Gioia et al. BMC Genomics (2018) 19:334 https://doi.org/10.1186/s12864-018-4718-6 RESEARCH ARTICLE Open Access A genome-wide survey of mutations in the Jurkat cell line Louis Gioia1* , Azeem Siddique2,StevenR.Head2, Daniel R. Salomon1ˆ and Andrew I. Su1 Abstract Background: The Jurkat cell line has an extensive history as a model of T cell signaling. But at the turn of the 21st century, some expression irregularities were observed, raising doubts about how closely the cell line paralleled normal human T cells. While numerous expression deficiencies have been described in Jurkat, genetic explanations have only been provided for a handful of defects. Results: Here, we report a comprehensive catolog of genomic variation in the Jurkat cell line based on whole-genome sequencing. With this list of all detectable, non-reference sequences, we prioritize potentially damaging mutations by mining public databases for functional effects. We confirm documented mutations in Jurkat and propose links from detrimental gene variants to observed expression abnormalities in the cell line. Conclusions: The Jurkat cell line harbors many mutations that are associated with cancer and contribute to Jurkat’s unique characteristics. Genes with damaging mutations in the Jurkat cell line are involved in T-cell receptor signaling (PTEN, INPP5D, CTLA4,andSYK), maintenance of genome stability (TP53, BAX,andMSH2), and O-linked glycosylation (C1GALT1C1). This work ties together decades of molecular experiments and serves as a resource that will streamline both the interpretation of past research and the design of future Jurkat studies. Keywords: Jurkat, Whole-genome sequencing, Cancer, T-cell, Genome stability, T-cell receptor, T-cell acute lymphoblastic leukemia Background Problems were first noticed in the form of gene expres- The Jurkat cell line was isolated in 1977 from the blood sion defects. The most publicized of these defects was of a fourteen-year-old boy with Acute Lymphoblastic aberrant PI3K signaling due to the absence of PTEN and Leukemia [1]. It was one of the first in vitro systems for INPP5D (SHIP) in Jurkat cells [2]. The loss of these two studying T-cell biology and helped to produce an incredi- central regulators of phosphatidylinositol signaling was ble number of discoveries and publications (Fig. 1)[2]. proposed as the cause of the previously-documented, con- As the workhorse behind a diverse array of molecular stitutive activation of PI3K signaling, a major mediator investigations, the Jurkat cell line revealed the founda- of downstream TCR signaling events [3]. This fundamen- tions for our modern understanding of multiple signaling tal TCR signaling defect in Jurkat led many researchers pathways. Most notably, studies of Jurkat cells established to question its validity as a model system for T-cell stud- the bulk of what is currently known about T-cell recep- ies [2]. Although the number of publications using Jurkat tor (TCR) signaling [2]. However, at the turn of the 21st dropped off over the following decade, it is still widely century, as the use of Jurkat as a model T-cell line was used in biomedical research (Fig. 1). reaching its height, some abnormalities in the cell line Defect detection up to now has been primarily based begantocometolight. on top-down approaches, requiring knowledge of signal- ing or expression defects, which leads to interrogations of *Correspondence: [email protected] specific coding sequences. While multiple genetic defects ˆDeceased have been described over the past few decades, these top- 1Department of Molecular Medicine, The Scripps Research Institute, La Jolla, down approaches are limited in scope and have failed to California 92037, USA Full list of author information is available at the end of the article provide a broader understanding of Jurkat biology. