2019_SDASD

The codes to process and analyze FASTQ files obtained from the MS2 pulldown ribosome profiling, standard ribosome profiling, and RNAseq in “A Genome-Wide Analysis of the Role of Shine-Dalgarno Sequences in Promoting Translation in E. coli”. First run codes for data processing, then run codes for data analyses.

Dependencies

• Tally
• skewer-0.2.2
• seqtk-1.0-r31
• bowtie-1.1.2
• free_align
• python 2.7
• Anaconda2-5.0.1
• Jupyter notebook
• pickle
• numpy
• scipy
• pandas
• matplotlib
• seaborn
• kpLogo-1.1
• R
• xtail

Data processing

Each notebook contains codes which performe UMI screening and trimming, linker trimming, chromsome mapping, and calculation of RPM and RPKM. For a library sequenced multiple times, merge fastq files before mapping by bowtie.

• SDASD_Genes_TranscriptionUnits.ipynb - creates a dictionary "Ecoli_Gene_TU.pickle", requied for calculation of RPKM during data processing, based on "coli.gff" and "mbo004141900st4_TranscriptionUnits.csv"
• Skewer_Bowtie_Density_RPKM.ipynb - for ribosome profiling libraries without UMI
• Tally_Skewer_Seqtk_Bowtie_Density_RPKM.ipynb - for ribosome profiling libraries with UMI
• Truseq_Bowtie_Density_RPKM.ipynb - for RNA-seq libraries

Data analyses

• SDfigure.csv - contains RPKM, RPC (read per codon), read counts from each library calculated in data processing; also contains SD-ASD affinity, GINI index, SHAPE reactivity
• SDASD_freealign.ipynb - calculates SD-ASD affinity of each gene
• SDASD_ShapeReactivity.ipynb - calculates SHAPE reactivity of each gene
• SDASD_Correlation.ipynb - calculates linear correlation and creates scatter plots
• SDASD_DensityPlot.ipynb - creates density plots
• SDASD_Stresses_Xtail.ipynb - run xtail to compare effect of SD motifs between optimal and stress conditions
• SDASD_Stresses_VolcanoPlot.ipynb - creates volcano plots based on the results of xtail
• SDASD_coupling_optimal.ipynb - analyzes distribution of RNA features between top and bottom 20% of SD effect in optimal condition
• SDASD_coupling_cold.ipynb - analyzes distribution of RNA features between top and bottom 20% of SD effect in cold shock
• SDASD_RET_DensityPlot.ipynb - creates density plots of retapamulin-treated ribosome profilnig
• SDASD_RET_AGP.ipynb - creates average gene plot of retapamulin-treated ribosome profilnig on annotated and non-annoated AUGs
• SDASD_RET_ViolinPlot.ipynb - calculates initiation scores and creates violin plots
• SDASD_kpLogo.ipynb - makes fasta files of specified AUGs then creates various Logo plots including probability logo plots
• SDASD_ShapeReactivity_StartVSNonstart.ipynb - compares median and IQR of SHAPE reactivity between annotated and non-annotated AUGs