Browse
Submit Data
Databases
API
Help

Dataset Information

10 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multisite verification of the accuracy of a multi-gene next generation sequencing panel for detection of mutations and copy number alterations in solid tumours.

ABSTRACT: Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and fusions can be detected in the clinical setting using deep targeted sequencing. These assays support low limits of detection using little genomic input material. They are gaining in popularity in clinical laboratories, where sample volumes are limited, and low variant allele fractions may be present. However, data on reproducibility between laboratories is limited. Using a ring study, we evaluated the performance of 7 Ontario laboratories using targeted sequencing panels. All laboratories analysed a series of control and clinical samples for SNVs/CNVs and gene fusions. High concordance was observed across laboratories for measured CNVs and SNVs. Over 97% of SNV calls in clinical samples were detected by all laboratories. Whilst only a single CNV was detected in the clinical samples tested, all laboratories were able to reproducibly report both the variant and copy number. Concordance for information derived from RNA was lower than observed for DNA, due largely to decreased quality metrics associated with the RNA components of the assay, suggesting that the RNA portions of comprehensive NGS assays may be more vulnerable to variations in approach and workflow. Overall the results of this study support the use of the OFA for targeted sequencing for testing of clinical samples and suggest specific internal quality metrics that can be reliable indicators of assay failure. While we believe this evidence can be interpreted to support deep targeted sequencing in general, additional studies should be performed to confirm this.

SUBMITTER: Bartlett J

PROVIDER: S-EPMC8486135 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours.

Project description:BackgroundIn a clinical diagnostic laboratory, we evaluated the applicability of the Ion Proton sequencer for screening 409 cancer-related genes in solid tumours.MethodsDNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue biopsy specimens of 55 solid tumours (20 with matched normal tissue) and four cell lines and screened for mutations in 409 genes using the Ion Proton system. The mutation profiles of these samples were known based on prior testing using the Ion Torrent Personal Genome Machine (46-gene hotspot panel), Sanger sequencing, or fluorescence in situ hybridisation (FISH). Concordance with retrospective findings and additional mutations were evaluated. Assay sensitivity and reproducibility were established. Gene copy number variations (CNVs) detected were confirmed by molecular inversion probe (MIP) array.ResultsThe average Ion Proton (409-gene panel) sequencing output per run was 8 gigabases with 128 million sequencing reads. Of the 15,992 amplicons in the 409-gene panel, 90% achieved a minimum average sequencing depth of 100X. In 59 samples, the Ion Proton detected 100 of 105 expected single-nucleotide variants (SNVs) and all expected deletions (n=8), insertions (n=5), and CNVs (n=7). Five SNVs were not detected due to failed amplification of targeted regions. In 20 tumours with paired normal tissue, Ion Proton detected 37 additional somatic mutations, several in genes of high prognostic or therapeutic significance, such as MET, ALK, TP53, APC, and PTEN. MIP array analysis confirmed all CNVs detected by Ion Proton.ConclusionsThe Ion Proton (409-gene panel) system was found to be well suited for use in a clinical molecular diagnostic laboratory. It can simultaneously screen 409 genes for a variety of sequence variants in multiple samples using a low input of FFPE DNA with high reproducibility and sensitivity.

| S-EPMC4229640 | biostudies-literature

Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data.

Project description:Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.

| S-EPMC5707205 | biostudies-literature

Simultaneous identification of clinically relevant single nucleotide variants, copy number alterations and gene fusions in solid tumors by targeted next-generation sequencing.

