In depth comparison of an individual’s DNA and its lymphoblastoid cell line using whole genome sequencing
© Nickles et al.; licensee BioMed Central Ltd. 2012
Received: 20 December 2011
Accepted: 5 September 2012
Published: 14 September 2012
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.
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).
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.
Epstein-Barr virus (EBV) is a herpesvirus that infects epithelial and B cells and has been associated with the development of various tumors, including Hodgkin’s and Burkitt’s lymphoma[1–4]. Since EBV is able to transform B cells into continuously proliferating lymphoblastoid cell lines (LCLs), it is commonly used as a tool in clinical research for creating an unlimited source of patients’ material[5–9]. Although LCLs have been used frequently as a source of DNA in genetic studies, controversy still exists about their reliability in faithfully replicating the variation present in the donor germ-line (e.g.[6, 9–13]).
In all latently infected LCLs, the EBV genome is present in the form of extra-chromosomal copies (episomes) from which the viral genome is replicated. Most research on the transforming abilities of EBV has been focused on the expression of viral gene products and the host’s transcriptional response. From this body of research it is now well understood that the viral transcription factor EBV nuclear antigen (EBNA)-2 activates the expression of several EBV proteins and non-coding RNAs - the growth transcription program - that interfere with the host’s signaling pathways[2, 14]. Specifically, the growth transcription program drives cell transformation by activating cellular proliferation, while suppressing growth inhibitors. Even though the viral gene products exert their transforming functions primarily by interacting with host proteins, recent evidence suggests that EBV also promotes genomic instability in the host. Furthermore, EBV has the potential to cause mutations through integration and disintegration into the host’s genome (e.g.[16, 17]). These putative early (pre-immortal) genetic consequences of EBV infection are less well studied. If ascertained, those structural genomic changes would have important implications for the interpretation of a large number of genetic studies that assume LCLs are a bona-fide source of genomic DNA.
In recent years, massively parallel DNA (i.e. next generation) sequencing has become increasingly affordable, enabling the discovery of causative DNA variants in rare genetic diseases[19–21] and providing new insights into carcinogenesis and autoimmunity[22–32]. A recent study reported the comparison of DNA isolated from peripheral lymphocytes and from an LCL from the same individuals by means of exome sequencing. This study concluded that the exome fractions of genomic and lymphoblastoid cell line DNA (roughly 1-2% of the genomes) are more than 99% identical.
Whole genome sequencing can, in addition to identifying variation in coding regions, reveal copy number variation (CNV) and chromosomal rearrangements at high resolution. A whole genome sequence data set is therefore multilayered and can be queried for different aspects of genomic organization, making it a valuable tool for our study. Here we report the analysis of complete genome sequencing (40X) of a single individual to gain a better understanding of putative genetic alterations introduced by EBV transformation.
Here we report the complete sequencing and analysis of the normal genomic and lymphoblastoid cell line DNA from the same individual. We organized our analysis so as to proceed from investigation of major chromosomal rearrangements to small sequence variation, to single base changes. As a preliminary step, we performed high resolution karyotyping of the cell line to identify major chromosomal abnormalities. Fifteen out of 20 analyzed metaphases showed a normal karyotype (Additional file1). Only 5 cells showed random changes in chromosome number, consistent with what would be expected for an early-passage LCL. Once sequencing data was obtained, we evaluated quality metrics of the two sequencing data sets, such as overall coverage, ratio of hetero-to-homozygous single nucleotide polymorphisms (SNPs) and the ratio of SNP transitions to transversions. These parameters are similar to those reported for previously sequenced genomes using the same technology (e.g.[20, 21, 34]) and other human datasets using next generation sequencing (Additional file2). We also assessed the presence of viral DNA. To this end, we extracted raw reads of sequences that did not map to the human genome and aligned these to the EBV genome. We found an average of 2 copies of EBV DNA reads in the cell line as compared to 0 copies in the genomic DNA (data not shown), confirming that viral DNA was present only in the cell line sample. Since EBV has been described to integrate into host DNA (e.g.[35–40]), we also checked for viral DNA in all longer (>8 nt) insertions and substitutions that passed a pre-determined quality filter (SomaticScore of at least 0.1), as well as in all “non-reference” DNA stretches joining the two arms of a chromosomal rearrangement in the cell line (transition sequences). None of these calls supported the presence of inserted EBV genomic information.
