Coffalyser software

Manufactured by MRC-Holland

Sourced in United States, Netherlands

Coffalyser is a software application developed by MRC-Holland for the analysis of data generated from their MLPA (Multiplex Ligation-dependent Probe Amplification) assays. The software is designed to process and interpret the raw data files produced by MLPA experiments, providing users with a comprehensive analysis of the target DNA sequences.

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Coffalyser.Net software (66 citations) Coffalyser (18 citations) Coffalyser.NET analysis software (3 citations) Coffalyser software v.8 (3 citations) Coffalyser v6 MLPA Analysis Software (2 citations) Coffalyser® NET Software (v.210604.1451) (2 citations) COFFALYSER Software version (2 citations)

59 protocols using coffalyser software

Karyotype, MLPA, and SNP-Array Analysis

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The karyotype test was performed in metaphase from cultures of peripheral blood lymphocytes of the patients and their parents with standard methodology for GTG banding (∼500 bands). MLPA reaction was performed using the SALSA MLPA kit P290-B2 and P249 (MRC-Holland, Amsterdam, Netherlands) following supplier instructions. The results were analyzed with the Coffalyser software (MRC-Holland, Amsterdam, Netherlands). The SNP-array analysis was carried out using the CytoScan HD matrix (SNP-array) following the protocol provided by the supplier (Affymetrix, Santa Clara, California, USA). The SNP-array was scanned with the Affymetrix GeneChip Scanner 3000 7G, and the data were analyzed with GTYPE (GeneChip Genotyping Analysis Software, version 1.0.12) to detect aberrations in the number of copies. The resolution of this procedure was estimated at 1.15 kb. The Research Committee of the Institution approved the protocol and the parents gave their informed consent for the participation in the study.

Toral‐López J., González-Huerta L.M., Martínez‐Saucedo M., Messina‐Baas O., Manuel-Valdes J., & Cuevas‐Covarrubias S.A. (2021). Characterization of two children with tetrasomy 18p syndrome through multiplex ligation-dependent probe amplification and single nucleotide polymorphism-array: expanding phenotype?. Revista Médica Del Hospital General De México, 84(1).

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Exon-Level Aberration Detection in DMD

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Multiple ligation‐dependent probe amplification was used to detect exon‐level aberrations in six patients with clinical diagnosis of Duchenne muscular dystrophy (DMD). MLPA analysis was performed using the P034 DMD mix 1 and P035 DMD mix 2 kit (CE‐IVD; MRC‐Holland, the Netherlands) following manufacturer's instruction. Data were visualized and analyzed with Coffalyser software (MRC‐Holland, the Netherlands).

Yao R., Yu T., Qing Y., Wang J, & Shen Y. (2018). Evaluation of copy number variant detection from panel‐based next‐generation sequencing data. Molecular Genetics & Genomic Medicine, 7(1), e00513.

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MLPA Protocol for PMS2 Gene Analysis

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MLPA was performed according to manufacturer’s protocol (MRC Holland, probemix P008-C1 PMS2 protocol issued 12/11/17 and MLPA General Protocol issued on 3/23/18). Generally, genomic DNA was covered with mineral oil to reduce evaporation during hybridization and ligation; next, DNA was denatured for 5 min at 98 °C and then held at 25 °C. Hybridization reagents and probemix were added to the samples and incubated at 95 °C for 1 min followed by 16–20 h at 60 °C. Probe pairs that bind target DNA at adjacent positions were ligated for 15 min at 54 °C and then amplified via PCR for 35 cycles. Amplified probes were mixed with ROX ladder and formamide and then separated on a capillary electrophoresis instrument. Coffalyser software (MRC Holland) normalized PMS2 probe intensities to those of the reference probes first within each sample and then among samples. Normalized probe intensities of each sample were compared to the average intensities of the reference samples; Coffalyser emitted CNV calls in the region.

Gould G.M., Grauman P.V., Theilmann M.R., Spurka L., Wang I.E., Melroy L.M., Chin R.G., Hite D.H., Chu C.S., Maguire J.R., Hogan G.J, & Muzzey D. (2018). Detecting clinically actionable variants in the 3′ exons of PMS2 via a reflex workflow based on equivalent hybrid capture of the gene and its pseudogene. BMC Medical Genetics, 19, 176.

