Nano-DMS-MaP

Code to analyze and visualize RNA structural probing by Nanopore DMS mutational profiling data.

Note: The code accompanying the Nature Methods Nano-DMS-MaP paper is located at this branch: https://github.com/smyth-lab/Nano-DMS-MaP/tree/NMethods-10.1038/s41592-023-01862-7

Core Workflow

(numbers in square brackets indicate in which jupyter notebook the process is documented)

1. [1] Basecalling (and demultiplexing) of fast5 files from native ligation barcoded SQK-NBD114 (Q20+ kits) runs of specific amplicons.
2. [1] Read-to-isoform assignment with Isoquant, followed by sorting.
3. [1] Per isoform alignment with LAST.
4. [1] RNA Framework analysis
   • rf-count with optimized settings on LAST-aligned BAM files.
   • rf-norm to quantify reactivity rates (we recommend having a non-modified control to evaluate DMS-induced mutation rates and for normalization)
   • rf-correlate and rf-combine to evaluate the reproducability of reactivity (>0.9 expected) and calculate the mean reactivity of replicates.
5. [4] Plotting DMS reactivities per sample and isoforms and generating varna files of known or predicted structures colored by DMS reactivities.
6. [4] Performing de novo RNA structure prediction using EternaFold (or rf-fold)

Optional (QC) steps

• [0] Generating a custom gtf file for Isoquant to take into account primer binding sites
• [2] Visualization of Isoquant's isoform assignment
• [3] Mutation profile analysis of BAM alignment files using perbase
• [5] Subsampling with sambamba to evaluate whether resequencing is worthwhile

Installation instructions

Prerequisites:

• Linux system
• Ana-/Miniconda: https://docs.conda.io/projects/miniconda/en/latest/
• Java (for Varna)

1. Clone the github repository into the current location. This will automatically clone additional tools available on GitHub, i.e. RNAFramework and EternaFold (into the tools folder)

git clone --recursive https://github.com/smyth-lab/Nano-DMS-MaP

2. Move into the Nano-DMS-MaP folder

cd Nano-DMS-MaP

3. Create a conda environment that contains the majority of tools required for analysis. This may take a while ...

conda env create -f environment.yml

4. Activate the conda environment

conda activate Nano-DMS-MaP

5. Compile the EternaFold binary

cd tools/EternaFold/src
make multi
cd ../../../

7. Download the most current version of VARNA (needs to be located in the working directory to function).

wget https://varna.lisn.upsaclay.fr/bin/VARNAv3-93.jar

8. Start the jupyter lab server. If running on a cluster connected via ssh, add the no-browser and port options.

jupyter lab (--no-browser --port=XXXX)

If launching jupyter lab on a cluster, run the following command on your local computer to create an SSH tunnel.

(ssh -N -L XXXX:localhost:XXXX user@server)

10. Start analysis in jupypter lab (either opened automatically when launched on local machine, or open by copying the URL shown after the jupyter lab command.

References

Tool	Citation	License
LAST	Michiaki Hamada, Yukiteru Ono, Kiyoshi Asai, Martin C Frith, Training alignment parameters for arbitrary sequencers with LAST-TRAIN, Bioinformatics, Volume 33, Issue 6, March 2017, Pages 926–928, https://doi.org/10.1093/bioinformatics/btw742	GNU GPLv3
Isoquant	rjibelski, A.D., Mikheenko, A., Joglekar, A. et al. Accurate isoform discovery with IsoQuant using long reads. Nat Biotechnol 41, 915–918 (2023). https://doi.org/10.1038/s41587-022-01565-y	GNU GPLv2
RNA Framework	Incarnato D, Morandi E, Simon LM, Oliviero S. RNA Framework: an all-in-one toolkit for the analysis of RNA structures and post-transcriptional modifications. Nucleic Acids Res. 2018 Sep 19;46(16):e97. doi: 10.1093/nar/gky486. PMID: 29893890; PMCID: PMC6144828.	GNU GPLv3
EternaFold	Wayment-Steele HK, Kladwang W, Strom AI, Lee J, Treuille A, Becka A; Eterna Participants; Das R. RNA secondary structure packages evaluated and improved by high-throughput experiments. Nat Methods. 2022 Oct;19(10):1234-1242. doi: 10.1038/s41592-022-01605-0	BSD-3-Clause
Perbase	Seth Stadick, Perbase, v0.9.0 released on 20.07.2023 https://github.com/sstadick/perbase	MIT license
Sambamba	Artem Tarasov, Albert J. Vilella, Edwin Cuppen, Isaac J. Nijman, Pjotr Prins, Sambamba: fast processing of NGS alignment formats, Bioinformatics, Volume 31, Issue 12, June 2015, Pages 2032–2034, https://doi.org/10.1093/bioinformatics/btv098	GNU GPLv2+
VARNA	Kévin Darty, Alain Denise, Yann Ponty, VARNA: Interactive drawing and editing of the RNA secondary structure, Bioinformatics, Volume 25, Issue 15, August 2009, Pages 1974–1975, https://doi.org/10.1093/bioinformatics/btp250	GNU GPLv3.0