Whole genome sequencing of tumor Met2 and paired germline DNA revealed extensive somatic alterations in tumor DNA. (A) A CIRCOS plot shows non-synonymous somatic mutations, copy number changes, lesser allele fraction, loss of heterozygosity (LOH), and abnormal junctions in the Met2 tumor genome. (B,C) Chromothripsis was evident by massive complex rearrangements detected at chromosomes 4q and 13p by whole genome sequencing. In the junction plots, black, green, and red lines represent deletions, tandem duplication, and inversion respectively. Each dot in the copy number and LAF plots represents a 2 kilobases DNA fragment, and red and green lines mark the average segmental copy number and LAF respectively. LAF, lesser allele fraction. doi: 10.1371/journal.pone.0077731.g001

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... we used ultra-deep sequencing to examine these somatic mutations in additional 5 tumor samples (a bone marrow metastasis at diagnosis and 4 different samples from the primary tumor removed by surgical resection) from the same patient. Finally, we performed transcriptome sequencing on the liver and bone marrow metastases and one of the primary tumors to identify potential driver oncogenes that have been present and expressed throughout the history of the tumor ( Figure S1). These massively parallel sequencing experiments revealed an accumulation of de novo mutations and an activating mutation of LPAR1 in this patient with metastatic neuroblastoma. ...

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... of variants from tumor and germline, we identified 61 non-synonymous mutations out of 8508 high- quality somatic small variants in the tumor (somatic scores ≥-10). Forty-four of the 61 non-synonymous mutations were confirmed experimentally using orthogonal re-sequencing methods including Sanger or semiconductor sequencing ( Figure 1A and Table S2), corresponding to an accuracy of 72% and a mutation rate of 0.85 per mega bases for somatic non-synonymous mutations, similar to what was reported in neuroblastoma [15,17]. Out of the 44 mutant genes, only 1 gene (NEB) was reported to be mutated in other neuroblastoma in a published whole genome sequencing study confirming a low frequency of recurrent mutations in this cancer [15]. ...

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... addition to the small variants, structural variant analysis of whole genome sequencing demonstrated extensive chromosomal structural and copy number alterations involving virtually every chromosome resulting in aneuploid (near- tetraploid) in Met2 ( Figure 1A). These changes consisted of gain and loss in the regions commonly seen in neuroblastoma including loss of heterozygosity (LOH) on chromosomes 1p, 3, 11, 14, and 19; gain of 1q, 2, 6, 7, 8, 12, 13, 17, 18, 20, and 22 [26]. ...

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... changes consisted of gain and loss in the regions commonly seen in neuroblastoma including loss of heterozygosity (LOH) on chromosomes 1p, 3, 11, 14, and 19; gain of 1q, 2, 6, 7, 8, 12, 13, 17, 18, 20, and 22 [26]. Chromothripsis [27] was also detected in this tumor, supported by complex massive rearrangements with only 2 major copy number states on chromosome arms 4p and 13q (135 and 24 high-confidence structural junctions in 8.5Mb and 1.5 Mb respectively) ( Figure 1B and C, Table S3). This was in keeping with a recent observation of chromothripsis in 18% of patients with high-stage neuroblastoma [15]. ...

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... the heterogeneous nature of neuroblastoma genome and the rarity of the recurrent mutations will make it challenging to identify druggable targets in an individual patient for treatment. Here, we demonstrated a systematic approach to identify driver oncogenes by sequencing both tumor genomic DNA and transcriptome in multiple tumor samples from the same patient ( Figure S1). Mutations in tumor suppressor genes usually result in loss of expression such as in nonsense-mediated decay of their mutation-harboring messenger RNAs. ...