Quality metrics of the sequenced genomes
Gross mapping yield (Gb)
SNP het/hom ratio
INS het/hom ratio
DEL het/hom ratio
SUB het/hom ratio
SNP total count
INS total count
DEL total count
SUB total count
SNP novel rate
INS novel rate
DEL novel rate
SUB novel rate
Fully called genome fraction
Partially called genome fraction
No-called genome fraction
Synonymous SNP loci
Missense SNP loci
Nonsense SNP loci
Nonstop SNP loci
Frame-shifting INS loci
Frame-shifting DEL loci
Frame-shifting SUB loci
Frame-preserving INS loci
Frame-preserving DEL loci
Frame-preserving SUB loci
Nonsyn/syn SNP ratio
Ins + del/SNP ratio
Coding insertion/deletions ratio
Coding SNP/all SNP ratio
Coding (ins + del)/all (ins + del) ratio
We next inspected ploidy (as a surrogate for CNVs) from genome coverage files in windows of 2 kb (Figure2B). Both genomes shared almost all CNVs throughout the genome; of note, most DNA stretches with ploidy > 2 were observed near telomers and centromers, likely reflecting the difficulty to properly align reads in these highly repetitive DNA regions. The cell line showed a decreased copy number in only 4 regions (3 haploid regions, one deletion) as compared to the genomic DNA (Additional file6). Four genes (KIAA0125, PRAME, ZNF280A, ZNF280B) and one pseudogene (ADAM6) were affected by the CNVs (Additional file6). None of these is reported to have a negative impact on cell proliferation. Hence, it is unclear whether their reduced ploidy plays any role in the transformation process. Notably, a 9-fold increase in copy number of mitochondrial DNA was observed for the cell line, likely reflecting the increased energy demand of the actively dividing transformed cells (Figure2B). This finding is consistent with a previous study.
Taken together, these results suggest that by using this technology at 40X resolution DNA from the cell line is mostly undistinguishable from genomic DNA from the same individual. The few putatively true differences are randomly distributed across the genome (Additional file8) and do not seem to drive the transformation process.
Here we report on the first genome-wide sequence-based analysis of the immediate genetic consequences of EBV transformation on a low-passage lymphoblastoid cell line from a subject with MS. While genomes from MS and healthy individuals may differ slightly, we deemed that this would not affect the conclusions of our study, which focused on characterizing the genomic consequences of EBV transformation. These effects should be clear-cut and insensitive to the source of the sample (with the exception of certain tumors, where DNA may contain abundant somatic mutations). For decades, the cell-transforming abilities of EBV have been used in genetic research to create repositories of subjects’ DNA. While the roles of viral gene products in the transformation process have been described in detail, whether genetic alterations are introduced as a consequence of EBV transformation is less well understood.
Several studies have systematically compared LCLs directly to their parental cells using traditional molecular techniques such as SNP typing, gene expression, and whole chromosome analysis[12, 44, 45]. The overall consensus is that no reproducible differences were identified[7, 45–47]. For instance, Redon et al. assessed differences between DNA from HapMap LCLs and their genomic counterparts and found that only 0.5% of observed CNVs were caused by transformation. Another study confirmed that most CNVs in LCLs can also be seen in normal B cells. Two of the CNVs reported in this latter study overlap with those detected in our LCL. This is not surprising since the genomic DNA was isolated from PBMCs, whereas the LCL is a B cell line – hence B cell specific CNVs were identified as differences to the genomic DNA in our study. The mitochondrial DNA CNV reported here was also seen as a “cell line-specific” CNV in a previous study.
Next generation sequencing was recently used to compare genomic and LCL DNA, although only the coding sequence (~1-2% of the genome) was assessed in that study. Specifically, authors used exome sequencing to compare DNA from four LCLs with their corresponding genomic DNA (extracted from peripheral blood mononuclear cells). Focusing their analysis on SNPs and small indels, authors reported a 99.82% concordance between the parental DNA and the cell lines, with all discordant calls stemming from a single LCL-donor pair. Given the relevance of non-coding regions in the modulation of gene expression and thus cell stability and function, whether the high level of concordance between genomic and LCL DNA extended to the whole genome remains an important question. By analyzing the whole genomes (at 40X) of an LCL and its genomic DNA counterpart, we identified 9,468 and 12,719 unique variants with respect to the reference genome, respectively. In-silico analysis reduced the number of differences to 417 (216 SNPs, 201 indels) in the LCL and 269 (15 SNP, 254 indels) in the genomic DNA. However, none of the 60 variants chosen for validation by Sanger sequencing were confirmed, thus suggesting that the number of real differences ought to be significantly smaller.
