Cell lines and cultivation
P-STS cells were cultured in Ham´s F12:M199 (PAA Laboratories, Pasching, Austria) (1:1) supplemented with 10% FBS (PAA). KRJ-I  were cultured in Ham’s F12 medium. The two metastatic cell lines L-STS and H-STS were cultured in serum free Quantum 263 Complete Medium (PAA). All cells were kept in a 5% CO2 atmosphere at 37°C. An adequate review of the immunocytochemical analysis for NET markers especially in the low passages was done previously (Pfragner et al. 2009). Cell cultures were periodically checked for mycoplasma contamination. During this study, cell line batches from low passage (< 15) and high passages (> 15) were used.
Confocal laser scanning microscopy
Suspensions of P-STS, L-STS, and H-STS were analyzed for serotonin (5_HT) immunoreactivity. The KRJ-I NET cell line served as positive control (data not shown). Negative controls were incubated in buffer instead of the primary anti-serotonin antibody. The primary anti-serotonin antibody was preadsorbed at a dilution of 1:2.000 in 1 mM serotonin creatinine sulphate complex (Sigma, Vienna, Austria) for 1h at 37°C. For immunofluorescence, cells were fixed in 2% paraformaldehyde for 15 min, incubated in.blocking solution for 30 min at RT and incubated for 16–24 hours at 4°C with the primary antibody (polyclonal rabbit-anti-human serotonin; Acris Immunostar, Hudson, WI). After incubation with goat-anti-rabbit-Cy3 antibody (Jackson, Suffolk, UK) for 2 h at RT in the dark the cells were analysed on a confocal laser scanning microscope (Leica TCS SP2; Leica Lasertechnik GmbH, Heidelberg, Germany). Scanning parameters were adjusted for each cell type using its pre-adsorption control.
Sequence capture array design
A custom tiling NimbleGen 385 k sequence capture array targeting the exonic sequences (n = 4,935) of 203 cancer associated genes and 16 collagen genes were designed and manufactured by Roche NimbleGen. The array was designed using NimbleGen’s standard 15-mer frequency masking to minimize repeat content within capture probes. The probe spacing, tiling overlap, and probe length were determined by NimbleGen using proprietary algorithms. A GFF- or BED-formatted file (Additional file 2 and 3) allowing visualization of the tiled intervals by the Genome Browser (http://genome.ucsc.edu/).
Sequence capture library construction
Genomic DNA (20 μg per sample) isolated from the snap-frozen primary tumor sample and the three corresponding cell lines (P-STS, H-STS and L-STS) was processed into a capture library according to the manufacturers protocol (Additional file 1: Procedure S1).
Capture array handling
Hybridization was performed using microarrays merged with X1 mixer on the NimbleGen Hybridization System for 3 days at 42°C following the manufacturer’s recommended conditions (Additional file 1: Procedures S2). Quantitative PCR (SYBR-Green based; LC480 instrument) using four internal NimbleGen control loci (NSC-0237, NSC-0247, NSC-0268, NSC-0272) was performed to estimate relative fold-enrichment (data not shown).
GS FLX sequencing
The amplified capture libraries were processed into sequencing libraries for the 454 GS-FLX using the Shotgun DNA Titanium Library Construction Kit and low-molecular-weight DNA (without the nebulisation step) protocols (454 Life Sciences, Branford, CT) according to the manufacturer’s recommended conditions. Each captured sample library was sequenced using a quarter of a Titanium PicoTiterplate (70x75) run on the GS-FLX platform.
Data analysis - Variant detection and annotation
High Confidence Differences were calculated using the GS Reference Mapper assembly package (version 2.0.00.20; Roche Diagnostics) and hg18 reference sequence and SNPdb built 130 (Additional file 1: Procedures S3).
Sequence variants observed by NGS sequencing were re-evaluated by Sanger sequencing. PCR products were purified using Nucleo Fast® (Macherey-Nagel, Düren, Gemany) clean up PCR plates according to the manufacturer´s protocol. Following purification, capillary sequencing reactions were performed using Applied Biosystems BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit (Foster City, CA). Sequencing reactions were purified using Sigma Spin Post Reaction Clean-up Plates (Sigma-Aldrich, Austria) and run on an Applied Biosystems 3730. Data files were analyzed using ABI SeqScape v2.5 software (Applied Biosystems).
High resolution melting curve analysis
High Resolution Melting Curve analysis for variant positions in RB1 exon 2 and MLL exon 32 mutations was performed using a LightCycler 480 system (Roche Diagnostics, Penzberg, Germany). Primers were designed using primer3 software to span the positions of interest with product sizes of 134 bp for RB1 and 185 bp for MLL. The 20 μl reaction mix for PCR amplification contained 20 ng genomic template DNA, 10 μl of LightCycler480 High Resolution Master Mix (Roche Diagnostics, Penzberg, Germany), 10 μM of each primer and MgCl2 in a final concentration of 2.5 mM. The reaction condition included a pre-incubation step at 95°C for 10 min for the activation of the polymerase, followed by 45 cycles of 95°C for 10 s, 58°C for 15 s and 72°C for 15 s. A melting pre-hold step was included to ensure that all PCR products have re-associated and encourage heteroduplex formation. The melting interval ranged from 65°C to 95°C and increased at 1°C per second with 35 acquisitions per degree. Normalized, temperature-shifted melting curves carrying sequence variation were analyzed using the automated grouping functionality provided by the LightCycler480 GeneScanning 1.5 Software (Roche Diagnostics, Penzberg, Germany).
Affymetrix SNP 6.0 array processing and analysis
Affymetrix GeneChip Human Mapping SNP 6.0 arrays were performed as described in the Genome-Wide Human SNP Nsp/Sty 6.0 User Guide (Affymetrix Inc., Santa Clara, CA, USA). Detailed information: Additional file 1: Procedures S4.
SNP 6.0 data were imported and normalized using the Genotyping Console 4.0 program default settings. All samples passing QC criteria were subsequently genotyped using the Birdseed (v2) algorithm. We used 60 raw HapMap data generated with the Affymetrix Genome-Wide Human SNP Array 6.0 as reference. Data were obtained from Affymetrix (Affymetrix, Santa Clara, CA) web site and used for normalization.