients; 99.9%; 95% CI, 99.6%-99.9%).Germline variants are common in individuals undergoing tumor-normal sequencing and may reveal otherwise unsuspected syndromic associations.

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.

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