Project Achilles Copy Number Paper

Data deposit for the publication:Aguirre &amp; Meyers, et al. Genomic copy number dictates a gene-independent cell response to CRISPR-Cas9 targeting. Cancer Discovery, 2016.<br>ABSTRACT: The CRISPR-Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy number gain, CRISPR-Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell cycle arrest. By examining single guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR-Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR-Cas9 elicits a geneindependent anti-proliferative cell response. This effect has important practical implications for interpretation of CRISPR-Cas9 screening data and confounds the use of this technology for identification of essential genes in amplified regions.<br>--------------------------------------------<br>GeCKOv2 Achilles dataset33 cell lines<br>Achilles_v3.3.8_rawreadcounts.gct contains the raw read counts for each replicate, including a column containing read counts for initial pDNA library representation.<br>Achilles_v3.3.8_lognorm.gct contains the replicate-level read counts per million<br>Achilles_v3.3.8a.gct is the guide-level log2FC data used for analyses in the publication. It is not normalized across cell lines, but the median of the negative control sgRNAs are subtracted in each sample (i.e. a score of 0 represents median of the negative controls).<br>Achilles_v3.3.8.gct is guide-level log2FC data, that has been z-score normalized per replicate and has had sgRNAs targeting more than one genomic perfect match site removed. This data was used to calculate gene-level essentiality.<br>Achilles_v3.3.8_gene-soln.gct is the version of the data used for gene-level analyses across cell lines in the publication. Samples are z-score normalized and sgRNAs with &gt;1 perfect match are removed. Gene solutions computed using ATARiS.<br>Achilles_v3.3.8_ABSOLUTE_CN_segtab.txt contains the ABSOLUTE copy number segments for each of the 33 samples.<br>Achilles_v3.3.8_sgRNA_mappings.txt contains the mapped genomic locus (loci) for each sgRNA.<br><br><br>Analysis steps v3.3.8a:<br>1. QC and identify reps with reproducibility &lt;0.8 or are outliers on PCA or fail FP2. remove sgRNAs(guides) with low counts in DNA samples:completely remove shRNAs with median &lt;=1 3. remove replicates that fail QC measures 4. fold change(FC) with DNA pool samples 5. zero-center median of negative controls6. collapse replicates per line 7. sgRNAs are mapped to genes using(Achilles_v3.3.8a.gct)<br>Analysis steps v3.3.8:<br>1. QC and identify reps with reproducibility &lt;0.8 or are outliers on PCA or fail FP2. remove sgRNAs(guides) with low counts in DNA samples:completely remove shRNAs with median &lt;=1 3. remove sgRNAs(guides) that have &gt;1 perfect match anywhere in the reference genome 4. remove replicates that fail QC measures 5. fold change(FC) with DNA pool samples 6. Z score normalize each cell line7. collapse replicates per line 8. sgRNAs are mapped to genes (Achilles_v3.3.8.gct)9. ATARiS (pval=0.05) was run on guide-level data (Achilles_v3.3.8_gene-soln.gct)<br><br><br>NOTE: There was a minor error in an earlier version (3.3.7) of the data posted on figshare. Please use this one.<br><br>本数据集配套发表论文：Aguirre、Meyers等学者的《基因组拷贝数决定CRISPR-Cas9（CRISPR-Cas9）靶向的基因非依赖性细胞应答》，发表于《Cancer Discovery》，2016年。 摘要：CRISPR-Cas9系统可实现哺乳动物细胞的基因组编辑与体细胞遗传筛选。本研究在33种癌细胞系中开展全基因组规模功能丧失筛选，以鉴定增殖与存活必需基因，并发现基因组编辑后基因拷贝数升高与细胞活力降低之间存在显著相关性。在拷贝数扩增区域内，CRISPR-Cas9靶向表达基因与非表达基因以及基因间位点，均可通过诱导G2细胞周期阻滞显著降低细胞增殖能力。通过分析靶向多个基因组位点的单引导RNA（single guide RNA, sgRNA），我们发现该CRISPR-Cas9编辑后的细胞应答与靶位点数量显著相关。上述结果表明，CRISPR-Cas9的基因组靶向可引发基因非依赖性的抗增殖细胞应答。该效应对解读CRISPR-Cas9筛选数据具有重要的实践意义，同时也会干扰该技术用于鉴定扩增区域内必需基因的应用。 -------------------------------------------- GeCKOv2 Achilles数据集包含33种细胞系相关数据： 1. Achilles_v3.3.8_rawreadcounts.gct：包含各重复样本的原始读取计数，其中一列记录了初始质粒DNA（pDNA）文库的读取计数。 2. Achilles_v3.3.8_lognorm.gct：包含以每百万读取数为单位的重复级读取计数数据。 3. Achilles_v3.3.8a.gct：为本研究分析所用的单引导RNA级log2倍数变化（log2 fold change, log2FC）数据。该数据未跨细胞系进行归一化，但每个样本中已减去负对照单引导RNA的中位数（即得分0代表负对照的中位数水平）。 4. Achilles_v3.3.8.gct：为单引导RNA级log2FC数据，已针对每个重复样本进行z-score归一化，且已移除靶向超过一个基因组完美匹配位点的单引导RNA。该数据用于计算基因水平的必需性评分。 5. Achilles_v3.3.8_gene-soln.gct：为本研究跨细胞系基因水平分析所用的数据版本。样本已进行z-score归一化，且移除了靶向超过一个完美匹配位点的单引导RNA。基因水平评分通过ATARiS工具计算得到。 6. Achilles_v3.3.8_ABSOLUTE_CN_segtab.txt：包含33个样本的ABSOLUTE拷贝数分段信息。 7. Achilles_v3.3.8_sgRNA_mappings.txt：包含每条单引导RNA对应的映射基因组位点信息。 分析流程v3.3.8a： 1. 质量控制（QC），筛选出重复性<0.8、主成分分析（PCA）中为异常值或未通过FP检验的重复样本； 2. 移除DNA样本中读取计数较低的单引导RNA：完全移除中位数≤1的单引导RNA； 3. 移除未通过质量控制的重复样本； 4. 以DNA文库池样本为参照计算倍数变化（FC）； 5. 以负对照的中位数进行零中心化处理； 6. 合并同一细胞系的重复样本； 7. 通过Achilles_v3.3.8a.gct将单引导RNA映射至对应基因。 分析流程v3.3.8： 1. 质量控制，筛选出重复性<0.8、主成分分析中为异常值或未通过FP检验的重复样本； 2. 移除DNA样本中读取计数较低的单引导RNA：完全移除中位数≤1的单引导RNA； 3. 移除在参考基因组中存在超过一个完美匹配位点的单引导RNA； 4. 移除未通过质量控制的重复样本； 5. 以DNA文库池样本为参照计算倍数变化； 6. 针对每个细胞系进行z-score归一化； 7. 合并同一细胞系的重复样本； 8. 通过Achilles_v3.3.8.gct将单引导RNA映射至对应基因； 9. 针对单引导RNA级数据运行ATARiS工具（显著性阈值p值=0.05），生成Achilles_v3.3.8_gene-soln.gct。 注意：此前发布于figshare平台的v3.3.7版本数据存在一处微小错误，请使用本版本数据。