Achilles_v3.3.7_README.txt

GeCKOv2 Achilles dataset33 cell lines<br>Achilles_v3.3.7_lognorm.gct contains the replicate-level read counts per million<br>Achilles_v3.3.7a.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_v3.3.7_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.<br>Achilles_v3.3.7_ABSOLUTE_CN_segtab.txt contains the ABSOLUTE copy number segments for each of the 33 samples.<br>Achilles_v3.3.7_sgRNA_mappings.txt contains the mapped genomic locus (loci) for each sgRNA.<br><br><br>Analysis steps v3.3.7a:<br> 1. QC and identify reps with reproducibility &lt;0.8 or are outliers on PCA or fail FP 2. 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 controls 6. collapse replicates per line 7. sgRNAs are mapped to genes using(Achilles_v3.3.7a.gct)<br>Analysis steps v3.3.7:<br> 1. QC and identify reps with reproducibility &lt;0.8 or are outliers on PCA or fail FP 2. 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 line 7. collapse replicates per line 8. sgRNAs are mapped to genes (Achilles_v3.3.7.gct) 9. ATARiS (pval=0.05) was run on guide-level data (Achilles_v3.3.7_gene-soln.gct)<br><br>

GeCKOv2 Achilles数据集（33株细胞系）
Achilles_v3.3.7_lognorm.gct 包含各重复样本的每百万读段计数
Achilles_v3.3.7a.gct 为本研究发表时所用分析的sgRNA水平log₂FC数据，该数据未跨细胞系标准化，但会在每个样本中减去阴性对照sgRNA的中位数（即得分0对应阴性对照的中位数水平）
Achilles_v3.3.7_gene-soln.gct 为本研究发表时用于跨细胞系基因水平分析的数据版本，该版本对样本进行了Z分数标准化，并移除了在参考基因组中存在≥2处完美匹配的sgRNA
Achilles_v3.3.7_ABSOLUTE_CN_segtab.txt 包含33个样本各自的ABSOLUTE拷贝数分段信息
Achilles_v3.3.7_sgRNA_mappings.txt 包含每个sgRNA所比对的基因组位点（单一位点或多位点）

分析流程v3.3.7a：
1. 开展质量控制（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样本中低计数的sgRNA：完全移除中位数≤1的短发夹RNA（shRNA）
3. 移除未通过QC标准的重复样本
4. 以DNA池样本为参照计算折叠变化（FC）
5. 对阴性对照的中位数进行零中心化处理
6. 合并每株细胞系的重复样本
7. 借助Achilles_v3.3.7a.gct将sgRNA比对至对应基因

分析流程v3.3.7：
1. 开展质量控制（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样本中低计数的sgRNA：完全移除中位数≤1的短发夹RNA（shRNA）
3. 移除在参考基因组任意位置存在≥2处完美匹配的sgRNA
4. 移除未通过QC标准的重复样本
5. 以DNA池样本为参照计算折叠变化（FC）
6. 对每株细胞系的数据进行Z分数标准化
7. 合并每株细胞系的重复样本
8. 将sgRNA比对至对应基因（对应文件：Achilles_v3.3.7.gct）
9. 针对sgRNA水平数据（Achilles_v3.3.7_gene-soln.gct）运行ATARiS分析，显著性阈值设为p=0.05