Copycaller software

Manufactured by Thermo Fisher Scientific

Sourced in United States

CopyCaller software is a tool that enables accurate quantification of nucleic acid copy number. It provides functionality for data analysis and visualization to support research applications.

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Lab products found in correlation

70 protocols using copycaller software

Quantitative Analysis of ABCB1 Copy Number

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ABCB1 (MDR1) copy number was assessed in A2780, A2780pacR and A2780olapR cells using a quantitative Taqman gene copy number assay (Taqman assay ID Hs04962504_cn), where ABCB1 copy number was compared with the copy number of the endogenous control gene RNAse P (Taqman Copy Number Reference Assay) by the comparative Ct method, and relative quantitation values obtained using CopyCaller Software (Life Technologies). As additional controls, ABCB1 and RNAse P copy numbers were assessed in peripheral blood samples (n=2) obtained from healthy volunteers, and copy numbers of two were confirmed.

Vaidyanathan A., Sawers L., Gannon A.L., Chakravarty P., Scott A.L., Bray S.E., Ferguson M.J, & Smith G. (2016). ABCB1 (MDR1) induction defines a common resistance mechanism in paclitaxel- and olaparib-resistant ovarian cancer cells. British Journal of Cancer, 115(4), 431-441.

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Quantifying CYP2D6 Copy Number

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To access CYP2D6 gene copy number, three TaqMan^® real-time PCR assays targeting different regions of the CYP2D6 gene were used. All TaqMan^® assays and reagents were purchased from Life Technologies (Carlsbad, CA), including three commercial quantitative TaqMan® copy number assays (assay IDs: Hs00010001_cn targeting exon 9, Hs04502391_cn targeting intron 6, and Hs04083572_cn targeting intron 2) and one TaqMan^® copy number reference assay, RNase P, human (assay ID: 4403326). All assays were performed in triplicate along with an internal control RNaseP assay according to the manufacturer’s protocol directly using genomic DNAs. Briefly, 10 ng of genomic DNA was used in the volume of 10 µl reaction and the PCR conditions were as follows: hold at 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 60 sby ViiA 7 Real-time PCR system (Life Technologies, Carlsbad, CA). Relative quantification of CYP2D6 copy number was performed using CopyCaller Software (Life Technologies, Carlsbad, CA) following the comparative delta-delta threshold cycle (ΔΔC_T) method. Each assay was repeated twice.

Fang H., Liu X., Ramírez J., Choudhury N., Kubo M., Im H.K., Konkashbaev A., Cox N.J., Ratain M.J., Nakamura Y, & O’Donnell P.H. (2014). Establishment of CYP2D6 Reference Samples by Multiple Validated Genotyping Platforms. The pharmacogenomics journal, 14(6), 564-572.

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CYP2D6 Gene Copy Number Quantification

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The CYP2D6 gene copy number for the patient DNA samples was determined by TaqMan^TM Copy Number Assay (Life Technologies, California, USA) according to the manufacturer’s recommendations. The genomic DNA samples were amplified in 96-well plates on QuantStudio^TM 12K Flex Real-Time PCR system (Applied Biosystems, Life Technologies Holding, Singapore) using comparative ΔΔC_T method. CYP2D6 exon 9 (TaqMan™ Copy Number Assay ID: Hs00010001_cn), and intron 6 (TaqMan™ Copy Number Assay ID: Hs04502391_cn) were used as primers. RNase P (ID: 431683) was used as the internal control for copy number analysis (Life Technologies). All the samples were run in triplicate. The final volume for each reaction was 10 μL, consisting of TaqMan™ Genotyping Master Mix (Applied Biosystems), TaqMan™ Copy Number Assay mix (20×, Applied Biosystems), TaqMan™ Copy Number Reference Assay mix (20×, Applied Biosystems). The PCR conditions consisted of an initial step at 60 °C for 30 s, hold stage at 95 °C for 10 min and PCR stage for 40 cycles step 1 with 95 °C for 15 and step 2 with 60 °C for 1 min and after read stage with 60 °C for 30 s. Relative quantification of CYP2D6 gene copy number was performed using CopyCaller™ Software (Life Technologies). Genomic DNA samples with known CYP2D6 gene copy number (0, 1, 2, 3 and 4 CYP2D6 gene copies) obtained from a previous study [15 (link)] were used as controls.

Ahmed J.H., Makonnen E., Fotoohi A., Aseffa A., Howe R, & Aklillu E. (2019). CYP2D6 Genotype Predicts Plasma Concentrations of Tamoxifen Metabolites in Ethiopian Breast Cancer Patients. Cancers, 11(9), 1353.

