Discovery of structural alterations in solid tumor oligodendroglioma by single molecule analysis

Optical map construction

We used the Optical Mapping (OM) system to explore the genomic landscape of a solid tumor. Optical Mapping creates high-resolution physical maps of genomes through the analysis of ensembles of single molecule ordered restriction maps. The tumor biopsies were disaggregated into single cells, then run through a Percoll gradient to enrich for cancer cells (methods). High molecular weight genomic DNA was extracted directly from these cells, stretched and immobilized in regular arrays on positively charged glass surfaces using a microfluidic device (Figure 1A, details in methods)[66]. After deposition, the DNA was digested with the restriction enzyme SwaI. The surface-bound restriction fragments remained in register, and were stained with a fluorescent dye and imaged by automated fluorescent microscopy (Figure 1B). Dedicated machine vision software calculated the size, in kilobase pairs (kb), of each fragment based on measurements of integrated fluorescent intensity, resulting in the high throughput, massively parallel generation of ordered restriction maps, or ‘Rmaps’, from individual genomic DNA molecules (Figure 1C)[66]. The oligodendroglioma datasets comprise close to 700,000 such Rmaps, with an average size of greater than 400 kb (Additional file1). The relative order and distance between successive restriction fragments in a single molecule optical map can be used to determine the precise location in the genome that gave rise to that molecule, by means of pair-wise alignment against an in-silico restriction map[67]. The HF087 tumor dataset comprised 235,026 aligned Rmaps, corresponding to 36.92 fold coverage of the human genome, while the HF1551 dataset comprised 167,012 aligned maps, representing 25.36 fold coverage of the human genome (Additional file1). In the absence of a karyotype, our assessment of ploidy is based on optical map coverage and Affymetrix array analysis. Both these platforms, discussed in detail in subsequent sections, calculate chromosome copy number relative to normal, diploid genomes, and are in agreement that neither tumor sample is polyploid. They do, however, display aneuploidy, due to allelic losses of specific chromosomes/chromosome arms (1p, 19q, 13 in HF087 and 1p, 19q, 14, 21 in HF1551), so if anything the coverage is likely to be higher than what we reported.

The Rmaps that cluster together upon pair-wise alignment were then assembled into consensus optical maps and analyzed for presence of structural variants using the bioinformatics pipeline described in[56]. The final consensus map contigs span 96.73% and 93.92% of the human genome for tumors HF087 and HF1551, respectively.

Optical map coverage analysis

Discernment of copy number variants

Copy number was inferred from aligned coverage of Rmaps, prior to assembly, in a manner analogous to read-depth based methods for detecting copy number variants from second generation sequencing data (methods). Briefly, Rmaps were aligned to the in silico reference map, and then partitioned into discrete windows spanning each chromosome. These alignments were then compared to alignments of a reference data set (comprising of a number of normal genomes) that was used to ‘normalize’ the observed coverage. This was necessary because the number of Rmaps that align to a particular region of the genome depends, in part, on the density of restriction sites in that region, which varies from chromosome to chromosome (ranging from a low of 2.5 cuts/100 kb on chromosome 22 to a high of 9.25 cuts/100 kb on chromosome 4). A Hidden Markov Model (HMM) was then fitted to this data, and copy number changes were detected (Figure 2A)[68, 69]. Optical map coverage analysis confirmed the allelic loss of chromosome arms 1p and 19q in HF087 and HF1551. The breakpoints appear to be very close to the centromere, consistent with the proposed mechanism of an unbalanced reciprocal translocation mediating the LOH event[70, 71]. Additionally, coverage analysis also detected allelic loss of chromosome 13 (HF087), 14 and 21 (HF1551), which are known to be rarer events associated with oligodendroglioma.

