Coffalyser net software

The MCR-Holland SALSA MLPA Kit P060-050R (MRC-Holland) was used to reidentify the exon 7 copy number according to the manufacturer’s directions. The sequence-specific probes that capture the SMN1 and SMN2 genes were included in the kit^{54 (link)}. The data were analyzed with Coffalyser.NET software (MRC-Holland). Samples with a copy number status were categorized as normal (2 copies), homozygous deletion (0 copies), heterozygous deletion (1 copy), heterozygous duplication (3 copies), homozygous duplication (4 copies) and ambiguous copy number by referring to the manufacturer’s manual.

When no pathogenic mutations were detected by both DNA and cDNA sequencing, the samples were analyzed using multiplex ligation-dependent probe amplification (MLPA) with the SALSA MLPA P081/P082 NF1 Kits to detect single and multiple exon deletions/duplications and the SALSA MLPA P122 NF1 AREA Kit to screen gross deletions in the NF1 chromosomal region, according to the manufacturer's instructions (MRC Holland, Amsterdam, The Netherlands). Denatured genomic DNA (100 ng/150 ng) was added to the MLPA mix and the probes were allowed to anneal overnight before the subsequent ligation reaction was performed. qPCR amplification was performed using 6-carboxyfluoescein (FAM)-labeled primers; products were sequenced by an ABI Prism 3130 automatic DNA sequencer (Life Technologies, Saint Aubin, France). Peak areas for each separated fragment were measured by using Coffalyser.NET software (MRC Holland). Each MLPA product was normalized by dividing each peak area by the total peak area of reference probes peak for the sample to obtain the relative peak area values. The change of the peak values greater than ±0.3 was considered a duplication (an increase in value) or a deletion (a decrease in value). Ratios of <0.65 and >1.35 indicated deletions and duplications, respectively.

Copy number ratios relative to the normal reference were calculated with Coffalyser.NET software (MRC-Holland) using the default settings. A numerical gain was scored when the ratios exceeded 1.2 and a loss when the ratios were lower than 0.8; all other values were considered to be normal diploid. For individual genes, aberrations were scored by the median ratio of the gene-specific probes. For 1p, 1q, and 16q, a gain or loss of at least two consecutive probed loci was required to score a chromosome arm aberration. Associations between copy number aberrations and histopathologic subtypes (Fig 1) were calculated by logistic regression, and survival analyses (Table 1; Fig 2; Data Supplement) were performed using the Kaplan-Meier estimator, log-rank test, and Cox proportional hazards regression model (Data Supplement Methods). For multivariable analyses, the factors considered are listed in the “Variable” column of Table 2.

The multiplex ligation-dependent probe amplification (MLPA) test was offered to patients with uninformative CNVs in NGS (six cases) or the presence of a pathogenic CNV in a close relative (seven cases).
CNVs in BRCA1 and BRCA2 were detected by MLPA using SALSA MLPA Probemix P002 BRCA1 or SALSA MLPA Probemix P045 BRCA2/CHEK2 (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer’s instructions. The PCR products were separated on 3500xL Genetic Analyzer (Applied Biosystems). The data analysis was performed using the Coffalyser.NET software (MRC-Holland, Amsterdam, The Netherlands).

Genomic DNA was isolated using High Pure PCR Template Preparation Kits (Roche Diagnostics, Indianapolis, IN, USA), quantified by a spectrophotometer at 260 nm and stored at −20 °C. CYP21A2 analysis was performed by using polymerase chain reaction (PCR) combined with Sanger sequencing and the multiplex ligation-dependent probe amplification (MLPA) assay. Briefly, an 8.5 kb PCR product, covering part of the RCCX region of the chromosome 6, was amplified (using CYP779f 5′-ccagaaagctgactctggatg-3′ and Tena32F 5′-ctgtgcctggctatagcaagc-3′ primers) and directly sequenced using the BigDye Terminator Cycle Sequencing Kit, Version 3.1 (Applied Biosystems, Waltham, MA, USA) and an ABI 3100 Avant Genetic Analyser (Applied Biosystems) according to the manufacturer’s instructions (internal sequencing primers are available on request). Sequencing electropherograms were analyzed against the reference sequence NM_000500.9 using the SeqScape Version 3.0 software package (Applied Biosystems).
MLPA was employed to establish the exact copy number of the CYP21A2 gene (SALSA MLPA Probemix P050 CAH by MRC Holland, Amsterdam, The Netherlands). Three healthy individuals were included in the analysis as controls. Results were analyzed by Coffalyser.NET Software (MRC Holland, Amsterdam, The Netherlands).

SALSA MLPA (Multiplex Ligation-dependent Probe Amplification) probe mixes P002-D1 and P090-C1 were used for large deletions/duplications analysis in BRCA1-2, according to the manufacturer’s instructions (MRC-Holland, Amsterdam, Netherlands). MLPA amplicons were run on ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA) while the collected data were analyzed using Coffalyser.net Software (MRC-Holland). Peak heights were normalized and deletions/duplications were defined as recommended by the manufacturer.