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Quantitative PCR Copy Number Analysis

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The amplification curves were analyzed using Viia7 RUO software v1.2 (Life Technologies). To determine the copy number for the targets in each sample, the real-time PCR data were exported into the CopyCaller® Software (Life Technologies). The CopyCaller® Software provides the calculated copy numbers and predicted copy numbers of each test sample. The software performs a comparative quantification cycle (Cq) relative quantification on the real-time data to determine the calculated copy numbers. First, the difference between the Cq of the target and reference assay is calculated (ΔCq) in each sample for both the patient sample and the calibrator sample. Then, the method compares the ΔCq values of the patient sample and the calibrator sample (ΔΔCq). With this approach, the predicted copy number of normal samples with two copies of each α-globin gene will be 2 for all assays. The predicted copy number of a sample with one gene deleted will be 1 in the respective assay, and a sample with a two-gene deletion will have a predicted copy number of 1 in all three assays, except for the HS-40 assay. Due to the mathematics of the 2^-ΔΔCq method, the calculated copy numbers deviates from whole numbers. Predicted copy numbers are based on the calculated copy numbers and are specified as whole numbers.

Grimholt R.M., Urdal P., Klingenberg O, & Piehler A.P. (2014). Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR. BMC Hematology, 14, 4.

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CDKN2A Copy Number Variation Assay

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All tissue samples were analyzed for CDKN2A copy number using a Taqman® copy number assay (Life Technologies, Carlsbad, CA) to measure copy number variation at the CDKN2A locus. The assay is a duplex polymerase chain reaction (PCR) for the CDKN2A gene and the reference gene, RNaseP (normalizer), using 10 ng DNA in quadruplicate PCR according to the manufacturer's protocol and run on the StepOnePlus real time PCR instrument (Life Technologies). The results were calculated as a ratio relative to a 2-copy control using the Copy Caller software (Life Technologies). Loss of CDKN2A was defined as ≤1.5 copies, no loss was >1.5 copies.

Stenzinger A., Endris V., Pfarr N., Andrulis M., Jöhrens K., Klauschen F., Siebolts U., Wolf T., Koch P.S., Schulz M., Hartschuh W., Goerdt S., Lennerz J.K., Wickenhauser C., Klapper W., Anagnostopoulos I, & Weichert W. (2014). Targeted ultra-deep sequencing reveals recurrent and mutually exclusive mutations of cancer genes in blastic plasmacytoid dendritic cell neoplasm. Oncotarget, 5(15), 6404-6413.

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Quantitative PCR for IGF1R Copy Number

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Genomic DNA from all samples and cell lines were extracted as described above and quantitated using Taqman RNaseP Detection Reagents (Life Technologies, Carlsbad, CA). To determine the IGF1R copy number, quantitative PCR was performed using three different Taqman copy number (CN) assays (Life Technologies, Carlsbad, CA; Assay IDs: Hs00401826_cn, Hs01239357_cn, Hs02543373_cn) targeting different locations on the chromosome 15 where the IGF1R gene spans. According to NCBI build 37 database, the three CN assay locations were at chr15∶99251313 (overlaps Exon 2 - Intron 2), chr15∶99460087 (overlaps Exon 10 - Intron 10) and chr15∶99491821 (overlaps Intron 19 - Exon 20), respectively. RNaseP was used as the reference internal control. Data analysis was done using CopyCaller software (Life Technologies, Carlsbad, CA), with the MSCs as the normal control.

Cao Y., Roth M., Piperdi S., Montoya K., Sowers R., Rao P., Geller D., Houghton P., Kolb E.A., Gill J, & Gorlick R. (2014). Insulin-Like Growth Factor 1 Receptor and Response to Anti-IGF1R Antibody Therapy in Osteosarcoma. PLoS ONE, 9(8), e106249.

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TaqMan Copy Number Assay for BP1-2 Region

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Before experimentation, a TaqMan assay Hs01476346_cn (Life Technologies) was used to determine copy number at the BP1-2 region in all lymphoblastoid and NPC lines used. Assays were performed in triplicate with TaqMan Gene Expression Master Mix (Life Technologies) following the standard TaqMan Copy Number Assay protocol. An RNase P probe (Cat# 4403326, Life Technologies) was used as a reference. Copy number variants were called using Copy Caller Software (v2.0, Life Technologies).

Nebel R.A., Zhao D., Pedrosa E., Kirschen J., Lachman H.M., Zheng D, & Abrahams B.S. (2016). Reduced CYFIP1 in Human Neural Progenitors Results in Dysregulation of Schizophrenia and Epilepsy Gene Networks. PLoS ONE, 11(1), e0148039.

