segSHAPE: RNA secondary structure prediction from nanopore direct RNA sequencing

End-to-end pipeline: raw nanopore direct-RNA signal → SHAPE reactivity → RNA secondary structure, supporting both RNA002 and RNA004 chemistries.

segSHAPE turns raw Oxford Nanopore direct-RNA signal into a SHAPE-constrained RNA secondary structure: it segments the signal into subevents, aligns them to a reference with an anchored Viterbi DP, calls per-position modification rates by comparing a SHAPE-treated sample against an untreated control, and feeds the resulting reactivity profile to ViennaRNA RNAfold.

Status: v0.1.0. All 7 pipeline steps are implemented end-to-end (pod5index → dorado-extract → segment → event-align → mod-calling → fold, plus evaluate / plot). Every command runs on built-in defaults that are the parameter set used in the segSHAPE paper, so a clean run reproduces the published results without extra tuning flags.

Install

Requires Python ≥ 3.10. The folding step (7) needs the ViennaRNA RNAfold binary on PATH.

# 1. Create an isolated environment (Python ≥ 3.10)
conda create -n segshape python=3.10
conda activate segshape

# 2. Install segSHAPE from PyPI
pip install segshape

# 3. Provide RNAfold for the folding step (7)
conda install -c bioconda viennarna

To install from source instead (for development):

git clone https://github.com/guangzhaocs/segSHAPE.git
cd segSHAPE
pip install -e .

The bundled ONT k-mer tables and RNAfold parameter file ship inside the package — nothing extra to download. Basecalling (step 2) uses Dorado and the pod5 CLI, installed separately from Oxford Nanopore.

Tutorial

End-to-end walkthrough — directory layout, per-step commands, and expected outputs — in docs/tutorial.md. Diagnostic plotting (segshape plot), with a tiny ready-to-run example fixture, is in docs/plot.md.

Public modules (CLI surface)

segshape <module> ... exposes 8 user-facing modules:

Run segshape <cmd> --help (and segshape <cmd> <step> --help) to see every parameter. Sub-command parsers register lazily, so segshape --help does not import numba / scipy / sklearn.

Pipeline overview

Raw signal → RNA secondary structure in 7 steps. Most map 1:1 onto a segshape module; step 2 wraps Dorado and step 7 wraps ViennaRNA RNAfold:

Steps 3–5 outputs all live under datasets/<DATASET>/<SAMPLE>/3_alignment/.

Quick start

ROOT=/path/to/data            # directory containing datasets/
DS=riboswitch_wt
REF=datasets/$DS/reference/ref_wt.fa
CONTIG=TETRA
CELL=rna004_de50_dk15_bc0.0_sp50_shift_only

for S in control treated; do
    segshape pod5index    datasets/$DS/$S/1_raw_signal/pod5 --force
    # (basecall with Dorado → 2_base_called/dorado-<VER>/dorado.sorted.bam)
    segshape dorado-extract --root-dir $ROOT --dataset $DS --sample $S
    segshape segment        --root-dir $ROOT --dataset $DS --sample $S
done

# control before treated (treated reuses control's k-mer table)
segshape event-align --root-dir $ROOT --dataset $DS --sample control \
    --reference-file $REF --contig $CONTIG
segshape event-align --root-dir $ROOT --dataset $DS --sample treated \
    --reference-file $REF --contig $CONTIG

segshape mod-calling --root-dir $ROOT --dataset $DS --sweep-cell $CELL --ref-fa $REF
segshape fold --mod-rate-csv \
    datasets/$DS/treated/3_alignment/$CELL/mod_rate/default_if-1D_c0.0050/mod_rate.csv \
    --ref-fa $REF --contig $CONTIG

See docs/tutorial.md for each step's inputs, outputs, and options.

Library use

Every cli.py is thin glue: each leaf module exports add_arguments(parser) and run(args), so the same code is reachable from the CLI and from Python.

from segshape.align import anchored

anchored.run(args)        # args: argparse.Namespace (or anything with the
                          # same attributes)

Repo layout

segSHAPE/
├── pyproject.toml
├── src/segshape/                # the importable package
│   ├── cli.py                   # `segshape` console-script entry
│   ├── io/                      # steps 1+3 — pod5 index, dorado-extract, readers
│   ├── segment/                 # step 4 — pod5 → peaks → subevents.parquet
│   ├── align/                   # step 5 — anchored alignment
│   ├── reactivity/              # step 6 — modification calling
│   ├── fold/                    # step 7 — SHAPE-constrained RNAfold
│   ├── evaluate/                # MCC / reactivity eval, LL filter, summary
│   ├── plot/                    # diagnostic plots
│   └── data/                    # bundled ONT k-mer tables + RNAfold params
├── tests/                       # smoke / unit tests
└── docs/tutorial.md             # end-to-end walkthrough

Tests

pip install -e .[dev]
pytest -q

Smoke tests verify package import, bundled-data presence, and that every leaf CLI sub-command responds to --help cleanly, alongside unit tests for the segmentation, calibration, and reactivity logic.

License

Apache-2.0 — see LICENSE.