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. 2011 Sep 26:12:464.
doi: 10.1186/1471-2164-12-464.

Whole-exome sequencing of DNA from peripheral blood mononuclear cells (PBMC) and EBV-transformed lymphocytes from the same donor

Eric R Londin  1 , Margaret A Keller, Michael R D'Andrea, Kathleen Delgrosso, Adam Ertel, Saul Surrey, Paolo Fortina
Affiliations

Whole-exome sequencing of DNA from peripheral blood mononuclear cells (PBMC) and EBV-transformed lymphocytes from the same donor

Eric R Londin et al. BMC Genomics. .

Abstract

Background: The creation of lymphoblastoid cell lines (LCLs) through Epstein-Barr virus (EBV) transformation of B-lymphocytes can result in a valuable biomaterial for cell biology research and a renewable source of DNA. While LCLs have been used extensively in cellular and genetic studies, the process of cell transformation and expansion during culturing may introduce genomic changes that may impact their use and the interpretation of subsequent genetic findings.

Results: We performed whole exome sequencing on a tetrad family using DNA derived from peripheral blood mononuclear cells (PBMCs) and LCLs from each individual. We generated over 4.7 GB of mappable sequence to a 125X read coverage per sample. An average of 19,354 genetic variants were identified. Comparison of the two DNA sources from each individual showed an average concordance rate of 95.69%. By lowering the variant calling parameters, the concordance rate between the paired samples increased to 99.82%. Sanger sequencing of a subset of the remaining discordant variants did confirm the presence of de novo mutations arising in LCLs.

Conclusions: By varying software stringency parameters, we identified 99% concordance between DNA sequences derived from the two different sources from the same donors. These results suggest that LCLs are an appropriate representation of the genetic material of the donor and suggest that EBV transformation can result in low-level generation of de novo mutations. Therefore, use of PBMC or early passage EBV-transformed cells is recommended. These findings have broad-reaching implications, as there are thousands of LCLs in public biorepositories and individual laboratories.

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Figures

Figure 1
Figure 1
Summary of the bioinformatic workflow followed. The raw sequence data was aligned to the hg19 human genome build (UCSC). Following the alignment of the sequence data, high-stringency parameters were used to make SNP and indel calls. Following the identification of genetic variants, the interpretation of our results included comparing the two-paired samples sequenced to determine if de novo mutations arise following EBV-transformation of B-lymphocytes.
Figure 2
Figure 2
Comparison of all variants identified from DNA derived from PBMCs and LCLs. Venn diagrams showing the distribution of variants (SNPs and indels) identified for the DNA samples (A, B) ND02537, (C, D) ND02538, (E, F) ND02539 and, (G, H) ND02540. The dark blue represents the total variants that were in concordance between the paired samples. The light blue and red regions represent the total variants that were identified in only the LCL or PBMC sample respectively. A, C, E and F represent the initial concordance rates between the two paired samples, and B, D, F and H represent the concordance rates following the examination of discordantly identified variants.
Figure 3
Figure 3
Validation of identified variants, which were observed in only one of the DNA samples. Each set of sequence chromatograms represents a different variant that was examined by Sanger sequencing. For each, the top panel is the sample with the SNP (highlighted with the red arrow), and the bottom is the corresponding sample with the non-variant allele. Below each chromatogram are the gene, putative amino acid change, and chromosome and bp location on the chromosome.
See this image and copyright information in PMC

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