The known error rate of the ligation-based sequencing technique we employed was empirically determined to be approximately 1 in 100–200 kb (SY, unpublished observations). Hence, we can expect 20,000 to 30,000 errors in each genome. In a previous comparison of two technical replicate samples (sequencing the same sample twice) using the same technology, 27,893 differences were detected (SY, unpublished observations); we observed a similar number of differences (22,187) between the genomic DNA and the cell line genome in our analysis. These numbers highlight how close the difference between the two genomes is to technical noise. We therefore chose to minimize false positives in our analysis by applying stringent filters to the lists of called differences. By these means we identified a number of SNPs that seemed enriched in the cell line (216 SNPs in the CT analysis versus 15 SNPs in the GT analysis). These variants represented silent mutations and appeared to be random mutational events, possibly resulting from the accelerated division rate of the transformed cells. However, we were not able to confirm a selected subset of those SNPs and further validation is needed to establish any true discrepancies between the genomic DNA and the cell line.
In conclusion, our results indicate that EBV transformed cell lines at low passage number/short time in culture are genetically indistinguishable to the parental cells, suggesting that discoveries in genetic studies conducted using low-passage LCLs with a normal karyotype can be extrapolated to the parental patient samples. We determined with high confidence that 99.2% of the genomes were identical, with no reproducible changes in structural variation (chromosomal rearrangements and CNV) or indels. While we identified 231 differences (216 from the LCL, 15 from the genomic DNA) in single nucleotide variants, none of the 60 randomly selected variants validated by Sanger sequencing. These findings suggest that the true differences between the two genomes ought to be less than 5.8% of the candidates identified by bioinformatics filters (proportions test; 95% confidence interval p-value: 1.3 x 10-14). We acknowledge that the sample size is a limiting factor of this study, and while similar results were reported by independent groups using different technologies[7, 9, 33, 45–47], larger studies will be needed to precisely determine the genomic effects of EBV transformation.
A lymphoblastoid cell line was established from whole blood from an adult male suffering from multiple sclerosis, essentially as described in (a detailed protocol can be found in Additional file10). Briefly, a buffy coat was obtained from freshly drawn blood and PBMCs were cultured with EBV supernatant. After infection, cells were kept in culture for 6 weeks. DNA was then extracted using standard salting-out procedure. DNA concentration was determined using the picogreen assay and adjusted to 0.1 μg/μl. DNA quality was assessed on an agarose gel prior to sequencing (Additional file11). 15 μg DNA were sent for sequencing. The study was approved by the Institutional Review Board at the University of California San Francisco (UCSF).
Genomes were sequenced and aligned by Complete Genomics Inc. (Mountain View, CA) (CGI, details on the technique in). Sequence reads were aligned to genome release hg19 and all annotations were performed based on this genome release. CGI provided 6 data files (evidence, variants, gene variant summary, gene, CNV segments, and high-confidence junction files) that were used in the analysis (software version 184.108.40.206, format version 1.5; Additional file7 panel A), most importantly the evidence and the variance files listing all sequenced loci that deviate from the reference genome. A position is either classified – or called – as “reference”, when the reads conform to the reference genome, or as “variant” if they do not accord. If a variant was called in this first round, corresponding reads are newly assembled to accurately determine the sequence of the variant locus; the resulting information is stored in the evidence file (assembled reads) and variance file (variant calls). Variants are classified into different classes, including SNP, deletion, insertion or substitution. All variants are assigned a score expressing the confidence of the variant call [this score was not used to prioritize variants for analysis; however, this score is incorporated into the SomaticScore (see below)]. Two other files, the gene variance summary file and the gene file, list all genes that are affected by a variant; the latter gives all variant positions that fall into genes, including untranslated regions, splice sites and introns, whereas the gene variance file specifies the number of mutations that can either be classified as “missense” (amino acid changing mutation), “nonsense” (creating a stop codon where there was none before), “nonstop” (removing a stop codon), “frameshift” (changing the reading frame of a gene) or “inframe”. Finally, the CNV segments file contains relative coverage and ploidy calls in windows of 2 kb, while the high-confidence junction file provides information on putative chromosomal rearrangement events. Genome data has been deposited at the European Genome-phenome Archive (EGA,http://www.ebi.ac.uk/ega/) which is hosted at the EBI under accession number EGAS00001000323.
