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In depth comparison of an individual's DNA and its lymphoblastoid cell line using whole genome sequencing.

Nickles D, Madireddy L, Yang S, Khankhanian P, Lincoln S, Hauser SL, Oksenberg JR, Baranzini SE - BMC Genomics (2012)

Bottom Line: Specifically, we sequenced the full genome (40X) of an individual using DNA purified from fresh whole blood as well as DNA from his LCL.We determined with high confidence that 99.2% of the genomes were identical, with no reproducible changes in structural variation (chromosomal rearrangements and copy number variations) or insertion/deletion polymorphisms (indels).Our results suggest that, at this level of resolution, the LCL is genetically indistinguishable from its genomic counterpart and therefore their use in clinical research is not likely to introduce a significant bias.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, University of California San Francisco, San Francisco, CA 94143-0435, USA.

ABSTRACT

Background: A detailed analysis of whole genomes can be now achieved with next generation sequencing. Epstein Barr Virus (EBV) transformation is a widely used strategy in clinical research to obtain an unlimited source of a subject's DNA. Although the mechanism of transformation and immortalization by EBV is relatively well known at the transcriptional and proteomic level, the genetic consequences of EBV transformation are less well understood. A detailed analysis of the genetic alterations introduced by EBV transformation is highly relevant, as it will inform on the usefulness and limitations of this approach.

Results: We used whole genome sequencing to assess the genomic signature of a low-passage lymphoblastoid cell line (LCL). Specifically, we sequenced the full genome (40X) of an individual using DNA purified from fresh whole blood as well as DNA from his LCL. A total of 217.33 Gb of sequence were generated from the cell line and 238.95 Gb from the normal genomic DNA. We determined with high confidence that 99.2% of the genomes were identical, with no reproducible changes in structural variation (chromosomal rearrangements and copy number variations) or insertion/deletion polymorphisms (indels).

Conclusions: Our results suggest that, at this level of resolution, the LCL is genetically indistinguishable from its genomic counterpart and therefore their use in clinical research is not likely to introduce a significant bias.

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Related in: MedlinePlus

Comparative display of structural variants of the two genomes. A: For the outer circle, the number of chromosomal rearrangements was assessed in bins of 5 Mb for both the genomic (transparent blue) and cell line DNA (transparent red). In the inner circle inter-chromosomal rearrangements are displayed. Because of the transparent colors, overlaps between the two genomes appear in purple. B: For comparison, a similar visualization is shown as a smaller inset, plotting chromosomal rearrangements of the genomic DNA (blue) and the genome of an unrelated individual (red). C: Genome ploidies for the genomic and cell line DNA are shown in the outer and inner circle, respectively. The curve is colored in orange in genome stretches with a ploidy of smaller than 1, the area under the curve is filled blue when ploidy exceeds 2. The axes are spaced in increments of 0.5.
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Figure 2: Comparative display of structural variants of the two genomes. A: For the outer circle, the number of chromosomal rearrangements was assessed in bins of 5 Mb for both the genomic (transparent blue) and cell line DNA (transparent red). In the inner circle inter-chromosomal rearrangements are displayed. Because of the transparent colors, overlaps between the two genomes appear in purple. B: For comparison, a similar visualization is shown as a smaller inset, plotting chromosomal rearrangements of the genomic DNA (blue) and the genome of an unrelated individual (red). C: Genome ploidies for the genomic and cell line DNA are shown in the outer and inner circle, respectively. The curve is colored in orange in genome stretches with a ploidy of smaller than 1, the area under the curve is filled blue when ploidy exceeds 2. The axes are spaced in increments of 0.5.

Mentions: First, we assessed whether the two genomes differed in structural variants, i.e. chromosomal rearrangements and copy number variations (CNVs). Chromosomal rearrangements can be inferred from reads spanning over a stretch of DNA that is not contiguous in the reference genome (discordant reads). For example, if one half of a sequence read maps to chromosome 1 and the other half to chromosome 3, this might be indicative of a translocation event. By visual inspection of reported DNA junctions, we found that both genomes exhibited a largely similar number of chromosomal rearrangements compared to the reference and that they shared most of the inter-chromosomal junctions as well (Figure 2A). Indeed, a similar but small number of unique chromosomal rearrangements were identified in each genome (131 in genomic, 78 in cell line; see Additional files 3, 4. 5); 34% and 45% of these unique junctions, respectively, had been detected in more than 75% of all other genomes sequenced by CGI at that time and hence might represent false positives (Additional files 4 and 5). To examine whether observed junctions might be linked to the transformation process, we reasoned that if the observed dif-ferences were of biological origin (e.g. driven by the transformation), a set of genes involved in cell cycle regulation would be among the affected loci. GO and KEGG analyses revealed very similar gene categories affected by chromosomal rearrangements in both genomes (data not shown), thus suggesting these differences were likely random or false discoveries.


