Quantitative analysis of differences in copy numbers using read depth obtained from PCR-enriched samples and controls

Reinecke, Frank; Satya, Ravi Vijaya; DiCarlo, John

doi:10.1186/s12859-014-0428-5

Cited by 14 publications

(11 citation statements)

References 24 publications

(24 reference statements)

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“…9,21,22 In addition, tumor size of NF2 type was larger than that of TRAKLS types, probably because of high proliferative ability. In fact, NF2 type meningioma showed higher Ki-67 LI compared with TRAKLS type, and this result is compatible with previous studies 14 has expanded the application of targeted amplicon sequencing. Current, reliable, clinically practical methods to investigate copynumber alterations include I-FISH and quantitative PCR analysis; however, mutations cannot be simultaneously confirmed with these methods.…”

Section: Discussionsupporting

confidence: 80%

Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system

et al. 2016

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Recent genetic analyses using next-generation sequencers have revealed numerous genetic alterations in various tumors including meningioma, which is the most common primary brain tumor. However, their use as routine laboratory examinations in clinical applications for tumor genotyping is not cost effective. To establish a clinical sequencing system for meningioma and investigate the clinical significance of genotype, we retrospectively performed targeted amplicon sequencing on 103 meningiomas and evaluated the association with clinicopathological features. We designed amplicon-sequencing panels targeting eight genes including NF2 (neurofibromin 2), TRAF7, KLF4, AKT1, and SMO. Libraries prepared with genomic DNA extracted from PAXgenefixed paraffin-embedded tissues of 103 meningioma specimens were sequenced using the Illumina MiSeq. NF2 loss in some cases was also confirmed by interphase-fluorescent in situ hybridization. We identified NF2 loss and/or at least one mutation in NF2, TRAF7, KLF4, AKT1, and SMO in 81 out of 103 cases (79%) by targeted amplicon sequencing. On the basis of genetic status, we categorized meningiomas into three genotype groups: NF2 type, TRAKLS type harboring mutation in TRAF7, AKT1, KLF4, and/or SMO, and 'not otherwise classified' type. Genotype significantly correlated with tumor volume, tumor location, and magnetic resonance imaging findings such as adjacent bone change and heterogeneous gadolinium enhancement, as well as histopathological subtypes. In addition, multivariate analysis revealed that genotype was independently associated with risk of recurrence. In conclusion, we established a rapid clinical sequencing system that enables final confirmation of meningioma genotype within 7 days turnaround time. Our method will bring multiple benefits to neuropathologists and neurosurgeons for accurate diagnosis and appropriate postoperative management.

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Section: Discussionsupporting

confidence: 80%

Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system

et al. 2016

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“…We determined regions of copy-number alteration based on their relative copy-number using the “copynumber” R package (64). For tumor-normal pairs without SNP array data, we estimated copy-number profiles based on sequencing data using Control-FREEC (65), Quandico (66), and Sequenza (67). We used GISTIC v2.0.22 (68) to determine regions of significant focal copy-number alterations, using ASCAT/Sequenza’s inferred copy-number segments, and associated copy-number values were defined as log 2 of the segment’s relative copy-number.…”

Section: Methodsmentioning

confidence: 99%

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma

et al. 2017

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Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters – Fluke-Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3′UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores – mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.

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“…Multiplex PCR-based enrichment focuses sequencing efforts on a very small fraction of the genome, and the observed read depth for each of the regions can differ due to varying number of PCR amplicons, sequence variation, or PCR enrichment efficiency. For CNV detection in our PCR-enriched amplicon sequencing data of the 20 genes, we used two algorithms, Quandico [ 49 ] and ONCOCNV [ 50 ], specifically developed for CNV analysis of amplicon sequencing data. The CNVs detected with these algorithms were then verified experimentally using the multiplex ligation-dependent probe amplification (MLPA) technique.…”

Section: Methodsmentioning

confidence: 99%

Germline breast cancer susceptibility gene mutations and breast cancer outcomes

et al. 2018

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BackgroundIt is unclear whether germline breast cancer susceptibility gene mutations affect breast cancer related outcomes. We wanted to evaluate mutation patterns in 20 breast cancer susceptibility genes and correlate the mutations with clinical characteristics to determine the effects of these germline mutations on breast cancer prognosis.MethodsThe study cohort included 480 ethnic Chinese individuals in Taiwan with at least one of the six clinical risk factors for hereditary breast cancer: family history of breast or ovarian cancer, young age of onset for breast cancer, bilateral breast cancer, triple negative breast cancer, both breast and ovarian cancer, and male breast cancer. PCR-enriched amplicon-sequencing on a next generation sequencing platform was used to determine the germline DNA sequences of all exons and exon-flanking regions of the 20 genes. Protein-truncating variants were identified as pathogenic.ResultsWe detected a 13.5% carrier rate of pathogenic germline mutations, with BRCA2 being the most prevalent and the non-BRCA genes accounting for 38.5% of the mutation carriers. BRCA mutation carriers were more likely to be diagnosed of breast cancer with lymph node involvement (66.7% vs 42.6%; P = 0.011), and had significantly worse breast cancer specific outcomes. The 5-year disease-free survival was 73.3% for BRCA mutation carriers and 91.1% for non-carriers (hazard ratio for recurrence or death 2.42, 95% CI 1.29–4.53; P = 0.013). After adjusting for clinical prognostic factors, BRCA mutation remained an independent poor prognostic factor for cancer recurrence or death (adjusted hazard ratio 3.04, 95% CI 1.40–6.58; P = 0.005). Non-BRCA gene mutation carriers did not exhibit any significant difference in cancer characteristics or outcomes compared to those without detected mutations. Among the risk factors for hereditary breast cancer, the odds of detecting a germline mutation increased significantly with having bilateral breast cancer (adjusted odds ratio 3.27, 95% CI 1.64–6.51; P = 0.0008) or having more than one risk factor (odds ratio 2.07, 95% CI 1.22–3.51; P = 0.007).ConclusionsWithout prior knowledge of the mutation status, BRCA mutation carriers had more advanced breast cancer on initial diagnosis and worse cancer-related outcomes. Optimal approach to breast cancer treatment for BRCA mutation carriers warrants further investigation.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4229-5) contains supplementary material, which is available to authorized users.

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Quantitative analysis of differences in copy numbers using read depth obtained from PCR-enriched samples and controls

Cited by 14 publications

References 24 publications

Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system

Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma

Germline breast cancer susceptibility gene mutations and breast cancer outcomes

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