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Quantification of EGFR Variant III

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DNA was obtained from cultured cells with QIAmp DNA Mini Kit (Qiagen, Inc., Valencia, CA, USA) and multiplex ligation-dependent probe amplification (MLPA) was performed to determine the number of copies of the mutant variant III of EGFR (EGFRvIII) by establishing a EGFRvIII ratio between the average ratio for exons 2–7 probes and the average ratio of exons 1, 8, 13, 17 and 22 probes. SALSA MLPA kits were used according to the manufacturer’s instructions (MRC-Holland, Amsterdam, Netherlands). Fragments were separated with a sequencer ABI 310 (Applied Biosystems Inc., Foster City, CA, USA). Analysis was performed with the Coffalyser software (MRC-Holland). Ratios under 0.8 units were related with the EGFRvIII deletion variant.

Muñoz-Hidalgo L., San-Miguel T., Megías J., Serna E., Calabuig-Fariñas S., Monleón D., Gil-Benso R., Cerdá-Nicolás M, & López-Ginés C. (2020). The Status of EGFR Modulates the Effect of miRNA-200c on ZEB1 Expression and Cell Migration in Glioblastoma Cells. International Journal of Molecular Sciences, 22(1), 368.

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Comprehensive CFTR Mutation Screening

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Genomic DNA was extracted from whole-blood samples. To detect CFTR gene mutations, we subjected all 27 exons and the intronic boundaries to PCR-Sanger sequencing. PCR was performed under the following conditions: initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. The list of primer pairs used for PCR was reported previously.^{10 (link)} The Sanger sequencing results were analyzed using CodonCode Aligner Software (CodonCode Aligner Corporation; Centerville, MA, USA). The presence of large CFTR rearrangements was tested in all 27 CFTR exons using a commercial kit (MLPA SALSA kit; MRC-Holland, Amsterdam, The Netherlands) and the generated results were analyzed with Coffalyser software (MRC-Holland, Amsterdam, The Netherlands).

Tian X., Liu Y., Yang J., Wang H., Liu T., Xu W., Li X., Zhu Y., Xu K.F, & Zhang X. (2016). p.G970D is the most frequent CFTR mutation in Chinese patients with cystic fibrosis. Human Genome Variation, 3, 15063-.

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Genetic Profiling of Glioblastoma Multiforme

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Tissue samples and DNA were prepared as previously described,²² and genetic alterations identified as having prognostic potential in GBM were analyzed.²⁴, ²⁵, ²⁸, ²⁹, ³⁰, ³¹ Hotspot mutations in the IDH1, IDH2, BRAF, H3F3A gene bodies, and TERT promoter were detected, and MGMT methylation status assessed as previously described.³², ³³, ³⁴ Copy number alterations (CNA), including those for genes EGFR, CDKN2A, PTEN, PDGFR, CDK4, TP53, were evaluated using a multiplex ligation‐dependent probe amplification (MLPA) kit (P105‐2; MRC‐Holland, Amsterdam, the Netherlands) containing PDGFRA, EGFR, CDKN2A, PTEN, CDK4, MDM2, NFKBIA, and TP53 specific probes, with six other probes used as control probes (http://www.mlpa.com). MLPA was performed according to manufacturer's protocol. Denatured fragments were separated and quantified by electrophoresis using an ABI 3730 capillary sequencer (Applied Biosystems Nieuwerkerk aan de Ijssel, the Netherlands) and analyzed using GeneMapper^® (Applied Biosystem) and Coffalyser^® software (MRC‐Holland). Based on previous studies, we used thresholds of 1.2 and 0.8 for the detection of gains and losses, respectively.³⁵, ³⁶ In addition, ratios below 0.4, and above 2.0 were considered homozygous deletions, or amplifications, respectively.³⁵

Funakoshi Y., Hata N., Takigawa K., Arita H., Kuga D., Hatae R., Sangatsuda Y., Fujioka Y., Sako A., Umehara T., Yoshitake T., Togao O., Hiwatashi A., Yoshimoto K., Iwaki T, & Mizoguchi M. (2021). Clinical significance of CDKN2A homozygous deletion in combination with methylated MGMT status for IDH‐wildtype glioblastoma. Cancer Medicine, 10(10), 3177-3187.