Discovery of optical structural alterations

The optical consensus maps generated by map assembler were aligned to the in silico restriction map (generated from build 35 human reference sequence), and by comparison of the order and sizing of the 219,224 restriction fragments (fragments smaller than 0.4 kb in size were merged) between the experimental and the reference map. Such comparisons revealed structural variants in the experimental genome that were classified as four types: extra cuts (EC), where the optical consensus map displays a restriction site that was not predicted by the reference sequence; missing cuts (MC), where a cut that was predicted was not observed in the experimental map; insertions (INS), where the size of a fragment in the consensus map was significantly larger than its counterpart in the reference map; deletions (DEL), where a fragment in the experimental map was smaller than the corresponding reference fragment (or missing altogether); and finally, complex events (OTHER) involving multiple cut or size differences (methods). Approximately a third of the ECs and MCs represent small indels that are below the resolution of Optical Mapping (~3 kb)[56]. Figure 3C shows an example of each class of variant detected by Optical Mapping.

At first glance, it might appear that any one of these variants could be attributed to errors inherent in Optical Mapping. For instance, a missing cut could be due to incomplete digestion, an extra cut could result from spurious cutting by the restriction enzyme, or physical breakage of the DNA molecule, and uneven staining could lead to inaccurate estimation of fragment size. However, the high throughput advantage of Optical Mapping allows us to distinguish such random errors from legitimate genomic events. Any alteration in the optical consensus map was supported by multiple single molecule maps (Rmaps), each representing an independent observation at that locus. The Optical Mapping error models estimated the statistical significance of each structural variant, after taking into account the quality and quantity of the data[56].

A total of 1081 and 1085 differences were detected in HF087 and HF1551 respectively (Figure 3A and B). The distribution of structural variants across the genome is uniform and the pattern is similar for both tumors (Figure 3A). Variants range in size from single base differences to complex genomic events spanning hundreds of kilobases (Figure 4). Approximately 800 single base changes were detected in each tumor, including point mutations (such as the example depicted in Figure 4A), polymorphic SNPs where only one allele has a SwaI restriction site (referred to as snip-SNPs[75]), and small indels that create or remove a SwaI cut site but are below the detection limit of Optical Mapping. 179 indels with a median size of 6.6 kb were detected in each sample (Figure 5). For comparison, the median size of indels reported in the Database of Genomic Variants is 2.3 kb (Figure 5, inset). ~70 complex events were found in each tumor, including known polymorphic loci such as the major histocompatibility complex (MHC), giving us confidence that these results are not spurious. Optical Mapping also discerns balanced genomic events, where there is no net gain or loss of genomic sequence. A putative inversion spanning 352 kb of chromosome 7 was observed in HF087 which appears to disrupt the ZNF92 gene (Figure 4C). Finally, the largest events detected by Optical Mapping include gains or losses of entire arms of chromosomes, for example, the allelic loss of chromosome 1 illustrated in Figure 4D, and discussed in detail previously. Intersection counts with genes, segmental duplications, published SNPs (dbSNP build 135,http://www.ncbi.nlm.nih.gov/projects/SNP/) and published structural variants (Database of Genomic Variants, November 2010 release) are shown in Additional file2. Comprehensive breakdown of the overlaps are shown in Additional file3.

Optical Mapping provides a comprehensive description of the vast and complex landscape of cancer genomes. The ability to study the genome in its entirety, including non-genic or repetitive regions using a single technology minimizes ascertainment bias. As detailed in subsequent sections, it is employed to generate a list of candidate cancer genes that is not hypothesis-limited, and elucidate their structure at sub-genic resolution.

Validation of copy number and structural variants

Experimental validation: PCR

The nature of many of the structural variants, being within repetitive portions of the genome, but detected by Optical Mapping unfortunately precludes their comprehensive validation by simple PCR techniques. Accordingly, we selected two variants that were amenable to PCR and overlapped genes that may offer insights into the chemo- and radio-sensitivity of oligodendroglioma. These loci were then PCR amplified, cloned and sequenced (methods).

The optical map shows an EC in the PARK2 gene in HF1551 (Figure 6A). PARK2 is a putative tumor suppressor, and mutations in this gene have been reported in multiple cancer types (detailed in ‘candidate mutations’ section of this document). An 848 bp amplicon spanning the predicted location of the EC was obtained (Figure 6B), and Sanger sequencing proved that a G to T transversion resulted in the creation of a new SwaI restriction site (Figure 6C).

