Analysis of Direct RNA Sequencing data for :

• Saccharomyces cerevisiae ribosomal RNAs (rRNAs)
• Saccharomyces cerevisiae small nuclear and small nucleolar RNAs (sn/snoRNAs)
• Saccharomyces cerevisiae messenger RNAs (mRNAs)

To reproduce the full results, you will need the FAST5 and FASTQ files related to yeast RNA analyses can be found here (total RNA) and here (polyA-selected RNA)

Demo/test data to run the scripts is included in each of the Analysis sections:

• Test data for NanoCMC-seq
• Test data for ncRNA analyses
• Test data for rRNA analyses

What's included:

Tools

• Epinano base frequency version in order to analyse direct RNA sequencing bam files
• Bam2Stats tool in order to analyse NanoCMC-Seq bam files]

Bash scripts

• Pre-process the files for R analysis

R scripts

• Process ribosomal RNA runs
• Process sn/snoRNA runs
• Process mRNA runs
• Process NanoCMC-Seq runs

Stoichiometry prediction

• If you'd like to predict stochiometry (difference in RNA modification levels) between two samples (e.g. wild type vs knockout, normal vs heat stress), you may wish to look at the NanoRMS GitHub repository.

Dependencies/requirements:

Following R Packages are needed to run the scripts:

R packages
plyr
stringr
reshape2
dplyr
ggplot2
ggbeeswarm
ggpubr
data.table

Citation:

Begik, O., Lucas, M.C., Pryszcz, L.P. et al. Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing. Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00915-6

Citation:

Please open an issue in the GitHub repo if you have any questions/doubts/suggestions about how to use this software. Thanks!