Analysis of variant calls
The variant files of the two genomes were compared to each other using the function calldiff from cgatools 1.3.0 package. An overview of overall differences was output by choosing the reports argument “SuperlocusStats”. For in-depth analysis of differences, the somatic output report was used (reports argument “SomaticOutput”). For this report, a “normal” and a “tumor” sample has to be specified. The tumor sample will be compared only to reference loci in the normal sample and the output will contain all non-reference loci in the tumor sample. Each call is assigned a “SomaticScore”, which indicates the reliability of the call. By using a SomaticScore of x, a sensitivity of 1-x is achieved. Being aware that the number of differences we observed was within the expected noise range, we chose a high SomaticScore of 0.5, knowingly losing some true positives, but minimizing false positives. From the resulting list of variants all loci that were not fully called were excluded. We did two analysis runs: (1) CT analysis (i.e. cell line is the tumor sample); (2) GT analysis, (i.e. genomic DNA is the tumor sample) as a comparison. Variants were mapped to genes using the CGI gene files.
Analysis of CNV calls
CNV calls were visually inspected by plotting the relative coverage of each genome using the Circos software. For an in-depth analysis, the presence of calls reported in one genome, the “reference”, was assessed in the other genome, taking both cell line and genomic DNA as reference. For this search, only calls with a ploidyScore greater than 40 in the reference were considered. The start and end points of the CNV call are not required to be an exact match, but have to fall within a 2 kb window around the start and end points in the reference. For each CNV call present in both genomes, ploidy calls were compared. When these differed between the two genomes, the locus was output for both genomes and visually assessed.
Analysis of chromosomal rearrangements
CGI provides a high confidence junction file for each sequenced genome, which lists events of discordant read pairs within a given DNA nanoball (DNB). In this file, sequences that are not adjacent in the reference genome are reported; these are defined as “junctions”, consisting of a “left arm” reference sequence, a breakpoint with an optional transition sequence (a stretch of sequence that is not contained in the reference genome) and a “right arm” reference sequence at a non-adjacent genome location. CGI also reports the frequency with which each identified junction was found in previously sequenced genomes (the higher the frequency, the more likely the reported junction might be an artifact of the sequencing technology). For the graphical (e.g. Circos) analysis, all high confidence junctions seen in 75% or more of CGI sequence data sets were removed. For visually contrasting structural variance calls of the two genomes, only inter-chromosomal high confidence junctions (as determined by CGI) were plotted. In addition, the total number of junctions in bins of 5 Mb was calculated with the help of the software tool binlinks that is distributed together with Circos. In-depth analysis was performed by comparing CGI’s high confidence junctions files for the different genomes using cgatools 1.3.0 junctiondiff. The program was run using the standard settings, i.e. the scoreThresholdA was set at 10, scoreThresholdB at 0, the maximum distance between the coordinates of the putatively compatible junctions at 200 and the minimum deletion length at 500. CGI provided accession numbers of genes that were affected by chromosomal rearrangements. Accession numbers affected by junctions specific to one of the genomes were translated into geneIDs and symbols using the Bioconductor/R package “biomaRt”. Then, enrichment for GO and KEGG categories was assessed using the “GOstats” R package.
Analysis of non-synonymous variants
For each class of non-synonymous variant - “missense”, “nonsense”, “nonstop” or “frameshift” -, all genes with at least one mutation in genomic and cell line DNA were determined, respectively. Of all genes that were affected by the same class of mutation in both genomes, mutations were compared between genomic and cell line DNA. If position or type of the variant was not identical in the two genomes, raw sequence reads were displayed using the Integrative Genomics Viewer and visually compared.
Detection of viral DNA
To assess the presence of viral DNA sequences within the whole genome data sets, all reads that could not be mapped to the human genome were extracted. Next, the reference genome sequence of EBV was downloaded in fasta format from the NCBI. Then, unmapped reads were aligned to this reference genome using bwa. Subsequently, samtools was used to convert aligned reads into bam format to enable display in IGV and calculate EBV genome coverage (44X). In addition, we extracted all transition sequences (sequences that join the two arms of chromosomal junctions, but are not present in the reference genome) as well as reported insertions and longer than 8 nucleotides from the junctions that were unique to the cell line and blasted them against the EBV genome (using NCBI BLAST).
Karyotyping and Sanger sequencing
Karyotyping was performed by the Cytogenetics Laboratory of UCSF. Sanger Sequencing was performed by the Genomics Core Facility of UCSF. Sequencing primers were designed based on a 200 bp region flanking the SNP that was inquired. 60 randomly selected discordant SNPs were sequenced using two independent primer pairs per position.
We thank R Lincoln, R Guerrero, H Mousavi, R Gomez, and A Santaniello for sample and database management. SEB is Harry Weaver Neuroscience Scholar of the US National MS Society. DN is a fellow from the Deutsche Forschungsgemeinschaft (DFG) Research Grant Program. This work was supported by National Institutes of Health [RO1NS26799]; and National Multiple Sclerosis Society [RG2901].
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