We also validated an EC in tumor HF087 that occurred in the STMN2 gene (Figure 7A). As discussed in subsequent sections, STMN2 regulates microtubule dynamics and is believed to be a target of beta-catenin/TCF signalling. We amplified a 1003 bp region around the putative mutation (Figure 7B), and were able to validate the alteration via sequencing (Figure 7C).

Candidate mutations

Candidates common to both HF087 and HF1551

Two candidate genes, NPAS3 and OSBPL3, harbored mutations in both tumor samples (Figure 8). NPAS3 (neuronal PAS domain protein 3) shows a complex event accompanied by a ~7 kb gain in the HF087 optical map, and a missing cut in the HF1551 optical map (Figure 8A). This neuronally expressed basic helix-loop-helix transcription factor has been implicated in schizophrenia[76, 77]and bipolar disorder[77], and is frequently deleted or inactivated in many cancers. Recently, it has been demonstrated that NPAS3 exhibits features of a tumor suppressor which drives late progression of malignant astrocytomas, and is a negative prognostic marker for survival[78].

Both tumor optical maps display cut differences in the OSBPL3 (oxysterol binding protein like-3) gene (Figure 8B). This gene plays a vital role in cell adhesion, cytoskeletal organization and lipid metabolism[79–81]. It is highly expressed in B-cell associated malignancies[82, 83], where it is one of the common sites of retroviral integration[84]. An independent study that used exon sequencing to study oligodendroglioma also found somatic mutations in OSBPL3[30].

Candidates observed in either HF087 or HF1551

In the HF1551 optical map, we observe a point mutation that creates a SwaI restriction site in the PARK2 gene (Figure 6). This gene encodes an E3 ubiquitin ligase, called Parkin that catalyzes the ubiquitination of a variety of target proteins for proteasome mediated degradation. Germline mutations in PARK2 have long been known to cause autosomal recessive juvenile Parkinson’s disease[85–87]. More recently, PARK2 has been identified as a tumor suppressor gene in Glioblastoma multiforme, breast, ovary, lung, colorectal and liver cancers[28, 88–94]. It encompasses most of FRA6E, the third most active common fragile site in the human genome[95], and shares the characteristics of other tumor suppressors such as FHIT and WWOX, that also occur in fragile sites. PARK2 is frequently deleted or inactivated in cancer cell lines and primary tumors[88, 92], and concomitantly, Parkin expression is either significantly diminished or absent[89, 92]. Unlike classical tumor suppressors where biallelic inactivation is necessary for oncogenesis, heterozygous mutations in PARK2 are sufficient to confer a growth advantage during tumor development[88, 92]. Restoring Parkin expression in Parkin-deficient cell lines reduces their profileration in vitro[92], while injection of Parkin-deficient cells into immunocompromised mice generate tumors in vivo[91]. Interestingly, PARK2 also mediates chemosensitivity in breast cancer via microtubule dependent mechanism[93, 96–98].

STMN2 (stathmin-like 2) is another interesting candidate gene. We observe a point mutation in this gene in tumor HF087 (Figure 7). STMN2 is a neuron specific member of the stathmin family of small regulatory phosphoproteins which control cell profileration and differentiation[99]. It is up-regulated in liver cancer and has been identified as a target of β-catenin/TCF-mediated transcription[100]. STMN2 sequesters soluble tubulin, forming a ternary complex, inhibits microtubule assembly and induces their disassembly[101]. Its highly similar, but more well-studied paralog STMN1, located on chromosome 1p, is known to sensitize cells to anti-microtubule drugs in glioma[102, 103], breast[104, 105] and prostate cancer[106].In light of recent studies demonstrating the synergistic epistasis between paralogous genes involved in essential cellular functions and its therapeutic implications[107, 108], we speculate that STMN1 and STMN2 might be functionally redundant, and inactivation of STMN2 might, in part, explain the treatment sensitivity of oligodendroglioma.

