DOI: 10.5281/zenodo.15223778
title: Worm Perturb-Seq: massively parallel whole-animal RNAi and RNA-seq

description: 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 It may also contain unpublished analyses and figures, which we did not intentionally delete and kept for records.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 Xuhang.Li@umassmed.edu.