Project description:In this study, we have set-up a routine pipeline to evaluate the clinical application of Oncomine™ Focus Assay, a panel that allows the simultaneous detection of single nucleotide hotspot mutations in 35 genes, copy number alterations (CNAs) in 19 genes and gene fusions involving 23 genes in cancer samples. For this study we retrospectively selected 106 patients that were submitted to surgical resection for lung, gastric, colon or rectal cancer. We found that 56 patients out of 106 showed at least one alteration (53%), with 47 patients carrying at least one relevant nucleotide variant, 10 patients carrying at least one CNA and 3 patients carrying one gene fusion. On the basis of the mutational profiles obtained, we have identified 22 patients (20.7%) that were potentially eligible for targeted therapy. The most frequently mutated genes across all tumor types included KRAS (30 patients), PIK3CA (16 patients), BRAF (6 patients), EGFR (5 patients), NRAS (4 patients) and ERBB2 (3 patients) whereas CCND1, ERBB2, EGFR and MYC were the genes most frequently subjected to copy number gain. Finally, gene fusions were identified only in lung cancer patients and involved MET [MET(13)-MET(15) fusion] and FGFR3 [FGFR3(chr 17)-TACC3(chr 11)]. In conclusion, we demonstrate that the analysis with a multi-biomarker panel of cancer patients after surgery, may present several potential advantages in clinical daily practice, including the simultaneous detection of different potentially druggable alterations, reasonable costs, short time of testing and automated interpretation of results.

| S-EPMC5978263 | biostudies-literature

Prevalence of actionable mutations and copy number alterations and the price of a genomic testing panel.

Project description:Interest in genomic testing for the selection of cancer therapy is growing. However, the cost of genomic testing has not been well studied. We sought to determine the price of identifying mutations and copy number alterations (CNAs) in theoretically actionable genes across multiple tumor types. We reviewed data from The Cancer Genome Atlas to determine the frequency of alterations in nine tumor types. We used price information from a commonly used commercial genomic testing platform (FoundationOne) to determine the price of detecting mutations and CNAs in different types of tumors. Although there are large variations in the prevalence by tumor type, when the detection of both mutations and CNAs was considered overall, most patients had at least one alteration in a potentially actionable gene (84% overall, range 51%- 98% among tumor types assessed). The corresponding average price of identifying at least one alteration per patient ranges from $5,897 to $11,572. Although the frequency of mutations and CNAs in actionable genes differs by tumor type, most patients have an actionable genomic alteration detectable by a commercially available panel. Determining CNAs as well as mutations improves actionability and reduces the price of detecting an alteration.

| S-EPMC5342111 | biostudies-literature

Functional copy-number alterations in cancer.

Project description:Understanding the molecular basis of cancer requires characterization of its genetic defects. DNA microarray technologies can provide detailed raw data about chromosomal aberrations in tumor samples. Computational analysis is needed (1) to deduce from raw array data actual amplification or deletion events for chromosomal fragments and (2) to distinguish causal chromosomal alterations from functionally neutral ones. We present a comprehensive computational approach, RAE, designed to robustly map chromosomal alterations in tumor samples and assess their functional importance in cancer. To demonstrate the methodology, we experimentally profile copy number changes in a clinically aggressive subtype of soft-tissue sarcoma, pleomorphic liposarcoma, and computationally derive a portrait of candidate oncogenic alterations and their target genes. Many affected genes are known to be involved in sarcomagenesis; others are novel, including mediators of adipocyte differentiation, and may include valuable therapeutic targets. Taken together, we present a statistically robust methodology applicable to high-resolution genomic data to assess the extent and function of copy-number alterations in cancer.

| S-EPMC2527508 | biostudies-literature

WaveCNV: allele-specific copy number alterations in primary tumors and xenograft models from next-generation sequencing.

Project description:MotivationCopy number variations (CNVs) are a major source of genomic variability and are especially significant in cancer. Until recently microarray technologies have been used to characterize CNVs in genomes. However, advances in next-generation sequencing technology offer significant opportunities to deduce copy number directly from genome sequencing data. Unfortunately cancer genomes differ from normal genomes in several aspects that make them far less amenable to copy number detection. For example, cancer genomes are often aneuploid and an admixture of diploid/non-tumor cell fractions. Also patient-derived xenograft models can be laden with mouse contamination that strongly affects accurate assignment of copy number. Hence, there is a need to develop analytical tools that can take into account cancer-specific parameters for detecting CNVs directly from genome sequencing data.ResultsWe have developed WaveCNV, a software package to identify copy number alterations by detecting breakpoints of CNVs using translation-invariant discrete wavelet transforms and assign digitized copy numbers to each event using next-generation sequencing data. We also assign alleles specifying the chromosomal ratio following duplication/loss. We verified copy number calls using both microarray (correlation coefficient 0.97) and quantitative polymerase chain reaction (correlation coefficient 0.94) and found them to be highly concordant. We demonstrate its utility in pancreatic primary and xenograft sequencing data.Availability and implementationSource code and executables are available at https://github.com/WaveCNV. The segmentation algorithm is implemented in MATLAB, and copy number assignment is implemented Perl.Contactlakshmi.muthuswamy@gmail.comSupplementary informationSupplementary data are available at Bioinformatics online.