In the HF1551 optical map, we see an extra cut in the gene ZFYVE26 (zinc finger, FYVE domain containing 26). Spastizin, the zinc finger protein encoded by ZFYVE26, causes the neurological disorder hereditary spastic paraplegia[109]. This gene binds to the tumor suppressor Beclin-1 and regulates cytokinesis[110, 111], and is recurrently mutated in breast cancer[29].

We detected a 485 kb inversion on 7q11.23 in tumor HF1551. Hemizygous deletions spanning a 1.5-1.8 MB region of this locus cause the neurodevelopmental disorder, Williams-Beuren (WB) syndrome[112]. However, inversion of this region is polymorphic, and is present in ~6% of the general population, and in ~25% of transmitting parents in WB families[113–115]. Given the disparity in size between the aberration detected by OM and reported instances of the WB inversion, it is possible that the event we observe arose de novo and is distinct from the ‘canonical’ inversion. To test this hypothesis, we ran several targeted assemblies on the WB region. The general strategy for this approach was to modify the reference map in-silico to reflect our hypothesized structure (Figure 9A), and then use the iterative assembly framework described earlier to pull out individual restriction maps and generate an optical consensus map (methods). Since our map assembly pipeline was designed to provide the single most conservative answer, this approach is helpful in detecting large-scale aberrations that are significantly different from the reference sequence. Of the eight modified reference maps we started with, the one that reflected the canonical WB inversion/deletion did not grow, while the one that reflected the 485 kb event successfully generated a consensus map that spanned it and flanked contiguous regions on chromosome 7 (Figure 9B). The optical consensus map also closes the putative sequence gap immediately to the right of the inversion, and in fact, approximately half of the sequence gaps in the reference genome (NCBI, build 35) are spanned by optical consensus maps. The inversion encompasses the genes GTF2IRD2, PMS2P5, WBSCR16, GTF2IRD2B and NCF1, and its breakpoints appear to disrupt the genes GTF2I and STAG3L2. In the absence of matched normal DNA, it is impossible to ascertain if the inversion we detected was inherited through the germline or somatically acquired, however, this is the first report, to the best of our knowledge, of inversions in the WB region in the context of cancer.

Biological significance of candidates identified by optical mapping

The aim of this study is to generate new hypotheses for oligodendroglioma genetics, and as such, functional studies are beyond the scope of this paper. However, by surveying publicly available data on the candidates discerned by Optical Mapping, we can gain some insight into the roles they might play in malignant transformation.

Since cells can employ a number of mechanisms to compensate for loss or mutational inactivation of genes, a more direct way of assessing the functional role of a given candidate gene is to analyze changes in its pattern of expression between normal and disease states. Array based expression profiling of tumors HF087 and HF1551 was performed by Fine et. al.[138], and is publicly available through the NCBI GEO database[139], accession GSE4290. Differential expression analysis carried out using the EBarrays algorithm[140] shows that 5 genes (NPAS3, STMN2, ZFYVE26, PHLDB2 and PLEKHM3) from our list of 24 candidates is significantly up or down-regulated (p-value 1E-03 or less). A complete list of differentially expressed genes can be found in Additional file8. To assay for functional effects in an even larger population of tumors, we queried for changes in expression of our candidate genes in REMBRANDT, a database of molecular data on brain tumors (National Cancer Institute, 2005, REMBRANDT home pagehttps://caintegrator.nci.nih.gov/rembrandt/, accessed 13^thAugust 2012). The results are reported for each gene as the number of oligodendroglioma samples in the database that are differential expression by at least two-fold (Additional file9). All but 3 of our candidate genes were differentially expressed in at least 10 tumor samples. Congruent with the previous analysis, NPAS3, STMN2, ZFYVE26 and PHLDB2 are the most frequently deregulated candidate genes.

