Worm Perturb-Seq: massively parallel whole-animal RNAi and RNA-seq

Description: This repository contains all source codes, necessary input and output data, and raw figures and tables for reproducing most figures and results published in the following study: Hefei Zhang#, Xuhang Li#, Dongyuan Song, Onur Yukselen, Shivani Nanda, Alper Kucukural, Jingyi Jessica Li, Manuel Garber, Albertha J.M. Walhout. Worm Perturb-Seq: massively parallel whole-animal RNAi and RNA-seq. (2025) Nature Communications, in press (# equal contribution, *: correspondng author) These include results related to method benchmarking and NHR data processing. Source data for figures that are not reproducible here have been provided with the publication. Files: This repository contains a few directories related to this publication. To deposit into Zenodo, we have individually zipped each subfolder of the root directory. There are three directories included: MetabolicLibrary contains files related to the benchmarking analyses using the metabolic gene WPS data. This folder is partially overlaped with the working directory of the sister paper deposited at 10.5281/zenodo.14198997 method_simulation contains files related to the simulation benchmarking NHRLibrary contains files related to the analyses of NHR gene WPS data Note: the parameter optimization output is deposited in a seperate Zenodo repository (10.5281/zenodo.15236858) for better oganization and easy usage. If you would like to reproduce results related to the "MetabolicLibrary" folder, please download and integrate the omitted subfolder "MetabolicLibrary/2_DE/output/" from this seperate repository. Please be advised that this repository contains raw codes and data that are not directly related to a figure in our paper. However, they may be useful to generate input used in the analysis of a figure, or to reproduce tables in our manuscript. It may also contain unpublished analyses and figures, which we did not intentionally delete and kept for records. Usage: Please refer to the table in below to locate a specific file for reproducing a figure of interest (also availabe in the METHOD_FIGURE_LOOKUP.xlsx under the root directory). Figure File Linesa Notes Fig. 2c MetabolicLibrary/1_QC_dataCleaning/2_badSampleExclusion_manual.R 65-235 output figure is selected from figures/met10_lib6_badSamplePCA.pdf Fig. 2d NHRLibrary/example_bams/* - load the bam files in IGV to make the figure Fig. 3a MetabolicLibrary/2_DE/2_5_vectorlike_re_analysis_with_curated_44_NTP_conditions.R 348-463 Fig. 3b,c MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 106-376 Fig. 3d MetabolicLibrary/2_DE/SUPP_extra_figures_for_rewiring.R 10-139 Fig. 3e MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 379-522 Fig. 3f,g MetabolicLibrary/2_DE/SUPP_extra_figures_for_rewiring.R 1-8 Fig. 3h method_simulation/Supp_systematic_mean_variation_example.R 1-138 Fig. 3i method_simulation/3_benchmark_DE_result_w_rep.R and 1_benchmark_DE_result_std_NB_w_rep.R 1-518 the example figure was from figures/GLM_NB_deltaMiu_k08_10rep/seed_12345_empircial_null_fit_simulated_data_NB_GLM.pdf and figures/GLM_NB_10rep/seed_1_empircial_null_fit_simulated_data_NB_GLM.pdf; Fig. 3j MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 2104-2106 load dependencies starting from line 1837 Fig. 3k MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 2053-2078 load dependencies starting from line 1837; the GSEA was performed using SUPP_supplementary_figures_for_method_noiseness_GSEA.R Fig. 4a,b method_simulation/3_benchmark_DE_result_w_rep.R 1-523 Fig. 4c method_simulation/3_benchmark_WPS_parameters.R 1-237 Fig. 4d MetabolicLibrary/2_DE/2_5_vectorlike_re_analysis_with_curated_44_NTP_conditions.R 1-346 output figure is selected from figures/0_DE_QA/vectorlike_analysis/2d_cutoff_titration_71NTP_rawDE_log2FoldChange_log2FoldChange_raw.pdf and 2d_cutoff_titration_71NTP_p0.005_log2FoldChange_raw.pdf. The "p0.005" in the second file name indicates the p_outlier cutoff used in the final parameter set for EmpirDE. Fig. 4e MetabolicLibrary/2_DE/SUPP_plot_N_DE_repeated_RNAi.R entire file Fig. 4f MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 1020-1407 Fig. 4g,h MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 529-851 Fig. 5d NHRLibrary/FinalAnalysis/2_DE_new/3_DE_network_analysis.R 51-69; 94-112 load dependencies starting from line 1 Fig. 5e NHRLibrary/FinalAnalysis/2_DE_new/5_GSA_bubble_plot.R 1-306 Fig. 5f NHRLibrary/FinalAnalysis/2_DE_new/5_GSA.R 1-1492 Fig. 6a NHRLibrary/FinalAnalysis/2_DE_new/4_DE_similarity_analysis.R 1-175 Fig. 6b NHRLibrary/FinalAnalysis/6_case_study.R 506-534 load dependencies starting from line 1 Fig. 6c NHRLibrary/FinalAnalysis/6_case_study.R 668-888 load dependencies starting from line 1 Supplementary Fig. 1e NHRLibrary/FinalAnalysis/5_revision/REVISION_gene_detection_sensitivity_benchmark.R 1-143 Supplementary Fig. 1f MetabolicLibrary/1_QC_dataCleaning/2_badSampleExclusion_manual.R 65-235 output figure is selected from figures/met10_lib6_badSampleCorr.pdf Supplementary Fig. 1g MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 