Abstract
U87MG is a commonly studied grade IV glioma cell line that has been analyzed in at least 1,700 publications over four decades. In order to comprehensively characterize the genome of this cell line and to serve as a model of broad cancer genome sequencing, we have generated greater than 30× genomic sequence coverage using a novel 50-base mate paired strategy with a 1.4kb mean insert library. A total of 1,014,984,286 mate-end and 120,691,623 single-end two-base encoded reads were generated from five slides. All data were aligned using a custom designed tool called BFAST, allowing optimal color space read alignment and accurate identification of DNA variants. The aligned sequence reads and mate-pair information identified 35 interchromosomal translocation events, 1,315 structural variations (>100 bp), 191,743 small (<21 bp) insertions and deletions (indels), and 2,384,470 single nucleotide variations (SNVs). Among these observations, the known homozygous mutation in PTEN was robustly identified, and genes involved in cell adhesion were overrepresented in the mutated gene list. Data were compared to 219,187 heterozygous single nucleotide polymorphisms assayed by Illumina 1M Duo genotyping array to assess accuracy: 93.83% of all SNPs were reliably detected at filtering thresholds that yield greater than 99.99% sequence accuracy. Protein coding sequences were disrupted predominantly in this cancer cell line due to small indels, large deletions, and translocations. In total, 512 genes were homozygously mutated, including 154 by SNVs, 178 by small indels, 145 by large microdeletions, and 35 by interchromosomal translocations to reveal a highly mutated cell line genome. Of the small homozygously mutated variants, 8 SNVs and 99 indels were novel events not present in dbSNP. These data demonstrate that routine generation of broad cancer genome sequence is possible outside of genome centers. The sequence analysis of U87MG provides an unparalleled level of mutational resolution compared to any cell line to date.
Highlights
Grade IV glioma, called glioblastoma multiforme (GBM), is the most common primary malignant brain tumor with about 16,000 new diagnoses each year in the United States
We performed an exon capture approach designed to sequence the exons of 5,253 genes (10.7Mb) annotated in the Wellcome Trust Sanger Institute Catalogue of Somatic Mutations in Cancer (COSMIC) V38 [8], Cancer Gene Census, Cancer Genome Project Planned Studies and The Cancer Genome Atlas (TCGA) [4] GBM gene list using a custom-created Agilent array
We explored the overlap of genes with mutations in GBMs according to the Cancer Genome Atlas (TCGA) with those we predicted are homozygously loss-of-function mutated in U87MG (Table S5)
Summary
Grade IV glioma, called glioblastoma multiforme (GBM), is the most common primary malignant brain tumor with about 16,000 new diagnoses each year in the United States. It is well recognized that cell line models of human disorders, especially cancers, are an important resource While these cell lines are the basis of substantial biological insight, experiments are currently performed in the absence of genome-wide mutational status as no cell line that models a human disease has yet had its genome fully sequenced. The U87MG cell line is known to have a highly aberrant genomic structure based on karyotyping, SKY [6], and FISH [7]. These methods neither provide the resolution required to visualize the precise breakpoint of a translocation event, nor are they generally capable of identifying genomic microdeletions (deletions on the order of a megabase or less in size) in a whole genome survey of structural variation. SNP genotyping microarrays can be used to detect regions of structural variation in the forms of loss of heterozygosity (LOH) and copy number (CN) based on probe intensity, but do Author Summary
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