Wrapper for running meta-gene analysis on microbial (Bacteria and Archaea) ribosome profiling data using modified scripts from Dr. Fuad Mohammad. v0.4 updated on 05/07/2020
To install locally:
git clone https://github.com/dgelsin/mRibo
then to make it executable from anywhere:
cp mRibo/mRibo/bin/* /usr/local/bin/
pip install multiprocess
pip install bcbio-gff
pip install biopython
conda install pandas
pip install numpy
pip install ipython
pip install collections-extended
pip install -U scikit-learn
pip install plotly==3.10.0
pip install seaborn
conda install bowtie
mRibo -U reads.fastq -R rRNA.fa -g genome.fa -a genome.gff -D genome_dict -C name_of_user -M name_of_microbe -o output_directory_name
mRibo core options:
-U STR ribosome profiling reads
-R SRT Ribosomal RNA and tRNA fasta file for filtering out corresponding reads
-g STR genome fasta file
-a STR genome annotation gtf/gff file
-D INT genome annotation dictionary file
-C STR user name
-M STR name of microbe being analyzed
-o STR output directory, do not name it "output"
Additional options:
-I INT min read length size for meta-gene analysis (default=10)
-X INT max read length size for meta-gene analysis (default=40)
-p INT number of threads to use for analysis (default=1)
-c INT number of cores to use for analysis (default=1)
-lu INT number of nucleotides to include upstream of the TSS (default=50)
-ld INT number of nucleotides to include downstream of the the TSS within the ORF, genes shorter than this are excluded (default=200)
-m INT style in which footprints are mapped for meta-gene analysis (5 prime or 3 prime) (default=5)
-d STR type of density to use for meta-gene plots (rpm or reads) (default=rpm)
-w STR style in which to weigh density at positions within ORFs, either equal weight (yes) or not (no) (default=yes)
-dg INT distance between genes, genes closer than this are removed from start and stop, or can choose 'no' (default=50)
-r INT rpkm threshold to use for meta-gene analysis, genes below will be removed from average (default=10)
-y INT maximum value for y-axis of avggenes meta-gene plot (default=0)
-s INT A-site shift value for pause score meta-gene analysis (default=-11)
-P STR Style in which to conduct pause score meta-gene analysis, either amino acids (aa) or codons (codon) (default=aa)
-Y INT maximum value for y-axis of pause score meta-gene plot (default=6)
--version | -v show current mRibo version
Example:
mRibo -U mRibo/mRibo/practice_files/FASTQ/practice.fq -R /Users/DRG/Desktop/mRibo/mRibo/practice_files/genomes/rRNA.fa -g mRibo/mRibo/practice_files/genomes/genome.fa -a /Users/DRG/Desktop/mRibo/mRibo/annotations/Volcanii.gff -D mRibo/mRibo/annotations/Volcanii_dict_for_pause -C DG -M Volcanii -o mRibo_run1
mRibo-build -a genome.gff -g genome.fa -C name_of_user -M name_of_microbe -o output_directory_name
Example:
mRibo-build -a mRibo/mRibo/practice_files/annotations/practice.gff -g mRibo/mRibo/practice_files/genomes/genome.fa -C DG -M Hv -o Hv_annotation
density_wrapper.py [FASTQ_directory] [GFF_file.gff] [GFF_DICTIONARY_file] [ALIGNMENT_directory] [NAME_OF_USER] [NAME_OF_MICROBE] [MIN_LEN] [MAX_LEN] [NUM_THREADS] [NUM_CORES]
Example:
density_wrapper.py input/FASTQ/ annotations/Volcanii.gff annotations/Volcanii_dict_for_pause input/alignment/ DG Volcanii 10 40 8 4
main_wrapper.py [GFF_DICTIONARY_file] [NAME_OF_USER] [NAME_OF_MICROBE] [MIN_LEN] [MAX_LEN] [NUM_THREADS] [NUM_CORES] [LENGTH_UPSTREAM] [LENGTH_DOWNSTREAM] [ALIGNMENT_STYLE] [DENSITY_STYLE] [WEIGHT_STYLE] [DISTANCE_GENES] [RPKM_THRESHOLD] [YMAX_AVGGENES] [ASITE_SHIFT] [PAUSE_STYLE] [YMAX_PAUSE]
Example:
main_wrapper.py annotations/Volcanii_dict_for_pause DG Volcanii 10 40 8 4 50 200 3 rpm yes 50 10 10 -11 aa 10
All input and output files generated by mRibo will be placed within the output_directory that you named with the -o option.