| S-EPMC3957071 | biostudies-literature

Germline copy number variants are not associated with globally acquired copy number changes in familial breast tumours.

Project description:A characteristic of sporadic and familial breast tumours is genomic instability, resulting from either inherited mutations in genes that control genome integrity or mutations that are acquired in somatic cells during development. It is well established that abnormal chromosome number and structural changes to chromosomes play an important role in the cause and progression of breast cancer. Familial BRCA1 breast tumours are characterised by basal-like phenotype and high-histological grade which are typically associated with increased genomic instability. Consistent with previous studies, the genomes with the greatest number of base pairs covered by copy number change were typically found in basal-like and/or high-histological grade breast tumours within our cohort. Moreover, we show that luminal A tumours that are high grade had significantly less copy number variant (CNV) coverage than the more clinically aggressive high-grade luminal B tumours, suggesting that chromosomal instability rather than cellular differentiation contributes to the aggressive nature of luminal B tumours. It has previously been proposed that germline CNVs may contribute to somatically acquired chromosome changes in the tumour, but this is the first study to address this idea in breast cancer. By comparing germline CNVs and tumour-specific CNVs in matched breast tumour and normal tissue using data from the Illumina Human CNV370 duo beadarray, we provide evidence that germline CNVs do not tend to act as a foundation on which larger chromosome copy number aberrations develop in tumour cells. Further studies are required with increased sequence resolution that will detect smaller CNVs and define CNV breakpoints to comprehensively assess the relationship between inherited genomic variation and genome evolution in breast cancer.

| S-EPMC3409366 | biostudies-literature

Identification of copy number alterations in colon cancer from analysis of amplicon-based next generation sequencing data.

Project description:The objective of this study was to determine the feasibility to detect copy number alterations in colon cancer samples using Next Generation Sequencing data and to elucidate the association between copy number alterations in specific genes and the development of cancer in different colon segments. We report the successful detection of somatic changes in gene copy number in 37 colon cancer patients by analysis of sequencing data through Amplicon CNA Algorithm. Overall, we have found a total of 748 significant copy number alterations in 230 significant genes, of which 143 showed CN losses and 87 showed CN gains. Validation of results was performed on 20 representative genes by quantitative qPCR and/or immunostaining. By this analysis, we have identified 4 genes that were subjected to copy number alterations in tumors arising in all colon segments (defined "common genes") and the presence of copy number alterations in 14 genes that were significantly associated to one specific site (defined "site-associated genes"). Finally, copy number alterations in ASXL1, TSC1 and IL7R turned out to be clinically relevant since the loss of TSC1 and IL7R was associated with advanced stages and/or reduced survival whereas copy number gain of ASXL1 was associated with good prognosis.

| S-EPMC5945505 | biostudies-literature

Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

Project description:BackgroundThe concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends.MethodAs a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB) in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP) microarray method entailing CNB-region refinement by competitor DNA.ResultUsing TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9%) were identified, and two polymerase chain reaction (PCR)-amplifiable rearrangements were obtained in six cases (66.7%). And significantly, TNGS-CNB, with its high positive identification rate (82.6%) of PCR-amplifiable rearrangements at candidate sites (19/23), just from filtering of aligned sequences, requires little effort for validation.ConclusionOur results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.

| S-EPMC4061055 | biostudies-literature

Simultaneous Detection of Both Single Nucleotide Variations and Copy Number Alterations by Next-Generation Sequencing in Gorlin Syndrome.

Project description:Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions.

| S-EPMC4636311 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data