Copycaller v2

Manufactured by Thermo Fisher Scientific

Sourced in United States

CopyCaller v2.0 is a software application developed by Thermo Fisher Scientific. The core function of CopyCaller v2.0 is to facilitate the analysis and quantification of nucleic acid samples.

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Lab products found in correlation

27 protocols using copycaller v2

Quantification of Genomic Alterations in FFPE Samples

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Genomic DNA was extracted from FFPE sections using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). To validate the results of QuantiGenePlex DNA Assay, the copy numbers of Cdk4, Ccnd1 and P16^INK4a were further quantified by TaqMan Copy Number Assays (Applied Biosystems of ThermoFisher, Waltham, MA). A TaqMan probe targeted on the Rnasep gene was used as a control. Quantitative real-time PCR was performed using the ABI 7500 FAST real-time PCR system (Applied Biosystems). Copy numbers were then determined by CopyCaller v2.0 software (Applied Biosystems) using the comparative Ct (ΔΔCt) method.

Xu L., Cheng Z., Cui C., Wu X., Yu H., Guo J, & Kong Y. (2019). Frequent genetic aberrations in the cell cycle related genes in mucosal melanoma indicate the potential for targeted therapy. Journal of Translational Medicine, 17, 245.

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Quantifying MAPK15 Copy Number in Gastric Cancer

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DNA copy number of MAPK15 in 16 gastric cancer cell lines and 48 gastric cancers were detected using commercially available, predesigned TaqMan Copy Number Assay (Assay ID: Hs00233516_cn, consisting of a pair of unlabeled primers and a FAM labeled MGB probe) and RNase P Copy Number Reference Assay (consisting of a pair of unlabeled primers and a VIC-labeled TAMRA probe) (Applied Biosystems) as previously described [34 (link), 35 (link)]. Genomic DNA was isolated using a DNA Mini Kit (Qiagen, Valencia, CA) for cell lines. The tissue sections were placed on slides and stained with H&E to evaluate the admixture of tumorous and non-tumorous tissues, before DNA was extracted from the fresh-frozen tissues. Tumor and non-tumorous areas were carefully microdissected under a microscope. The microdissected tissues were digested with proteinase K, and genomic DNA was isolated using DNeasy tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qPCR) was carried out in triplicate on an ABI 7900HT instrument (Applied Biosystems). The thermal cycling conditions were 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Analyses of the qPCR data were performed using the Copy Caller v2.0 software (Applied Biosystems).

Jin D.H., Lee J., Kim K.M., Kim S., Kim D.H, & Park J. (2015). Overexpression of MAPK15 in gastric cancer is associated with copy number gain and contributes to the stability of c-Jun. Oncotarget, 6(24), 20190-20203.

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Quantifying EZH2 Copy Number in Melanoma

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To validate the results of QuantiGenePlex DNA Assays, the copy numbers of EZH2 were further quantified by TaqMan Copy Number Assays (Applied Biosystems, ThermoFisher, Waltham, MA, USA). A TaqMan probe targeting the Rnasep gene was used as a control. The EZH2 Tagman probe (4400291) was purchased from Invitrogen (ThermoFisher). Quantitative real-time PCR was performed using an ABI 7500 FAST real-time PCR system (Applied Biosystems). Copy numbers were then determined using CopyCaller v2.0 software (Applied Biosystems) with the comparative Ct (ΔΔCt) method. The EZH2 copy numbers for melanoma samples were calculated by dividing the sample values by the control values (benign nevus). No gain was considered for copy number ≤ 2, whereas gain was considered for copy number ≥ 2 and high gain was considered for copy number ≥ 4.

Yu H., Ma M., Yan J., Xu L., Yu J., Dai J., Xu T., Tang H., Wu X., Li S., Lian B., Mao L., Chi Z., Cui C., Guo J, & Kong Y. (2017). Identification of coexistence of BRAF V600E mutation and EZH2 gain specifically in melanoma as a promising target for combination therapy. Journal of Translational Medicine, 15, 243.