Finally, we asked what biological processes and pathways are significantly enriched or depleted in our list of candidates. This can identify fundamental cellular mechanisms that contribute to cancer development. As a whole, these candidate genes are enriched for proteins involved in cytoskeletal organization (p-value = 0.00223, after correcting for multiple testing, Ontologizer[141]). Our candidate genes are also significantly enriched for microRNA binding targets (p-values between 0.0144-0.0198 after correcting for multiple testing, WebGestalt[142]). Approximately half of the over-represented sites have been associated with binding of cancer-related microRNAs[143], underscoring the importance of post-transcriptional control of expression in oligodendroglioma.

While these results are not a direct indicator of aberrant function, this is a demonstration that Optical Mapping results can be expanded to clinical samples and used to create direct functional hypotheses.

Selection of tumors

The tumors used in this study originated from the tissue bank at the Hermelin Brain Tumor Center/Department of Neurosurgery, Henry Ford Hospital (provided by Dr. Oliver Bogler). Freshly resected tumors were snap frozen in liquid nitrogen in the operating room. Samples were sectioned in a guillotine in frozen condition, and adjacent pieces prepared for Optical Mapping and for re-review by a neuropathologist.

Extraction of high molecular weight DNA from solid tumor biopsies

The tumor was sectioned into 1–2 mm slices under sterile conditions in a cell culture hood. Each slice was treated with 0.8% type IV collagenase (Sigma-Aldrich, St. Louis, MO) in PBS (Phosphate buffered saline, Life Technologies, Carlsbad, CA) for 15 minutes at 37°C. The tumor tissue was mechanically disaggregated into a homogeneous suspension by repeated pipetting. The cells were pelleted by centrifugation at 1,000 RPM with a Beckman GS-6R centrifuge (Beckman Instruments, Fullerton, CA), and then resuspended in 1X HBS (Hanks Balanced Salts, Life Technologies, Carlsbad, CA) in order to lyse red blood cells. Cell debris and HBS were removed by centrifugation at 1,000 RPM. Finally, the pellet was rinsed three times with 35 mL of PBS, and resuspended in 0.5 mL of PBS.

A three layer Percoll gradient was employed to enrich for cancer cells, and minimize stromal contamination[146]. First, a 100% solution was made by using 9 parts Percoll (Sigma-Aldrich, St. Louis, MO) and 1 part 10X HBS, which was subsequently diluted with PBS to prepare 10%, 30%, and 50% solutions. The gradient was prepared by layering 2 mL of 50% Percoll, 2 mL of 30% Percoll, and 1 mL of 10% Percoll in a 15 mL Falcon tube. The single cell suspension was then carefully layered on top, and the gradient was spun at 1,000 RPM for 10 minutes. Studies have shown that cellular debris and non-viable cells are unable to penetrate the 30% layer, while lymphocytes pelleted at the bottom of the tube. The 30% layer, containing viable cells, was carefully removed, rinsed three times with 10 mL of PBS and then resuspended in PBS at a final concentration of 1X10⁷ cells/mL. Next, this cell suspension was mixed 1:1 (v/v) with 1.6% low gelling temperature agarose, poured into a mold and cooled to 4°C so that the agarose solidified to for gel inserts (each ~100 μL in volume).

The inserts were treated with 0.5 mg/mL proteinase K (Bioline USA, Taunton, MA), 100 mM EDTA pH 8.0 (Sigma-Aldrich, St. Louis, MO), 0.5% N-lauroylsarcosine (Sigma-Aldrich, St. Louis, MO) and incubated at 55°C overnight to lyse the tumor cells and degrade cellular proteins[45, 147–150]. Embedding cells in agarose inserts eliminates shear induced breakage of genomic DNA molecules upon lysis[151].

Prior to use, the gel inserts were rinsed in TE twice for 1 hour and then a third time overnight to remove the detergent and excess EDTA. DNA was electrophoretically extracted by applying a cycle of 100 V for 30 seconds and -100 V for 6 seconds.