2191-2342 Supplementary Fig. 2a MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 1409-1822 Supplementary Fig. 2b method_simulation/Supp_systematic_mean_variation_example.R 1-138 Supplementary Fig. 2c method_simulation/Supp_systematic_mean_variation_example.R; 2_fit_logFC_distribution.R 141-231; 1-201 the middle panel was generated from Supp_systematic_mean_variation_example.R (lines 141-231) and right panel was from 2_fit_logFC_distribution.R (lines 1-201) Supplementary Fig. 2d method_simulation/1_benchmark_DE_result_std_NB_w_rep.R 1-518 the example figure was from figures/GLM_NB_10rep/seed_1_empircial_null_fit_simulated_data_NB_GLM.pdf; Supplementary Fig. 2e method_simulation/3_benchmark_DE_result_w_rep.R 1-518 the example figure was from figures/GLM_NB_deltaMiu_k08_10rep/seed_12345_empircial_null_fit_simulated_data_NB_GLM.pdf Supplementary Fig. 2f method_simulation/3_benchmark_DE_result_w_rep.R 528-573 may need to run the code from line 1 to load other variables needed Supplementary Fig. 3a,b method_simulation/1_benchmark_DE_result_std_NB_w_rep.R 1-523 Supplementary Fig. 3c method_simulation/3_benchmark_WPS_parameters.R 1-237 Supplementary Fig. 3d MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 3190-3300 Supplementary Fig. 3e 2_3_power_error_tradeoff_optimization.R entire file the figure was in figures/0_DE_QA/cleaning_strength_titration/benchmark_vectorlikes_titrate_cleaning_cutoff_DE_log2FoldChange_FDR0.2_FC1.pdf (produced in line 398); this script produced the titration plots for a series of thresholds, where we picked FDR0.2_FC1 for presentation in the paper Supplementary Fig. 3f 2_3_power_error_tradeoff_optimization.R entire file the figure was in figures/0_DE_QA/cleaning_strength_titration/benchmark_independent_repeats_titrate_cleaning_cutoff_FP_log2FoldChange_raw.pdf (produced in line 195). The top line plot was from figures/0_DE_QA/cleaning_strength_titration/benchmark_independent_repeats_titrate_cleaning_cutoff_FP_log2FoldChange_raw_summary_stat.pdf (produced in line 218). Supplementary Fig. 4 MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 853-898 please run from line 529 to load dependencies Supplementary Fig. 5a,b MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 2590-3185 Supplementary Fig. 5c MetabolicLibrary/2_DE/SUPP_supplementary_figures_for_methods.R 2430-2521 Supplementary Fig. 6a NHRLibrary/FinalAnalysis/2_DE_new/3_DE_network_analysis.R 1-31 Supplementary Fig. 6b NHRLibrary/FinalAnalysis/2_DE_new/3_DE_network_analysis.R 234-251 load dependencies starting from line 1 Supplementary Fig. 6c NHRLibrary/FinalAnalysis/2_DE_new/3_DE_network_analysis.R 257-268 load dependencies starting from line 1 Supplementary Fig. 6d NHRLibrary/FinalAnalysis/2_DE_new/5_GSA_bubble_plot.R 1-308 Supplementary Fig. 6e NHRLibrary/FinalAnalysis/2_DE_new/5_GSA.R 1495-1526 load dependencies starting from line 1 Supplementary Fig. 6f NHRLibrary/FinalAnalysis/6_case_study.R 610-666 load dependencies starting from line 1 Supplementary Fig. 6h NHRLibrary/FinalAnalysis/2_DE_new/3_DE_network_analysis.R 168-231 load dependencies starting from line 1 Supplementary Fig. 7c,e NHRLibrary/FinalAnalysis/2_DE_new/5_GSA_bubble_plot.R 312-313; 319-320 please run lines 1-286 to load dependencies Supplementary Fig. 7d,f NHRLibrary/FinalAnalysis/2_DE_new/5_GSA.R 1563-1600; 1620-1629 load dependencies starting from line 1 Supplementary Fig. 8 (upper left) NHRLibrary/FinalAnalysis/2_DE_new/4_DE_similarity_analysis_control_no_clean.R entire file Supplementary Fig. 8 (lower left) NHRLibrary/FinalAnalysis/2_DE_new/4_DE_similarity_analysis.R 1-152 Supplementary Fig. 8 (lower right) NHRLibrary/FinalAnalysis/2_DE_new/4_DE_similarity_analysis_control_no_clean.R 668-929 load dependencies starting from line 1; the first randomized heatmap in the output pdf file was used as the example heatmap in the figure Supplementary Fig. 9a NHRLibrary/FinalAnalysis/6_case_study.R 261-394 load dependencies starting from line 1 Supplementary Fig. 9b NHRLibrary/FinalAnalysis/6_case_study.R 261-448 load dependencies starting from line 1 Supplementary Fig. 10a NHRLibrary/FinalAnalysis/5_revision/REVISION_pairwise_modularity_follow_up.R 1-105 Supplementary Fig. 10b NHRLibrary/FinalAnalysis/5_revision/REVISION_pairwise_modularity_follow_up.R 1-304 a: The lines indicate the chunk of codes to reproduce the corresponding figure; The figure is reproduced at the end of the referred codes. Please note that you may have to run the codes above the referred chunk (i.e., from the first line) to load dependent variables to execute the referred codes. However, you should be able to reproduce the figure only with the codes within the referred script. Notice: We advise you download the entire project working directory (including all zip files and unzip them into corresponding folders), for reproducing any analysis. If you only download the zip file relevant to your figure of interest, you may or may not run into issues due to the missing files in another folder. Contact: For any questions, please contact Xuhang (Hang) Li at [e-mail].