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Comprehensive MLPA Screening for DMD Gene

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A total of 102 DMD/BMD/Carrier subjects were screened through MLPA for all exon deletions and duplications in the human DMD gene. MLPA was performed using SALSA MLPA kit P034/P035 (MRC-Holland, Netherlands) as per the manufacturer’s instructions. Fragment analysis was performed on the 3500xL Genetic Analyzer (Applied Biosystems, United States) and MLPA data were analysed using Coffalyser Software (MRC-Holland, Netherlands).

Patel K.M., Bhatt A.D., Shah K., Waghela B.N., Pandit R.J., Sheth H., Joshi C.G, & Joshi M.N. (2021). Molecular Diagnosis of Muscular Dystrophy Patients in Western Indian Population: A Comprehensive Mutation Analysis Using Amplicon Sequencing. Frontiers in Genetics, 12, 770350.

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Whole-genome sequencing and CNV analysis

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Following DNA isolation from whole blood, sequencing libraries were generated using TruSeq kits (Illumina). Whole-genome sequencing was performed on NovaSeq 6000 instruments (Illumina), generating paired 101-nt reads with an average coverage of 39.5× for the proband and 52.9× for the mother. Alignment and SNV calling were performed using the DRAGEN hardware and software platform, yielding 4,913,732 calls for the proband and 5,041,148 for the mother.
CNV calling was performed with CNVnator and Manta (Abyzov et al. 2011 (link); Chen et al. 2016 (link)). Variant call format (VCF) files incorporating SNV and CNV calls were annotated and analyzed using Fabric Enterprise version 6.5.0 (Fabric Genomics) according to standard guidelines (Coonrod et al. 2013 (link); Richards et al. 2015 (link)).
Multiplex ligation-dependent probe amplification (MLPA) was performed on the patient and parental samples and three unrelated control samples, using two custom-designed probes falling within the deleted region. The MLPA products were separated using a 3500 Genetic Analyzer (ThermoFisher), and the results were analyzed using Coffalyser software (MRC-Holland). For each probe, patient to averaged control peak intensity ratios of 0.40–0.65 were considered indicative of a heterozygous deletion, and ratios of 0 were considered indicative of a homozygous deletion.

James K.N., Lau M., Shayan K., Lenberg J., Mardach R., Ignacio R J.r., Halbach J., Choi L., Kumar S, & Ellsworth K.A. (2021). Expanding the genotypic spectrum of ACTG2-related visceral myopathy. Cold Spring Harbor Molecular Case Studies, 7(3), a006085.

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Identification of Globin Gene Rearrangements

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Two MLPA kits (MRC-Holland, Amsterdam, the Netherlands), for the identification of rearrangement in the alpha-globin gene cluster (SALSA MLPA KIT P140 HBA) and in the beta-globin gene cluster (SALSA MLPA KIT P102 HBB), were used according to the manufacturer's recommendations and as previously reported. 14 MLPA products were separated by ABI-3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), quantified with the Coffalyser software (MRC-Holland), and compared with a pool of normal subjects (Fig. 1A). qRT-PCR was performed with ABI 7900HT System and the Power SYBR-Green PCR-Master mix (Applied Biosystems) with primers chosen outside repeated sequences (Table 1 and Supplementary Data). The beta-2-microglobulin was used as the reference gene.

Cardiero G., Prezioso R., Dembech S., Del Vecchio Blanco F., Scarano C, & Lacerra G. (2016). Identification and molecular characterization of a novel 163 kb deletion: The Italian (ϵγδβ)(0)-thalassemia. Hematology (Amsterdam, Netherlands), 21(5).

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Methylation-Specific MLPA for Prader-Willi Syndrome

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MS-MLPA ME028 kits (MRC-Holland) were used for the diagnosis of PWS following the manufacturer's instructions. Denaturing and hybridization were the same as the MLPA assay. The hybridized samples underwent ligation with or without methylation-sensitive restriction enzyme Hha I at 48 °C for 30 min, and were amplified by PCR following the same procedure as MLPA. The fragments were separated using the 3730 Genetic Analyzer (Applied Biosystems) and assayed with the Coffalyser software (MRC-Holland).

Zhang L., Liu X., Zhao Y., Wang Q., Zhang Y., Gao H., Zhang B., Cui W, & Zhao Y. (2022). Genetic subtypes and phenotypic characteristics of 110 patients with Prader-Willi syndrome. Italian Journal of Pediatrics, 48, 121.

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