In depth comparison of an individual's DNA and its lymphoblastoid cell line using whole genome sequencing.

Nickles D, Madireddy L, Yang S, Khankhanian P, Lincoln S, Hauser SL, Oksenberg JR, Baranzini SE - BMC Genomics (2012)

Comparative display of structural variants of the two genomes. A: For the outer circle, the number of chromosomal rearrangements was assessed in bins of 5 Mb for both the genomic (transparent blue) and cell line DNA (transparent red). In the inner circle inter-chromosomal rearrangements are displayed. Because of the transparent colors, overlaps between the two genomes appear in purple. B: For comparison, a similar visualization is shown as a smaller inset, plotting chromosomal rearrangements of the genomic DNA (blue) and the genome of an unrelated individual (red). C: Genome ploidies for the genomic and cell line DNA are shown in the outer and inner circle, respectively. The curve is colored in orange in genome stretches with a ploidy of smaller than 1, the area under the curve is filled blue when ploidy exceeds 2. The axes are spaced in increments of 0.5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473256&req=5

Figure 2: Comparative display of structural variants of the two genomes. A: For the outer circle, the number of chromosomal rearrangements was assessed in bins of 5 Mb for both the genomic (transparent blue) and cell line DNA (transparent red). In the inner circle inter-chromosomal rearrangements are displayed. Because of the transparent colors, overlaps between the two genomes appear in purple. B: For comparison, a similar visualization is shown as a smaller inset, plotting chromosomal rearrangements of the genomic DNA (blue) and the genome of an unrelated individual (red). C: Genome ploidies for the genomic and cell line DNA are shown in the outer and inner circle, respectively. The curve is colored in orange in genome stretches with a ploidy of smaller than 1, the area under the curve is filled blue when ploidy exceeds 2. The axes are spaced in increments of 0.5.
Mentions: First, we assessed whether the two genomes differed in structural variants, i.e. chromosomal rearrangements and copy number variations (CNVs). Chromosomal rearrangements can be inferred from reads spanning over a stretch of DNA that is not contiguous in the reference genome (discordant reads). For example, if one half of a sequence read maps to chromosome 1 and the other half to chromosome 3, this might be indicative of a translocation event. By visual inspection of reported DNA junctions, we found that both genomes exhibited a largely similar number of chromosomal rearrangements compared to the reference and that they shared most of the inter-chromosomal junctions as well (Figure 2A). Indeed, a similar but small number of unique chromosomal rearrangements were identified in each genome (131 in genomic, 78 in cell line; see Additional files 3, 4. 5); 34% and 45% of these unique junctions, respectively, had been detected in more than 75% of all other genomes sequenced by CGI at that time and hence might represent false positives (Additional files 4 and 5). To examine whether observed junctions might be linked to the transformation process, we reasoned that if the observed dif-ferences were of biological origin (e.g. driven by the transformation), a set of genes involved in cell cycle regulation would be among the affected loci. GO and KEGG analyses revealed very similar gene categories affected by chromosomal rearrangements in both genomes (data not shown), thus suggesting these differences were likely random or false discoveries.

Bottom Line: Specifically, we sequenced the full genome (40X) of an individual using DNA purified from fresh whole blood as well as DNA from his LCL.We determined with high confidence that 99.2% of the genomes were identical, with no reproducible changes in structural variation (chromosomal rearrangements and copy number variations) or insertion/deletion polymorphisms (indels).Our results suggest that, at this level of resolution, the LCL is genetically indistinguishable from its genomic counterpart and therefore their use in clinical research is not likely to introduce a significant bias.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, University of California San Francisco, San Francisco, CA 94143-0435, USA.

ABSTRACT

Background: A detailed analysis of whole genomes can be now achieved with next generation sequencing. Epstein Barr Virus (EBV) transformation is a widely used strategy in clinical research to obtain an unlimited source of a subject's DNA. Although the mechanism of transformation and immortalization by EBV is relatively well known at the transcriptional and proteomic level, the genetic consequences of EBV transformation are less well understood. A detailed analysis of the genetic alterations introduced by EBV transformation is highly relevant, as it will inform on the usefulness and limitations of this approach.

Results: We used whole genome sequencing to assess the genomic signature of a low-passage lymphoblastoid cell line (LCL). Specifically, we sequenced the full genome (40X) of an individual using DNA purified from fresh whole blood as well as DNA from his LCL. A total of 217.33 Gb of sequence were generated from the cell line and 238.95 Gb from the normal genomic DNA. We determined with high confidence that 99.2% of the genomes were identical, with no reproducible changes in structural variation (chromosomal rearrangements and copy number variations) or insertion/deletion polymorphisms (indels).

Conclusions: Our results suggest that, at this level of resolution, the LCL is genetically indistinguishable from its genomic counterpart and therefore their use in clinical research is not likely to introduce a significant bias.

Show MeSH
Related in: MedlinePlus