Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here we use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, we uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.

In a genetic screen in a drnl-2 background, we isolated a loss-offunction allele in miR319a (miR319a 129 ). Previously, miR319a has been postulated to play a role in leaf development based on the dramatic curled-leaf phenotype of plants that ectopically express miR319a (jaw-D). miR319a 129 mutants exhibit defects in petal and stamen development; petals are narrow and short, and stamens exhibit defects in anther development. The miR319a 129 loss-of-function allele contains a single-base change in the middle of the encoded miRNA, which reduces the ability of miR319a to recognize targets. Analysis of the expression patterns of the three members of the miR319 gene family (miR319a, miR319b, and miR319c) indicates that these genes have largely non-overlapping expression patterns suggesting that these genes have distinct developmental functions. miR319a functions by regulating the TCP transcription factors TCP2, TCP3, TCP4, TCP10, and TCP24; the level of RNA expression of these TCP genes is down-regulated in jaw-D and elevated in miR319a 129 . Several lines of evidence demonstrate that TCP4 is a key target of miR319a. First, the tcp4 soj6 mutant, which contains a mutation in the TCP4 miRNAbinding site complementary to the miR319a 129 mutation, suppresses the flower phenotype of miR319a 129 . Second, expression of wild-type TCP4 in petals and stamens (i.e., AP3:TCP4) has no effect on flower development; by contrast, a miRNA-resistant version of TCP4, when expressed in petals and stamens (i.e., pAP3:mTCP4) causes these organs not to develop. Surprisingly, when AP3:TCP4 is present in a miR319a 129 background, petal and stamen development is severely disrupted, suggesting that proper regulation by miR319a of TCP4 is critical in these floral organs.DRNL ͉ flower development ͉ forward genetics ͉ microRNA ͉ stamen M icroRNAs (miRNAs) are 21-24 nucleotide regulatory RNAs that function in diverse aspects of plant biology (1) such as biotic and abiotic stress responses (2), metabolism (3), hormone signaling (4), transcription (5), development (6), and the regulation of miRNA machinery itself (7,8). Most studies of plant miRNAs focus on alleles that ectopically express the miRNA. In miRNA overexpression lines, the RNA levels of target genes are downregulated resulting in a phenotype that mimics the loss-of-function phenotypes of miRNA target mutants (9, 10). Loss-of-function alleles in miRNA genes are rare because most often miRNAs are redundantly encoded in plant genomes. Thus, mutation of a single miRNA gene usually does not result in a mutant phenotype. In the few miRNA loss-of-function mutants that are available (5,6,11,12), the RNA levels of target genes are elevated, which is expected if the miRNA can no longer function to degrade target mRNAs. miR319a was initially characterized based on the dramatic leaf phenotype that results from overexpression of miR319a, as in the jaw-D allele that results in a jagged and wavy (jaw) leaf-phenotype (13). The best-characterized targets of miR319 are a subset of TCP transcription factors...

Cas9 is commonly introduced into cell lines to enable CRISPR/Cas9-mediated genome editing. Here we studied the genetic and transcriptional consequences of Cas9 expression per se . Gene expression profiling of 165 pairs of human cancer cell lines and their Cas9-expressing derivatives revealed upregulation of the p53 pathway upon Cas9 introduction, specifically in TP53 -WT cell lines. This was confirmed at the mRNA and protein levels. Moreover, elevated levels of DNA repair were observed in Cas9-expressing cell lines. Genetic characterization of 42 cell line pairs showed that Cas9 introduction can lead to the emergence and expansion of p53-inactivating mutations. This was confirmed by competition experiments in isogenic TP53 -WT/ TP53 -null cell lines. Lastly, Cas9 was less active in TP53 -WT than in TP53 -mutant cell lines, and Cas9-induced p53 pathway activation affected cellular sensitivity to both genetic and chemical perturbations. These findings may have broad implications for the proper use of CRISPR/Cas9-mediated genome editing.

BackgroundSample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk.ResultsCross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative.ConclusionsFor sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4428-5) contains supplementary material, which is available to authorized users.