Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods

AbstractQuantitative PCR is the diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of HHV-6 is important for detection of viral reactivation and inherited chromosomally integrated HHV-6 in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies. 11 of 17 (65%) HHV6 candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1254 bp and 983 bp, respectively. Copy number measured between 4-10X copies relative to the rest of the genome. We also report the first interspecies recombinant HHV-6 strain with a HHV-6A GS backbone and >5.5kb region from HHV-6B Z29 from U41-U43 that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at UL1/UL2, U87, and U89, as well as deletion in the U12-U24 region and U94/95 genes. HHV-6 strains derived from cord blood mononuclear cells from different labs on different continents revealed no copy number differences throughout the viral genome. These data indicate large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within theBetaherpesvirinae.ImportanceAnything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very usefulness through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well-known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6 biology and the standard selection process.

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Next Generation Sequencing of Circulating Cell-Free DNA for Evaluating Mutations and Gene Amplification in Metastatic Breast Cancer

Clinical Chemistry ◽

10.1373/clinchem.2016.261834 ◽

2017 ◽

Vol 63 (2) ◽

pp. 532-541 ◽

Cited By ~ 41

Author(s):

Karen Page ◽

David S Guttery ◽

Daniel Fernandez-Garcia ◽

Allison Hills ◽

Robert K Hastings ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Next Generation Sequencing ◽

Gene Amplification ◽

Somatic Mutations ◽

Metastatic Breast ◽

Healthy Controls ◽

Next Generation ◽

Free Dna ◽

Generation Sequencing

Abstract BACKGROUND Breast cancer tissues are heterogeneous and show diverse somatic mutations and somatic copy number alterations (CNAs). We used a novel targeted next generation sequencing (NGS) panel to examine cell-free DNA (cfDNA) to detect somatic mutations and gene amplification in women with metastatic breast cancer (MBC). METHODS cfDNA from pretreated patients (n = 42) and 9 healthy controls were compared with matched lymphocyte DNA by NGS, using a custom 158 amplicon panel covering hot-spot mutations and CNAs in 16 genes, with further validation of results by droplet digital PCR. RESULTS No mutations were identified in cfDNA of healthy controls, whereas exactly half the patients with metastatic breast cancer had at least one mutation or amplification in cfDNA (mean 2, range 1–6) across a total of 13 genes. Longitudinal follow up showed dynamic changes to mutations and gene amplification in cfDNA indicating clonal and subclonal response to treatment that was more dynamic than cancer antigen 15-3 (CA15-3). Interestingly, at the time of blood sampling disease progression was occurring in 7 patients with erb-b2 receptor tyrosine kinase 2 (ERBB2) gene amplification in their cfDNA and 3 of these patients were human epidermal growth factor receptor 2 (HER2) negative at diagnosis, suggesting clonal evolution to a more aggressive phenotype. Lastly, 6 patients harbored estrogen receptor 1 (ESR1) mutations in cfDNA, suggesting resistance to endocrine therapy. Overall 9 of 42 patients (21%) had alterations in cfDNA that could herald a change in treatment. CONCLUSIONS Targeted NGS of cfDNA has potential for monitoring response to targeted therapies through both mutations and gene amplification, for analysis of dynamic tumor heterogeneity and stratification to targeted therapy.

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Comparison of MET gene amplification analysis by next-generation sequencing and fluorescence in situ hybridization

Oncotarget ◽

10.18632/oncotarget.28092 ◽

2021 ◽

Vol 12 (22) ◽

pp. 2273-2282

Author(s):

Christina Schmitt ◽

Anna-Alice Schulz ◽

Ria Winkelmann ◽

Kevin Smith ◽

Peter J. Wild ◽

...

Keyword(s):

In Situ Hybridization ◽

Next Generation Sequencing ◽

Fluorescence In Situ Hybridization ◽

Gene Amplification ◽

Next Generation ◽

Generation Sequencing

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A novel multiplexed 11 locus HLA full gene amplification assay using next generation sequencing

HLA ◽

10.1111/tan.13729 ◽

2019 ◽

Vol 95 (2) ◽

pp. 104-116

Author(s):

Linh Truong ◽

Benedict Matern ◽

Lloyd D'Orsogna ◽

Patricia Martinez ◽

Marcel G. J. Tilanus ◽

...

Keyword(s):

Next Generation Sequencing ◽

Gene Amplification ◽

Next Generation ◽

Amplification Assay ◽

Generation Sequencing

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Evaluating Targeted Next-Generation Sequencing (NGS) Assays and Reference Materials for NTRK Fusion Detection

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2021.09.008 ◽

2021 ◽

Author(s):

Christina Bormann Chung ◽

Jeeyun Lee ◽

Marc Barritault ◽

Pierre-Paul Bringuier ◽

Zhaolin Xu ◽

...

Keyword(s):

Next Generation Sequencing ◽

Reference Materials ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Fusion Detection

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Quantitative and Qualitative QC of Next-Generation Sequencing for Detecting Somatic Variants: An Example of Detecting Clonal Hematopoiesis of Indeterminate Potential

Clinical Chemistry ◽

10.1093/clinchem/hvaa088 ◽

2020 ◽

Vol 66 (6) ◽

pp. 832-841

Author(s):

Young Kyu Min ◽

Young Kee Lee ◽

Seong-Hyeuk Nam ◽

Jae Kyung Kim ◽

Kyung Sun Park ◽

...

Keyword(s):

Next Generation Sequencing ◽

Reference Materials ◽

Limit Of Detection ◽

Read Depth ◽

Analytical Sensitivity ◽

Next Generation ◽

Clonal Hematopoiesis ◽

Fold Reduction ◽

The Cost ◽

Generation Sequencing

Abstract Background Because next-generation sequencing (NGS) for detecting somatic mutations has been adopted in clinical fields, both qualitative and quantitative QC of the somatic variants through whole coding regions detected by NGS is crucial. However, specific applications or guidelines, especially for quantitative QC, are currently insufficient. Our goal was to devise a practical approach for both quantitative and qualitative QC using an example of detecting clonal hematopoiesis of indeterminate potential (CHIP). Methods We applied the QC scheme using commercial reference materials and in-house QC materials (IQCM) composed of haplotype map and cancer cell lines for monitoring CHIP. Results This approach efficiently validated a customized CHIP NGS assay. Accuracy, analytical sensitivity, analytical specificity, qualitative precision (concordance), and limit of detection achieved were 99.87%, 98.53%, 100.00%, 100.00%, and 1.00%, respectively. The quantitative precision analysis also had a higher CV percentage at a lower alternative read depth (R2 = 0.749∼0.858). Use of IQCM ensured more than 100-fold reduction in the cost per run compared with that achieved using commercial reference materials. Conclusion Our approach determined the general analytical performance of NGS for detecting CHIP and recognized limitations such as lower precision at a lower level of variant burden. This approach could also be theoretically expanded to a general NGS assay for detecting somatic variants. Considering the reliable NGS results and cost-effectiveness, we propose the use of IQCM for QC of NGS assays at clinical laboratories.

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