Generation of single molecule optical maps

Optical Mapping surfaces were prepared as described earlier[152]. Briefly, acid-cleaned glass coverslips (22 × 22 mm, Fisher's Finest, Fisher Scientific) were treated with a mixture of N-trimethoxylsilylpropyl-N,N,N-trimethylammonium chloride and vinyltrimethoxysilane (Gelest, Morrisville, PA) rendering a positive charge to the surface. Genomic DNA, mixed with a sizing standard, was elongated via c apillary flow in a microfluidic device[66], and immobilized by electrostatic interactions with the positively charged surface, creating arrays of stretched, biochemically accessible substrates. The surface was then washed with TE (10 mM Tris–HCl, 1 mM EDTA, pH8.0) twice, equilibrated with digestion buffer (NEB buffer 3), then incubated with the restriction endonuclease SwaI (New England Biolabs, Beverly, MA), which cleaves the genomic DNA at its cognate site. Since the elongated DNA molecule is under slight tension, upon cleavage its ends relax, creating a 1–2 micron gap, readily detected by microscopy. The resulting restriction fragments remain adsorbed to the surface, aided by a polyacrylamide overlay, and hence retain their order creating, in essence, a barcode from each genomic DNA molecule. Restriction fragments were then stained with the DNA intercalating dye YOYO-1 (0.2 μM in β-mercaptoethanol/TE, Life Technologies, Carlsbad, CA) and imaged by automated fluorescence microscopy.

The images were collected on an Optical Mapping workstation, which consists of Zeiss 135M inverted microscope (Carl Zeiss, Thornwood, NY), illuminated by 488 nm argon ion laser (Spectra Physics, Santa Clara, CA) equipped with 63X oil immersion objective. Fully automated image acquisition software, referred to as Channel Collect[66], takes multiple overlapping images to span the entire length of each microchannel. The images were analyzed by custom machine vision software, called Pathfinder, which identifies DNA molecules on the surface and calculates the size of each restriction fragment based on integrated fluorescence intensity measurements relative to a sizing standard. Previous studies have shown that integrated fluorescence intensity scales with fragment mass, and is independent of stretch of the DNA molecule. The end result of these operations is the high throughput, massively parallel generation of single molecule ordered restriction maps, or optical maps, containing information about both the size and order of its restriction fragments.

Pipeline for optical Map assembly and identification of structural variants

The analytical framework for assembly of optical maps is analogous to sequence assembly. First, our pipeline automatically aligned optical maps against a SwaI restriction map created in silico from the human genome reference sequence (NCBI build 35) via SOMA (Software for Optical Map Alignment) using gapped global pair wise alignment[56, 67]. SOMA uses a scoring function that assigns penalties for differences in the optical map and the reference map, including missing or extra restriction sites, or differences in the size of the fragments that could represent insertions or deletions. The parameters of SOMA were set so that we are accurately aligning the molecule to the correct location, but loose enough for allow for a small number of differences that result from the mutations or polymorphisms present in the genome. The aligned maps were then partitioned into smaller bins (1 Mb windows spanning across each chromosome, with 500 kb overlap between adjacent windows) based on their location. The optical maps in each bin were assembled into optical consensus maps by a map assembler program, using a Bayesian inference algorithm[153]. Because some structural polymorphisms and mutations represent large-scale changes from the reference map, an iterative assembly process was used for the analysis of human data sets. The consensus map constructed in the previous step was used in place of the reference for seven more iterations of alignment and assembly, after which it was aligned to the reference sequence using SOMA. Using this strategy, Rmaps harboring major alterations that preclude alignment to the reference were gradually incorporated into the consensus map, extending it into regions that contain more complex rearrangements.

Lastly, the pipeline automatically performed analysis that tabulated structural variants using the final consensus map to the reference (derived from NCBI build 35 of the human genome[56]) and identified five classes of differences: missing cuts, extra cuts, insertions, deletions, and ‘other’ (multiple cut and/or size differences) across each cancer genome. Each of these differences, which are largely structural variants, has to satisfy certain statistical and empirical criteria. These parameters have been detailed in Teague et al.[56]. The only difference being the indel calling threshold, which was increased to a 13% change relative to the reference, with a 4.5 kb minimum.

