RNAnue is a comprehensive analysis tool to detect RNA-RNA interactions from Direct-Duplex-Detection (DDD) data.
Note: This is an independently maintained fork. The original project was developed at Ibvt/RNAnue and is now continued by the original group at RNABioInfo/RNAnue.
RNAnue has the following dependencies. Make sure the requirements are satisfied by your system.
- C++20 compiler — GCC >= 12 (SeqAn3 does not support Clang)
- CMake (>= 3.22.1)
- Boost C++ Libraries (>= 1.56.0) —
program_optionscomponent only - SeqAn3 (v3.3.0) — expected in
./seqan3/in the repo root - HTSlib — linked at build time, found via
pkg-config(htslib.pc) - ViennaRNA Package (v2.6.4 recommended) — linked at build time, found via
pkg-config(RNAlib2.pc). Not packaged on Ubuntu/Debian; build from source with./configure --without-swig --without-doc --without-tutorial && make && sudo make install. - Segemehl (v0.3.4) — runtime dependency for the
alignsubcall, must be in$PATH
Clone the repository and retrieve SeqAn3:
git clone https://github.com/riasc/RNAnue.git
cd RNAnue
# download SeqAn3 into the repo root
curl -L https://github.com/seqan/seqan3/releases/download/3.3.0/seqan3-3.3.0-Source.tar.xz -o seqan3.tar.xz
tar -xf seqan3.tar.xz && mv seqan3-3.3.0-Source seqan3 && rm seqan3.tar.xzBuild using CMake (out-of-source build):
mkdir -p build && cd build
cmake .. -DCMAKE_BUILD_TYPE=Release
makeThe RNAnue binary will be in the build/ directory.
A ready-to-use Docker container is available:
docker pull riasc/rnanue:latest
docker run -v /path/to/data:/data riasc/rnanue RNAnue <subcall> --config /data/params.cfgThe -v flag mounts your local data directory into the container at /data. Paths in your config file should refer to /data/... (the in-container path), not the host path.
The Docker container can also be used with Singularity:
singularity pull docker://riasc/rnanue:latest
singularity exec --bind /path/to/data:/data rnanue_latest.sif RNAnue <subcall> --config /data/params.cfg
RNAnue provides different subcalls for individual procedures:
| Subcall | Description |
|---|---|
preproc |
Adapter trimming, quality filtering, PE merging |
align |
Read alignment via segemehl |
detect |
Split read detection from aligned BAM files |
clustering |
Merge overlapping split reads into clusters |
analysis |
Annotation, statistical scoring, interaction tables |
complete |
Run the full pipeline in sequence |
The pipeline order (also what complete runs) is: preproc → align → detect → clustering → analysis.
RNAnue <subcall> --config /path/to/params.cfgRun RNAnue --help for the full list of options.
End-to-end example using a config file:
RNAnue complete --config params.cfgOr with the most common parameters on the command line:
RNAnue complete \
--trtms ./trtms \
--ctrls ./ctrls \
--outdir ./results \
--dbs reference.fa \
--features annotation.gff \
--threads 8RNAnue requires sequencing files in a specific folder structure. The root folders for treatments (--trtms) and controls (--ctrls) contain subfolders with arbitrary condition names, each holding the read files:
trtms/
condition1/ # FASTQ files
condition2/
ctrls/ # optional
condition1/
condition2/
The --trtms parameter is required. --ctrls is optional.
RNAnue accepts parameters from the command line and through a configuration file:
RNAnue <subcall> --config /path/to/params.cfgCommand-line parameters take precedence over the config file.
tests/humanSE.cfg is a complete annotated config file you can copy as a starting template.
Results are stored in the specified output folder and its subfolders (./preproc, ./align, ./detect, ./clustering, ./analysis).
RNAnue reports detected splits in BAM format (detect subcall). Pairs of rows represent the split reads consisting of individual segments:
A00551:...:10645 16 gi|...|NC_010473.1| 3520484 22 1X51= * 0 0 AGGG...TCAA * XA:Z:TTTCTGG XC:f:0.714 XE:f:-15.6 ...
A00551:...:10645 16 gi|...|NC_010473.1| 3520662 22 11=5S * 0 0 TTCG...GAAC * XA:Z:GAAGAAC XC:f:0.714 XE:f:-15.6 ...