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Copy Number Analysis of Fragile Sites

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Taqman copy number analysis was carried out as multiplex PCR in duplicates with 20 ng DNA per reaction and Ttert as internal reference according to the manufacturer’s protocol. Wwox, Spata22, Fhit were selected as described markers from common fragile sites in humans and Fgfr1 and Fgr were selected as genes of interest in previously published areas of genetic instability in Tak1^Δhep-/- mice (Bettermann et al., 2010 (link)). Data analysis was performed using Copy Caller Software (Life Technologies).

Boege Y., Malehmir M., Healy M.E., Bettermann K., Lorentzen A., Vucur M., Ahuja A.K., Böhm F., Mertens J.C., Shimizu Y., Frick L., Remouchamps C., Mutreja K., Kähne T., Sundaravinayagam D., Wolf M.J., Rehrauer H., Koppe C., Speicher T., Padrissa-Altés S., Maire R., Schattenberg J.M., Jeong J.S., Liu L., Zwirner S., Boger R., Hüser N., Davis R.J., Müllhaupt B., Moch H., Schulze-Bergkamen H., Clavien P.A., Werner S., Borsig L., Luther S.A., Jost P.J., Weinlich R., Unger K., Behrens A., Hillert L., Dillon C., Di Virgilio M., Wallach D., Dejardin E., Zender L., Naumann M., Walczak H., Green D.R., Lopes M., Lavrik I., Luedde T., Heikenwalder M, & Weber A. (2017). A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development. Cancer Cell, 32(3), 342-359.e10.

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Detecting CpG Islands and Copy Number Variations in GPR155 Promoter

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We used CpG Island Searcher software (http://cpgislands.usc.edu/)43 (link)44 (link) to detect predicted CpG islands in the GPR155 promoter region (chr2:174486637–174487426). Genomic DNAs of the cell lines were treated with bisulfite, and bisulfite sequence analysis was performed as previously described (Table 4)45 (link). Using purified genomic DNA isolated from GC cell lines, DNA copy numbers were determined using TaqMan Copy Number Assays (Applied Biosystems). The assays were as follows: upstream (assay ID: Hs01092594_cn, 175351658 within exon 1), midstream (assay ID: Hs01971174_cn, 175335170 within exon 6), and downstream (assay ID: Mn00059996_cn, 73351855 overlaps intron 14 and exon 14). Copy number alteration in the GPR155 locus were determined using CopyCaller™ Software (Life Technologies, Carlsbad, CA, USA)⁴⁶.

Shimizu D., Kanda M., Tanaka H., Kobayashi D., Tanaka C., Hayashi M., Iwata N., Niwa Y., Takami H., Yamada S., Fujii T., Nakayama G., Fujiwara M, & Kodera Y. (2017). GPR155 Serves as a Predictive Biomarker for Hematogenous Metastasis in Patients with Gastric Cancer. Scientific Reports, 7, 42089.

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Validation of GFP Cassette Insertion

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To detect the presence or absence of correctly targeted insertion of the GFP reporter cassette, PCR was performed using GoTaq Long PCR Master Mix (Promega), primers flanking the 5′ and 3′CRX homology arm sequences (see Supporting Information Table 1) and genomic DNA extracted (Quick‐gDNA MiniPrep, Zymo Research) from puromycin‐resistant colonies. PCR products were resolved on a 1% agarose gel. Sequencing was performed by Eurofins Genomics on genomic DNA extracted from puromycin‐resistant colonies and using primers flanking and residing across the donor construct (for primer details see Supporting Information Table 1).
Copy number analysis of GFP puromycin‐resistant clone 1 genomic DNA was carried out using a quantitative PCR (qPCR) approach with TaqMan Copy Number Assays and Genotyping Master Mix (Life Technologies) following manufacturers guidelines. Briefly, a duplex qPCR reaction was performed with an RNase P reference and GFP assay (Supporting Information Table 1) to quantify the copy number of GFP using clone 1 genomic DNA alongside untreated wild‐type H9 hESC genomic DNA. Four technical replicates were performed for each sample. Reactions were run on a 7500 Fast Real‐Time PCR System (Life Technologies) and data analyzed in Copy Caller software (Life Technologies).

Collin J., Mellough C.B., Dorgau B., Przyborski S., Moreno‐Gimeno I, & Lako M. (2015). Using Zinc Finger Nuclease Technology to Generate CRX‐Reporter Human Embryonic Stem Cells as a Tool to Identify and Study the Emergence of Photoreceptors Precursors During Pluripotent Stem Cell Differentiation. Stem Cells (Dayton, Ohio), 34(2), 311-321.

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