Additionally, each structural variant was manually curated to ensure that the most conservative decision has been made at every locus. The genomic locations of the variants were converted to NCBI build 37 co-ordinates using the Batch Coordinate Conversion (liftover) tool from the University of California Santa Cruz Genome Browser[154] (http://genome.ucsc.edu).

Optical map coverage analysis

Variations in depth of coverage of optical maps aligned by SOMA across the genome can be used to detect copy number alterations. Intuitively, if a region of the tumor sample has increased (or decreased) copy number relative to the ‘normal’ reference genome, more (or less) maps will originate from it on an average. This is formalized as described. Pair-wise alignments of optical maps to an in silico reference were summarized by a single number (midpoint) representing location. These locations were modeled as realizations of a non-homogeneous Poisson process. The non-homogeneity arises from the fact that the likelihood of a map aligning to a genomic region depends on the density of restriction sites, and was accounted for using alignment data from a normal genome, which are used to define random intervals with counts that follow a negative binomial distribution. These counts were then modeled by a Hidden Markov Model, incorporating spatial dependence in the data and allowing more natural estimation of certain parameters[68, 69].

Affymetrix genome wide human SNP array 6.0

DNA was prepared for hybridization using the Blood and Cell Culture Kit (Qiagen, Valencia, CA), starting from frozen cells (HF087), or tumor tissue (HF1551), disaggregated into single cells as described previously. The HF087 cells were derived from the same slice used for Optical Mapping. However, since the same was not available for HF1551, a slice adjacent to the one used for mapping was used.

The DNA was digested with NspI and StyI restriction enzymes and ligated to adaptors that recognize the 4 bp overhangs. A generic primer that anneals to the adaptor sequence was then used to amplify adaptor-ligated DNA fragments, under PCR conditions optimized to preferentially amplify fragments in the 200 to 1,100 bp size range. The amplified DNA was then fragmented, labelled, and hybridized to a Genome-Wide Human SNP 6.0 Array (experiments were performed by the DNA Facility at the Carver College of Medicine, University of Iowa). Data analysis was performed using Genotyping Console 2.0 (Affymetrix, Santa Clara, CA). CNVs were called using either the Affymetrix algorithm (with default parameters) or five different algorithms (GLAD, Circular Binary Segmentation, Fused Lasso, Gaussian Model with Adaptive Penalty, Forward-Backward Fragment Annealing Segmentation) from CGHweb (http://compbio.med.harvard.edu/CGHweb/)[155]. Only CNV calls made by two or more algorithms were considered for comparison.

Parameters for comparing oligodendroglioma structural variants

PCR validation

Template for PCR was prepared by whole genome amplification of tumor DNA using the REPLI-g Mini kit (Qiagen Inc., Valencia, CA) as per the protocol provided by the manufacturer. Pooled normal DNA from 6 individuals (Promega Corporation, Madison, WI) was used for control reactions. Primers were designed using freely available software Primer 3 Plus[157]. PCR reactions were performed using reagents from the Expand Long Template PCR System (Roche Applied Science, Indianapolis, IN) following the protocol supplied by the manufacturer. PCR reactions were digested with appropriate restriction enzymes to establish that the correct region had been amplified. The amplicon was then cloned in E. coli using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA), plasmid DNA was purified using the Qiagen Plasmid Mini Kit (Qiagen Inc., Valencia, CA), and sequenced using Sanger biochemistry.

Targeted assemblies on Williams-Beuren chromosomal region

The SwaI in-silico restriction map from the Williams-Beuren region on chromosome 14 was modified to reflect one of eight alterations: 4 possible inversions, each with unique start/end locations and spans (including the ‘canonical’ inversion), and 4 possible deletions, each with unique start/end locations and sizes (including the ‘canonical’ deletion). These modified in-silico maps were subjected to 8 rounds of iterative assembly, using the collection of HF1551 Rmaps, with the same parameters as the genome-wide assembly. The results were manually curated to rule out assembly errors.

Ethics statement

This study was approved by the Institutional Review Board of the University of Wisconsin-Madison.

Availability of supporting data

All structural variation calls and analysis are contained within the additional files.