Limits...
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

Coverage of the two genomes. A: Number of reads is plotted against the number of bases for which that number of reads was observed. The proportion of reads with a higher coverage (more reads) is slightly higher for the genomic DNA (black line) than for the cell line (grey line). B: Proportions of the two genomes at a certain X coverage. C: Regional plot of averaged normalized coverages of the genomic DNA (black line) and the cell line (grey line). The axes are spaced in increments of 10.
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Figure 1: Coverage of the two genomes. A: Number of reads is plotted against the number of bases for which that number of reads was observed. The proportion of reads with a higher coverage (more reads) is slightly higher for the genomic DNA (black line) than for the cell line (grey line). B: Proportions of the two genomes at a certain X coverage. C: Regional plot of averaged normalized coverages of the genomic DNA (black line) and the cell line (grey line). The axes are spaced in increments of 10.

Mentions: A total of 217.33 Gb of sequence were generated from the cell line and 238.95 Gb from the genomic DNA. This difference in coverage did not alter most of the parameters analyzed, as these were highly similar between the two genomes (Table 1). While the slightly deeper coverage of the genomic DNA did result in more variants being called in this sample, the overall depth of coverage was highly similar for the two genomes (~ 80% of both genomes were covered at least 40 times, Figure 1A and C). Also, the regional distribution of reads was highly concordant (Figure 1B). Once we established that quality measures were not significantly different, we set out to make a detailed comparison between the two genomes.


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)

Coverage of the two genomes. A: Number of reads is plotted against the number of bases for which that number of reads was observed. The proportion of reads with a higher coverage (more reads) is slightly higher for the genomic DNA (black line) than for the cell line (grey line). B: Proportions of the two genomes at a certain X coverage. C: Regional plot of averaged normalized coverages of the genomic DNA (black line) and the cell line (grey line). The axes are spaced in increments of 10.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Coverage of the two genomes. A: Number of reads is plotted against the number of bases for which that number of reads was observed. The proportion of reads with a higher coverage (more reads) is slightly higher for the genomic DNA (black line) than for the cell line (grey line). B: Proportions of the two genomes at a certain X coverage. C: Regional plot of averaged normalized coverages of the genomic DNA (black line) and the cell line (grey line). The axes are spaced in increments of 10.
Mentions: A total of 217.33 Gb of sequence were generated from the cell line and 238.95 Gb from the genomic DNA. This difference in coverage did not alter most of the parameters analyzed, as these were highly similar between the two genomes (Table 1). While the slightly deeper coverage of the genomic DNA did result in more variants being called in this sample, the overall depth of coverage was highly similar for the two genomes (~ 80% of both genomes were covered at least 40 times, Figure 1A and C). Also, the regional distribution of reads was highly concordant (Figure 1B). Once we established that quality measures were not significantly different, we set out to make a detailed comparison between the two genomes.

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