In the input/ directory you will find a .sam bowtie alignment to the genome in the alignment/ directory, the reads after rRNA filtering used to map to the genome in the FASTQ/ directory, the rRNA bowtie index and .sam rRNA alignment in the bowtie_rRNA_alignment/ directory, and lastly the genome bowtie index in the bowtie_genome_alignment/ directory.
4 figures are generated as output from mRibo. They can be found in output_directory_name/output/reads/figures/user_name/
- aa_pausescore_.pdf - a plot of pause scores for each amino acid at the A-site (large colored dot), P-site (gray dot), and E-site (black dot). Next are individual plots for each amino acid of read density along the length within ribosome footprints (top plot) with a heatmap underneath plotting footprint size (bottom plot). Alternatively, if -P is set to codon, this will plot individual codons instead.
- asymmetry_score_.pdf - boxplot of asymettry score for the library used in the analysis.
- avggene_.pdf - meta-gene plots of ribosome density along ORFs (top plots). Underneath are heatmaps of ribosome footprint lengths and where they map along ORFs (bottom plots). The right plot corresponds to the start of ORFs where 0 is x nt upstream of the TSS (5' UTRs), set by the -lu option, and the TSS is marked by the number set by the -lu option. -ld option sets how far to plot downstream within ORFs. The left plot corresponds to the stop of ORFs where 0 is x nt upstream of the stop codon, set by the -ld option, and the stop codon is marked by the number set by the -ld option. -lu option sets how far to plot downstream of ORF stop codons (3' UTRs). The same plot but with a legend (scale) for the heatmap is provided as a second pdf.
- frame_.pdf - Various plots of reading frame distribution for all footprint lengths on average (left plot), each individual footprint (middle), and each gene (right plot). 0 corresponds to the 0t position in an in-frame ORF codon, 1 is +1 position, and 2 the +2 position.
Various data files are generated as output from mRibo for visualization and further analysis by the user.
- wig files for viewing ribosome density per nt on a genome viewer are provided in output_directory_name/output/reads/density/density/wigfiles/
- data used to generate avggene plots are provided in output_directory_name/output/reads/analysis/individual/user_name/avggenes/ for either the start of ORFs (start_all.csv) or stop of ORFs (stop_all.csv)
- pause score values tp generate aa_pausescore figures are provided in output_directory_name/output/reads/analysis/individual/user_name/pause_score/ for either amino acids (aa_scores.csv) or individual codons (codon_scores.csv). Pause scores are provided for the -1, -2, A, P, E, +1, and +2 sites of the ribosome in both csv files. aa_plot_values.csv contains pause scores for all amino acids along the length of ORFs.
- Expression values (rpkm) of genes are found in output_directory_name/output/reads/analysis/individual/user_name/*_genelist.csv
If mRibo helped you in an analysis for a paper, please cite my NAR paper for the wrapper tool:
Gelsinger DR, Dallon E, Reddy R, Mohammad F, Buskirk AR, DiRuggiero J. Ribosome profiling in archaea reveals leaderless translation, novel translational initiation sites, and ribosome pausing at single codon resolution. Nucleic Acids Research. Volume 48, Issue 10, 04 June 2020, Pages 5201–5216, https://doi.org/10.1093/nar/gkaa304
and Dr. Fuad Mohammad's Elife paper for the core python scripts:
Mohammad F, Green R, Buskirk AR. A systematically-revised ribosome profiling method for bacteria reveals pauses at single-codon resolution. Elife. 2019;8:e42591. Published 2019 Feb 6. doi:10.7554/eLife.42591
I also recommend citing any other tools used in this program as well (i.e. bowtie).
Author of pipeline: Diego Rivera Gelsinger
Principal investigators: Jocelyne DiRugierro & Allen Buskirk
Institution: Johns Hopkins, Department of Cell, Molecular, Developmental Biology, and Biophysics
For general feedback you can contact me at [email protected]. For issues with mRibo please post it on the Issues tab of this github repository. Please include the mRibo version number, the entire command and options you inputted as a user, and the full stdout error.