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U87MG decoded: the genomic sequence of a cytogenetically aberrant human cancer cell line.

Clark MJ, Homer N, O'Connor BD, Chen Z, Eskin A, Lee H, Merriman B, Nelson SF - PLoS Genet. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University of California Los Angeles, Los Angeles, California, United States of America.

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 30x 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.

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Related in: MedlinePlus

Increased resolution of structural variations by sequencing.Resolution of karyotyping and SKY approaches is not high enough to see the complex nature of this translocation event between chr1 and chr16. With high-resolution whole-genome sequencing, the true structure of the translocation is revealed as mutual translocations between a small fragment of chr2 with chr1 and chr16 on either end.
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pgen-1000832-g007: Increased resolution of structural variations by sequencing.Resolution of karyotyping and SKY approaches is not high enough to see the complex nature of this translocation event between chr1 and chr16. With high-resolution whole-genome sequencing, the true structure of the translocation is revealed as mutual translocations between a small fragment of chr2 with chr1 and chr16 on either end.

Mentions: The resolution of genome-wide chromosomal rearrangements is substantially improved by the mate-pair strategy, coupled with sensitive and independent alignment of the short 50-base reads (Figure 5). Based on published SKY data, we anticipated 7 interchromosomal breakpoints [6]. However, whole-genome mate-paired sequence data revealed the precise chromosomal joins of 35 interchromosomal events, which account for previously observed chromosomal abnormalities in U87MG but at additional finer scale resolution (Figure 5, Figure 6, Figure 7). The translocation events were enriched in genic regions with 32/35 (91.4%) occurring within 1kb of genes. A weaker, but still noticeable enrichment over genes occurs with microdeletions as well, which are generally missed by other experimental techniques like DNA microarrays. Thus, within the overall mutational landscape of this cancer cell line, translocations and structural variants preferentially occurred over genes, supporting a model where cancer mutations occur via structural instability rather than novel point mutations.


U87MG decoded: the genomic sequence of a cytogenetically aberrant human cancer cell line.

Clark MJ, Homer N, O'Connor BD, Chen Z, Eskin A, Lee H, Merriman B, Nelson SF - PLoS Genet. (2010)

Increased resolution of structural variations by sequencing.Resolution of karyotyping and SKY approaches is not high enough to see the complex nature of this translocation event between chr1 and chr16. With high-resolution whole-genome sequencing, the true structure of the translocation is revealed as mutual translocations between a small fragment of chr2 with chr1 and chr16 on either end.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2813426&req=5

pgen-1000832-g007: Increased resolution of structural variations by sequencing.Resolution of karyotyping and SKY approaches is not high enough to see the complex nature of this translocation event between chr1 and chr16. With high-resolution whole-genome sequencing, the true structure of the translocation is revealed as mutual translocations between a small fragment of chr2 with chr1 and chr16 on either end.
Mentions: The resolution of genome-wide chromosomal rearrangements is substantially improved by the mate-pair strategy, coupled with sensitive and independent alignment of the short 50-base reads (Figure 5). Based on published SKY data, we anticipated 7 interchromosomal breakpoints [6]. However, whole-genome mate-paired sequence data revealed the precise chromosomal joins of 35 interchromosomal events, which account for previously observed chromosomal abnormalities in U87MG but at additional finer scale resolution (Figure 5, Figure 6, Figure 7). The translocation events were enriched in genic regions with 32/35 (91.4%) occurring within 1kb of genes. A weaker, but still noticeable enrichment over genes occurs with microdeletions as well, which are generally missed by other experimental techniques like DNA microarrays. Thus, within the overall mutational landscape of this cancer cell line, translocations and structural variants preferentially occurred over genes, supporting a model where cancer mutations occur via structural instability rather than novel point mutations.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, University of California Los Angeles, Los Angeles, California, United States of America.

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 30x 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.

Show MeSH
Related in: MedlinePlus