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Quantification of UBE3A Copy Number

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The copy number of UBE3A in each iPSC line was analyzed by qPCR using genomic DNA purified from two biological replicates and TaqMan Copy Number Assays (Hs01665678_cn and Hs03908756_cn, Life Technologies, Grand Island, NY, USA). The RNase P Copy Number Reference Assay was used as an endogenous reference gene to allow for quantification of UBE3A copy number. Data were analyzed using the CopyCaller v2.0 software from Applied Biosystems (Life Technologies, Grand Island, NY, USA).

Germain N.D., Chen P.F., Plocik A.M., Glatt-Deeley H., Brown J., Fink J.J., Bolduc K.A., Robinson T.M., Levine E.S., Reiter L.T., Graveley B.R., Lalande M, & Chamberlain S.J. (2014). Gene expression analysis of human induced pluripotent stem cell-derived neurons carrying copy number variants of chromosome 15q11-q13.1. Molecular Autism, 5, 44.

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Quantifying CNV of MCM2 and MCM3 Genes

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Genomic DNA was isolated as described [31] (link) from set 1 and set 3. CNV was measured using PCR-Taqman Copy Number Reference Assay from Life Technologies according to the manufacturer's instructions using the primer sets Hs00575269_cn and Hs02422238_cn (MCM2) and Hs00378664_cn (MCM3). CNVs of each gene were normalized to telomerase reverse transcriptase. The results were analyzed with CopyCaller v2.0 software (www.appliedbiosystems.com).

Schimmack S., Lawrence B., Kenney B., Schmitz-Winnenthal H., Modlin I.M, & Kidd M. (2016). Minichromosome Maintenance Expression Defines Slow-Growing Gastroenteropancreatic Neuroendocrine Neoplasms. Translational Oncology, 9(5), 411-418.

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Validating Copy Number Variations

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CNV results were validated using pre-designed TaqMan Copy Number (CN) Assays (Applied Biosystems). Up to two CN assays were selected within the CNV region indicated by the Cyto2.7 M array and CN assays, proximal but external to the region were also selected as controls (assay information summarized in Additional file 1: Table S4). A total of 11 samples were run in triplicate comprised of the sample(s) of interest, a calibrator (control) sample with known CN for the region of interest and a no-template-control (NTC). Real-time PCR was conducted according to manufacturer’s protocols using 10 ng of DNA sample in a final reaction volume of 20 μL. The assay was run on the real-time PCR machine (Applied Biosystems 7500; SDS software Version v1.4) according manufacturer’s protocols. The results were exported to CopyCaller v2.0 software (Applied Biosystems) for analysis.
Three CNVs were validated using this secondary independent assay (Additional file 1: Table S5). The CNVs included a CN gain and a CN loss in the WWOX gene as well as a CN loss in the FHIT gene. Given the high concordance between the CNV calling within the experimental parameters set for this study and the independent copy number assays we considered that it was not necessary to confirm all CNVs using a second independent assay.

Masson A.L., Talseth-Palmer B.A., Evans T.J., Grice D.M., Hannan G.N, & Scott R.J. (2014). Expanding the genetic basis of copy number variation in familial breast cancer. Hereditary Cancer in Clinical Practice, 12(1), 15.

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Quantitative Real-Time PCR for SMN Genes

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The copy number of two SMN genes, SMN1 and SMN2, was determined by a quantitative real-time PCR assay as previously described (15 (link)). Briefly, 50ng of genomic DNA was amplified in the presence of 300 nM of forward and reverse primers and 100 nM of FAM dye-labeled MGB probes (Applied Biosystems, Carlsbad, CA). The PCR was conducted as described previously (15 (link)), with an exception in using RNase P as the internal endogenous control (Applied Biosystems, Carlsbad, CA). The copy number of SMN genes was determined by using the ABI7500 SDS software and the CopyCaller v2.0 software from Applied Biosystems.

Kariya S., Sampson J.B., Northrop L.E., Luccarelli C.M., Naini A.B., Re D.B., Hirano M, & Mitsumoto H. (2014). Nuclear localization of SMN and FUS is not altered in fibroblasts from patients with sporadic ALS. Amyotrophic lateral sclerosis & frontotemporal degeneration, 15(0), 581-587.

