Merge pull request #83 from pinellolab/v218

Release v218

This PR introduces major improvements in off-target detection, annotation efficiency, and command-line usability.

Key Updates:

- Comprehensive off-target nomination; integrated support for alignments featuring gaps in both gRNA and spacer sequences, as well as spacer alignments initiating with gaps. (Requires CRISPRitz v2.7.0)

- Refined CFD scoring; adjusted the CFD (Cutting Frequency Determination) computation to correctly account for the expanded alignment configurations introduced in this version.

- Enhanced annotation pipeline; annotation now supports compressed and indexed BED files, leveraging samtools indexing for substantially faster processing particularly beneficial for genome-wide searches and large variant datasets such as gnomAD.

- Fix bug in gnomAD VCF conversion pipeline

- Improved CLI help for complete-search; updated and clarified help messages, providing detailed flag descriptions and emphasizing input format requirements for annotation files.

Summary:
This version extends the accuracy and scalability of CRISPRme’s off-target search and improves usability through optimized annotation handling and clearer documentation.

Author: ManuelTgn

.gitignore

@@ -1,9 +1,177 @@
+# Byte-compiled / optimized / DLL files
+__pycache__/
+*.py[cod]
+*$py.class
+
+# C extensions
+*.so
+
+# Distribution / packaging
+.Python
+build/
+develop-eggs/
+dist/
+downloads/
+eggs/
+.eggs/
+lib/
+lib64/
+parts/
+sdist/
+var/
+wheels/
+share/python-wheels/
+*.egg-info/
+.installed.cfg
+*.egg
+*.pyc
+MANIFEST
+
+# PyInstaller
+#  Usually these files are written by a python script from a template
+#  before PyInstaller builds the exe, so as to inject date/other infos into it.
+*.manifest
+*.spec
+
+# Installer logs
+pip-log.txt
+pip-delete-this-directory.txt
+
+# Unit test / coverage reports
+htmlcov/
+.tox/
+.nox/
+.coverage
+.coverage.*
+.cache
+nosetests.xml
+coverage.xml
+*.cover
+*.py,cover
+.hypothesis/
+.pytest_cache/
+cover/
+
+# Translations
+*.mo
+*.pot
+
+# Django stuff:
+*.log
+local_settings.py
+db.sqlite3
+db.sqlite3-journal
+
+# Flask stuff:
+instance/
+.webassets-cache
+
+# Scrapy stuff:
+.scrapy
+
+# Sphinx documentation
+docs/_build/
+
+# PyBuilder
+.pybuilder/
+target/
+
+# Jupyter Notebook
+.ipynb_checkpoints
+
+# IPython
+profile_default/
+ipython_config.py
+
+# pyenv
+#   For a library or package, you might want to ignore these files since the code is
+#   intended to run in multiple environments; otherwise, check them in:
+# .python-version
+
+# pipenv
+#   According to pypa/pipenv#598, it is recommended to include Pipfile.lock in version control.
+#   However, in case of collaboration, if having platform-specific dependencies or dependencies
+#   having no cross-platform support, pipenv may install dependencies that don't work, or not
+#   install all needed dependencies.
+#Pipfile.lock
+
+# poetry
+#   Similar to Pipfile.lock, it is generally recommended to include poetry.lock in version control.
+#   This is especially recommended for binary packages to ensure reproducibility, and is more
+#   commonly ignored for libraries.
+#   https://python-poetry.org/docs/basic-usage/#commit-your-poetrylock-file-to-version-control
+#poetry.lock
+
+# pdm
+#   Similar to Pipfile.lock, it is generally recommended to include pdm.lock in version control.
+#pdm.lock
+#   pdm stores project-wide configurations in .pdm.toml, but it is recommended to not include it
+#   in version control.
+#   https://pdm.fming.dev/latest/usage/project/#working-with-version-control
+.pdm.toml
+.pdm-python
+.pdm-build/
+
+# PEP 582; used by e.g. github.com/David-OConnor/pyflow and github.com/pdm-project/pdm
+__pypackages__/
+
+# Celery stuff
+celerybeat-schedule
+celerybeat.pid
+
+# SageMath parsed files
+*.sage.py
+
+# Environments
+.env
+.venv
+env/
+venv/
+ENV/
+env.bak/
+venv.bak/
+
+# Spyder project settings
+.spyderproject
+.spyproject
+
+# Rope project settings
+.ropeproject
+
+# mkdocs documentation
+/site
+
+# mypy
+.mypy_cache/
+.dmypy.json
+dmypy.json
+
+# Pyre type checker
+.pyre/
+
+# pytype static type analyzer
+.pytype/
+
+# Cython debug symbols
+cython_debug/
+
+# PyCharm
+#  JetBrains specific template is maintained in a separate JetBrains.gitignore that can
+#  be found at https://github.com/github/gitignore/blob/main/Global/JetBrains.gitignore
+#  and can be added to the global gitignore or merged into this file.  For a more nuclear
+#  option (not recommended) you can uncomment the following to ignore the entire idea folder.
+.idea/
+
+# OSX
+.DS_store
+__MACOSX
+
+# crisprme
 Genomes/
 genome_library/
 Results/
 Dictionaries/
 VCFs/
-*.pyc
 Gencode/
 Cache/
 *nohup*