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gioia et al. BMC Genomics (2018) 19:334 Page 2 of 13 Fig. 1 Jurkat publication trends. Yearly publication counts for PubMed queries. Representative queries are given in the legend. Note that these query descriptions are abbreviations of more detailed search terms, which are provided in the “Methods”section Modern sequencing technology allows for interrogation small insertions and deletions, as well as inversions, tan- of the entire genome. In contrast to top-down techniques, dem duplications, and inter-chromosomal translocations. whole-genome sequencing (WGS) allows us to investigate BreakDancer compares the distance between aligned read genetic defects from the bottom up, with the potential pairs to the insert size distribution from the sequencing to extend our understanding of abnormalities in Jurkat. library in order to find large structural variants. CNVnator Thus, in this study, we used shotgun sequencing to per- uses read-depth information and a mean-shift algorithm form genome-scale characterization of genomic variants to assign copy number levels across the genome and in this commonly-used cell line. identify deletion and duplication events. In order for GATK to call small variants, it must be Results told how many alleles to expect at each position. As such, Sequencing and variant callers an accurate estimate of Jurkat ploidy is required before Whole-genome sequencing of the Jurkat cell line pro- GATK can be used. While both the original 1977 publica- duced over 366 million 100bp paired-end reads and over tion and the American Type Culture Collection (ATCC) 531 million 150bp paired-end reads, totaling over 116 bil- report that Jurkat is diploid, other publications refute this lion sequenced bases. More than 98% of the reads were description. The first karyotypes of the Jurkat cell line successfully aligned to the hg19 human reference genome were published by Snow and Judd in 1987, who found with the Burrows-Wheeler Aligner [4], totaling over 110 that Jurkat was hypotetraploid, possessing fewer than four billion aligned bases. This gave an average coverage of times the haploid number of chromosomes [9]. A few ∼ 36x across the hg19 reference sequence, with over 10x years later, tetraploidy was corroborated by an investiga- depth of coverage for 78.8% of the genome. The aligned tion of p53 mutations, which found that the Jurkat cell line reads were then used to detect both small and large contained 4 separate p53 alleles [10]. More recent reports genomic variants in the Jurkat genome. confirm Jurkat tetraploidy. The German Collection of In order to utilize all of the information available in the Microorganisms and Cell Cultures (DSMZ) describes the WGS data, we employed a suite of variant calling tools Jurkat karyotype as a "human flat-moded hypotetraploid for the identification of all major types of genomic vari- karyotype with 7.8% polyploidy." In addition, a multicolor- ants. Each tool uses a certain type of sequence information Fluorescence In Situ Hybridization study from 2013 found to identify specific categories of variants. Our variant within-culture mosaicism on a tetraploid background caller suite consisted of four distinct tools and algorithms: [11]. The Genome Analysis Toolkit, Pindel, BreakDancer, and CNVnator [5–8]. Variant calls The Genome Analysis Toolkit (GATK) from the Broad Given the previous reports of tetraploidy, we ran GATK Institute uses De Bruijn graph-based models to iden- with a ploidy count of 4. GATK identified nearly 5 mil- tify single-nucleotide substitutions and small insertions lion variants, comprising ∼ 3.5 million single-nucleotide and deletions. Pindel’s split-read approach can also detect substitutions, ∼ 1.0 million small deletions, and ∼ 357 Gioia et al. BMC Genomics (2018) 19:334 Page 3 of 13 thousand small insertions, across over 4.6 million variant depth density is provided in Additional file 1:FigureS1. loci. Basic metrics for the GATK variant calls are consis- CNVnator reported a modal copy number of 4 in Jurkat, tent with normal human samples. The ratio of homozy- representing over 65% of the genome and corroborat- gous to heterozygous variant loci is 0.635, which is in ing reports of tetraploidy. From the CNVnator results, the range of previously reported ratios, and the ratio of we identified 2499 deletion sites (CN ≤ 1), of which transitions to transversions is 2.10, which is the expected 218 were homozygous (CN = 0), and 1863 duplication value for human genomes [12].Thenumberofsingle- sites (CN ≥ 5). nucleotide substitutions is similar to previously reported The structural variant calls from each tool were com- values. However, the total number of indels is higher pared and merged with specific considerations made for than published values from human WGS studies, which each category of variant and each detection tool (see generally detect fewer than 700 thousand indels by shot- “Methods” section). Short and long insertions and dele- gun sequencing [12]. To date, the highest number of tions were defined using a cutoff of 50 bp, in accordance indels identified in a single human genome was