Custom SAM tags reported in split reads:
| Tag | Description |
|---|---|
| XC:f | Complementarity score |
| XE:f | Hybridization energy (MFE in kcal/mol) |
| XA:Z | Alignment of sequence |
| XM:i | Matches in alignment |
| XL:i | Length of alignment |
| XR:f | Site length ratio |
| XS:i | Alignment score |
| XD:Z | MFE structure in dot-bracket notation |
| XH:i | Number of splits (SAM record) |
| XJ:i | Number of splits (whole read) |
| XX:i | Split boundary start |
| XY:i | Split boundary end |
The clustering subcall produces a clusters.tab file — a tab-delimited file where each line represents a cluster of overlapping split reads:
| Field | Description |
|---|---|
| clustID | Unique identifier of the cluster |
| fst_seg_chr | Chromosome (accession) of the first segment |
| fst_seg_strd | Strand of the first segment |
| fst_seg_strt | Start position of the first segment |
| fst_seg_end | End position of the first segment |
| sec_seg_chr | Chromosome (accession) of the second segment |
| sec_seg_strd | Strand of the second segment |
| sec_seg_strt | Start position of the second segment |
| sec_seg_end | End position of the second segment |
| no_splits | Number of split reads in the cluster |
| fst_seg_len | Length of the first segment |
| sec_seg_len | Length of the second segment |
The analysis subcall generates _interactions files for each library. Each line represents an annotated split read mapped to a transcript interaction:
| Field | Description |
|---|---|
| qname | Read/template identifier |
| fst_seg_strd | Strand of the first segment |
| fst_seg_strt | Start position of the first segment |
| fst_seg_end | End position of the first segment |
| fst_seg_ref | Reference name of the first segment |
| fst_seg_name | Gene name/symbol of the first segment |
| first_seg_bt | Biotype of the transcript |
| fst_seg_anno_strd | Strand of the overlapping annotation |
| fst_seg_prod | Description of the transcript |
| fst_seg_ori | Orientation (sense/antisense) |
| sec_seg_strd | Strand of the second segment |
| sec_seg_strt | Start position of the second segment |
| sec_seg_end | End position of the second segment |
| sec_seg_ref | Reference name of the second segment |
| sec_seg_name | Gene name/symbol of the second segment |
| sec_seg_bt | Biotype of the transcript |
| sec_seg_anno_strd | Strand of the overlapping annotation |
| sec_seg_prod | Description of the transcript |
| sec_seg_ori | Orientation (sense/antisense) |
| cmpl | Complementarity score |
| fst_seg_compl_aln | Complementarity alignment of the first segment |
| sec_seg_cmpl_aln | Complementarity alignment of the second segment |
| mfe | Hybridization energy |
| mfe_struc | MFE structure in dot-bracket notation |
The main results are transcript interactions stored in allints.txt:
| Field | Description |
|---|---|
| fst_rna | Gene/transcript name of the first partner |
| sec_rna | Gene/transcript name of the second partner |
| fst_rna_ori | Orientation of the first partner |
| sec_rna_ori | Orientation of the second partner |
| <sample>_supp_reads | Number of supporting split reads |
| <sample>_ges | Global energy score |
| <sample>_ghs | Global hybridization score |
| <sample>_pval | P-value (binomial test) |
| <sample>_padj | Benjamini-Hochberg adjusted p-value |
Additional output options:
--outcnt 1— generatescounts.txt(count table for differential expression analysis)--outjgf 1— generatesgraph.json(interactions in JSON graph format)--stats 1— generatesstats.txt(basic statistics for each step)
Run the test suite:
./build/RNAnue_testsTest data is available in tests/data/.
Please create an issue if you encounter any problems.
If you use RNAnue, please cite the original paper:
Schäfer, R. A., & Voß, B. (2021). RNAnue: efficient data analysis for RNA-RNA interactomics. Nucleic Acids Research, 49(10), 1–10. https://doi.org/10.1093/nar/gkab340
See CITATION.cff for a machine-readable citation entry.
RNAnue is licensed under the GNU General Public License v3.0. See LICENSE for the full text.