CGI_nature_micro_2022

Code and commands used in the Shuqi & Poulton et al. Nature Micro 2022 paper

Requirements

Python code:

• MAGeCK: (Version 0.5.7): https://sourceforge.net/p/mageck/wiki/install/
• pandas (Version 1.4.1)
• ast (Version 3.10.4)
• snippy (Version 3.2-dev): https://github.com/tseemann/snippy

R code:

• R (Version 4.1.0)
• readr (Version 1.4.0)
• ggplot2 (Version 3.3.3)
• reshape2 (Version 1.4.4)
• gplots (Version 3.1.1)
• dendextend (Version 1.14.0)
• RColorBrewer (Version 1.1-2)
• stringr (Version 1.4.0)
• parallel (Version 2.14.0)

Directory Structure:

./

Contains the scripts used to analyze counts and plot results.

Results

Contains results and temporary files

data

• Final_PreDepletion_Grouped_Split.csv: Table summarizing MAGeCK analysis results
• list_of_H37Rv_orfs_in_library.txt: List of genes in M. Tuberculosis strain H37Rv
• Weiz_wLFC.csv: Cleaned copy of Supplementary Data from Weizhen et al 2017.
• Volcano_Plot_Axes_Table.csv: Table read in volcano plot generating script to specify desired data, axes, and title.
• counts:
• • Raw counts obtained from the sequencing pipeline described in Bosch & DeJesus et al. 2021.
• mageck:
• • Contains MAGeCK result files

MAGECK Analysis

All MAGECK analysis were done using this command with the following flags:

mageck test -k <path to file with combined control and experimental counts> -c 0,1,2 -t 3,4,5 -n <output file name> --gene-lfc-method alphamedian --norm-method control --control-sgrna <file with negative control IDs>

Gene-level result outputs are provided in the repository, as well as files with read-counts for each of the conditions.

Snippy Analysis

Raw reads were downloaded from NCBI and run through snippy using the following command:

snippy --ref <NC_018143.2.gb> --prefix <sample ID> --outdir <path to output directory> --pe1 <path to read 1 FASTQ> --pe2 <path to read 2 FASTQ>

Heatmap

Read through the MAGeCK result files to generate a table (./Results/Heatmap/htmp_data_gt1Tmt_D5.csv) of log-fold change values as described in the paper.

python Data_Prep_Heatmap_Script.py

Take the log-fold change data table and create the Heatmap (./Results/Heatmap/htmp_Ward_D_gt1Tmt_D5.png) from Supplementary material.

Rscript Heatmap_Script_D5.R

TnSeq Comparison

Read cleaned supplementary data from Weizhen et al 2017 (./data/Weiz_wLFC.csv) and the table summarizing MAGeCK results (./data/Final_PreDepletion_Grouped_Split.csv) to generate Supplementary Data 2 (./Results/Supplemental_Data_2/Supplemental_Data_2.xlsx).

python Gen_Weizhen_Overlap_Supplemental_Fig2.py

Volcano Plots

Read through the MAGeCK result files to generate volcano plots in Figure 1 (./Results/Volcano_Plots).

Rscript Volcano_Plot_Condition_Level.R