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GSTT1 Copy Number Genotyping

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DNA was extracted from blood leukocytes using a QiaAmp DNA Blood extraction kit (Qiagen, Germantown MD). The samples were genotyped using a predesigned TaqMan GSTT1 copy number assay (Hs00010004_cn) and run on the 7900HT Fast Real-Time System (Life Technologies, Foster City, CA). Copy number counts were calculated using Life Technologies CopyCaller v2.0 software. Approximately 5% of blind duplicates were included for quality control. Test for Hardy Weinberg Equilibrium was met for all three populations (p>0.05).

Park S.L., Kotapati S., Wilkens L.R., Tiirikainen M., Murphy S.E., Tretyakova N, & Marchand L.L. (2014). 1,3 Butadiene Exposure and Metabolism among Japanese American, Native Hawaiian, and White Smokers. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 23(11), 2240-2249.

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Genotyping GSTT1 and GSTM1 Deletions

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DNA was extracted from blood leukocytes by lysing any red blood cells present in the sample with Qiagen RBC lysis solution prior to using a QIAmp Blood Mini Extraction kit (Qiagen, Germantown, MD). GSTT1 and GSTM1 gene deletions were genotyped for each sample using a predesigned TaqMan GSTT1 copy number assay (Hs00010004_cn) and run on the 7900HT Fast Real-Time System (Life Technologies, Foster City, CA). Copy number counts were calculated using Life Technologies CopyCaller v2.0 software.

Hepler J.R., Berman D.M., Gilman A.G, & Kozasa T. (1997). RGS4 and GAIP are GTPase-activating proteins for Gqα and block activation of phospholipase Cβ by γ-thio-GTP-Gqα. Proceedings of the National Academy of Sciences of the United States of America, 94(2), 428-432.

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Quantifying RANKL Transgene Copy Number

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Mouse tails (0.5 cm or less) were clipped and frozen; genomic DNA (gDNA) was isolated from the samples with proteinase K digestion and phenol/chloroform extraction. We obtained four custom TaqMan primers from Applied Biosystems for quantifying the copy number of the RANKL transgene: one targeted to the deleted region of the Tnfsf11 gene and therefore was absent in the RANKL^−/− mice (forward 5′-GGTCTAACCCCTGGACATGTG-3′; reverse 5′-CTTTGCAATGACATGGCATCCT-3′; probe FAM-5′-CTGAGAACCTTGAAATTAAG-3′-NFQ), three targeted to the RANKL enhancer regions D2 (forward 5′-CATCTTCCAGGCCAGGCT-3′; reverse 5′-CAGAATTTGGTTTGCTGGTCTACAA-3′; probe FAM-5′-CAGGCTTCCTATGCAAATAA-3′-NFQ), D5 (forward 5′-ACTTCAACCAGAACCCATTCG-3′; reverse 5′-CTCGCCTCAT TGTGCAATTG-3′; probe FAM-5′-AACCACGGGCTTCACTGTCCTC-3′-NFQ) and T1 (forward 5′-TTTCCACATACCATCACATTTTCTG-3′; reverse 5′-CGGTTAATGCAGCCAACTG-3′; probe FAM-5′-AAACCACTT CCTCTCCCTGCTCC-3′-NFQ) (see Fig. 1). PCR was assembled as a multiplex reaction with FAM-labeled target probes and VIC-labeled TaqMan Copy Number Reference probes, mouse Tfrc (Life Technologies) according to the manufacturer’s directions. Copy number was calculated using Copy Caller v2.0 software (Life Technologies).

Onal M., Bishop K.A., St. John H.C., Danielson A.L., Riley E.M., Piemontese M., Xiong J., Goellner J.J., O’Brien C.A, & Pike J.W. (2015). A DNA Segment Spanning the Mouse Tnfsf11 Transcription Unit and Its Upstream Regulatory Domain Rescues the Pleiotropic Biologic Phenotype of the RANKL Null Mouse. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 30(5), 855-868.

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