G418_readthrough

All code used in analysis of G418 data is available here.

Exact code for generating figures is found in G418_readthrough/figures/figscripts, Example figures are found in G418_readthrough/figures/output_figures

Clonning this repository and running Wangen_G418_workflow.py will regenerate the figures from Wangen and Green. 2019. (https://elifesciences.org/articles/52611)

In Wangen_g418_workflow.py, Change threadNumb to the desired number of threads, default is 40

*** WARNING ***

Running this workflow requires at least 1.1 Terabytes of free storage space to generate all necessary files

The pipeline is designed to be run on a computational server with at least 40 threads, and may take an extremely long time to complete if run locally. STAR also requires a considerable amount of memory to run this workflow with default settings. If memory is limiting, increase the --genomeSAsparseD value

Overview of Wangen_G418_workflow.py

1. Generate Genomes
   • Download hg38 and Gencode annotations
   • Parse the GTF file and choose single isoform for each gene
   • Build all other necessary annotation files from parsed GTF
   • Parse the GTF file and create a second GTF file that contains all valid transcripts
   • Build non codon RNA depletion annotation files
   • Build indexes for STAR alignments
2. Raw Sequencing Data
   • Download FASTQ files from SRA Pending release of data
   • create merged FASTQ files of replicates for select ribosome profiling experiments
3. Ribosome Profiling data processing
   • run main analysis pipeline on ribosome profiling data, building all files required for generation of figures
   • mapping to all possible transcripts is only performed for select datasets, as this takes a long time
4. RNAseq data processing
   • run main analysis pipeline on RNAseq data, building all files required for generation of figures
5. Plot Figures
   • Run individual scripts that generate all figures in the manuscript
   • Figures can be compared to examples in output_figures to validate successful completion of analysis

Description of subfolders:

• Data contains raw luciferase measurments. All raw and processed data files will be saved here
• figures contains scripts for generating all figures in figures/figscripts and example output figures in figures/output_figures
• genomes contains currated refseq annotations of rRNA in fasta format. All annotation files will be created in this directory
• GFF contains a python module that must be coppied to python2.7/site-packages for import into python. Can also be found here: https://github.com/chapmanb/bcbb/tree/master/gff/BCBio/GFF
• riboseq contains scripts for processing ribosome profiling data
• RNAseq contains scripts for processing RNAseq data
• utils contains scripts for generating annotation files

To run the analysis pipeline, clone the repository and run the Wangen_G418_workflow.py script

All command line utilities must be downloaded and added to $PATH

REQUIREMENTS:

Command Line Utilities, added to $PATH:

• tally, 15-065: (https://www.ebi.ac.uk/research/enright/software/kraken)
• seqtk, 1.0-r31: (https://github.com/lh3/seqtk)
• skewer, 0.2.2: (https://github.com/relipmoc/skewer)
• STAR, STAR_2.5.3a_modified: (https://github.com/alexdobin/STAR)
• pigz, 2.3.1: (https://zlib.net/pigz/)
• samtools, 0.1.19-96b5f2294a:(https://github.com/samtools/samtools)
• kpLogo (http://kplogo.wi.mit.edu/)
• kentUtils (https://github.com/ENCODE-DCC/kentUtils)
• sra-tools (https://github.com/ncbi/sra-tools)

python 2.7, install using pip:

built in modules do not have version number listed

• os
• sys
• ftplib
• subprocess
• glob
• struct
• ast
• time
• datetime
• collections
• importlib
• math
• multiprocessing
• urllib
• argparse 1.1
• pandas 0.22.0
• numpy 1.14.0
• pysam 0.13
• scipy 1.0.1
• statsmodels 0.9.0
• twobitreader 3.1.5
• Bio 1.58
• pathos 0.2.1
• matplotlib 2.2.2
• seaborn 0.9.0
• csv 1.0
• lifelines 0.19.5
• logomaker 0.8
• scikit-learn 0.20.3
• GFF copy and paste GFF folder into ~/lib/python2.7/site-packages/

R 3.4.3:

• ggplot2 2.2.1
• plyr 1.8.4
• reshape2 1.4.3
• scales 0.5.0
• xtail 1.1.5
• DESeq2 1.18.1
• glue 1.3.1