.vscode/settings.json

@@ -0,0 +1,175 @@
+""" """
+
+from pysam import TabixFile
+from pysam.utils import SamtoolsError
+from typing import List, Tuple, Optional
+from time import time
+
+import pysam
+import sys
+import os
+
+TBI = "tbi"
+
+def parse_commandline(args: List[str]) -> Tuple[str, str, str]:
+    """Parses and validates command line arguments for the annotation script.
+
+    Ensures the correct number of arguments are provided and that input files exist. 
+    Returns the file paths for the offtargets, annotation, and output files.
+
+    Args:
+        args: List of command line arguments.
+
+    Returns:
+        Tuple containing the offtargets file name, annotation file name, and output 
+            file name.
+
+    Raises:
+        ValueError: If the number of arguments is incorrect.
+        FileNotFoundError: If any of the specified files do not exist.
+    """
+    if len(args) != 3:  # check input arguments consistency
+        raise ValueError("Too many/few input arguments for annotation script")
+    offtargets_fname = args[0]  # offtargets table report
+    if not os.path.isfile(offtargets_fname):
+        raise FileNotFoundError(f"Cannot find off-targets file {offtargets_fname}")
+    annotation_fname = args[1]
+    if os.path.basename(annotation_fname) != "vuoto.txt" and not os.path.isfile(annotation_fname):
+        raise FileNotFoundError(
+            f"Cannot find annotation files {annotation_fname}"
+        )
+    offtargets_out_fname = args[2]  # annotated offtargets table report
+    assert offtargets_out_fname
+    return offtargets_fname, annotation_fname, offtargets_out_fname
+
+
+def load_annotation_bed(annotation_fname: str) -> TabixFile:
+    """Loads a BED annotation file and ensures a Tabix index is present.
+
+    Checks for the existence of a Tabix index for the given annotation BED file 
+    and creates one if necessary. Returns a TabixFile object for querying the 
+    annotation data.
+
+    Args:
+        annotation_fname: Path to the annotation BED file.
+
+    Returns:
+        TabixFile object for the annotation BED file.
+
+    Raises:
+        SamtoolsError: If the annotation BED file cannot be loaded or indexed.
+    """
+    # check that tabix index is available for all annotation bed
+    pysam.tabix_index(annotation_fname, force=True, preset="bed")
+    try:  # return tabix indexes for each annotation bed
+        return pysam.TabixFile(annotation_fname)
+    except (SamtoolsError, Exception) as e:
+        raise SamtoolsError(
+            "An error occurred while loading Annotation BED files"
+        ) from e
+
+
+def compute_target_coords(fields: List[str]) -> Tuple[str, int, int]:
+    """Computes the genomic coordinates for a target from a list of fields.
+
+    Returns the chromosome, start, and stop positions for the target based on 
+    the provided fields.
+
+    Args:
+        fields: List of string fields containing target information.
+
+    Returns:
+        Tuple containing the chromosome (str), start (int), and stop (int) 
+            positions.
+    """
+    start = int(fields[5])  # retrieve target start position
+    stop = start + len(fields[1].replace("-", ""))  # compute target stop
+    return fields[4], start, stop
+
+
+def annotate_target(chrom: str, start: int, stop: int, annotation: TabixFile) -> str:
+    """Annotates a genomic target using a Tabix-indexed annotation file.
+
+    Retrieves and returns a comma-separated list of annotation features overlapping 
+    the specified genomic region.
+
+    Args:
+        chrom: Chromosome name.
+        start: Start position of the target.
+        stop: Stop position of the target.
+        annotation: TabixFile object for annotation data.
+
+    Returns:
+        Comma-separated string of annotation features for the target.
+    """
+    target_anns = {
+        feature.split()[3]
+        for feature in annotation.fetch(chrom, start, stop)
+    }  # retrieve annotations for current target
+    return ",".join(sorted(target_anns))  # report as comma-separated list
+
+
+def annotate_offtargets(
+    offtargets_fname: str, annotation: Optional[TabixFile], offtargets_out_fname: str,
+) -> None:
+    """Annotates a genomic target using a Tabix-indexed annotation file.
+
+    Retrieves and returns a comma-separated list of annotation features overlapping 
+    the specified genomic region.
+
+    Args:
+        chrom: Chromosome name.
+        start: Start position of the target.
+        stop: Stop position of the target.
+        annotation: TabixFile object for annotation data.
+
+    Returns:
+        Comma-separated string of annotation features for the target.
+    """
+    try:
+        with open(offtargets_fname, mode="r") as infile, open(
+            offtargets_out_fname, mode="w"
+        ) as outfile:
+            header = infile.readline().strip()  # preserve header in annotated report
+            outfile.write(f"{header}\n")
+            for offtarget in infile:  # read offtargets
+                fields = offtarget.split()  # split offtarget individual fields
+                # annotate current off-target using input annotation datasets
+                fields[14] = (
+                    "n" if annotation is None else annotate_target(
+                        *compute_target_coords(fields), annotation
+                    )
+                )
+                offtarget_annotated = "\t".join(fields)
+                outfile.write(f"{offtarget_annotated}\n")  # write annotated offtarget
+    except (IOError, Exception) as e:
+        raise OSError(f"Annotation failed on off-targets in {offtargets_fname}") from e
+
+
+def main() -> None:
+    """Annotates a list of off-targets using an optional annotation dataset.
+
+    Reads off-targets from a file, annotates each entry with features from the 
+    provided annotation dataset, and writes the results to an output file.
+
+    Args:
+        offtargets_fname: Path to the input file containing off-targets.
+        annotation: TabixFile object for annotation data, or None if no annotation.
+        offtargets_out_fname: Path to the output file for annotated off-targets.
+
+    Raises:
+        OSError: If annotation fails due to file I/O or processing errors.
+    """
+    # parse input command line arguments
+    offtargets_fname, annotation, offtargets_out_fname = parse_commandline(sys.argv[1:])
+    start = time()  # track annotation time
+    empty = os.path.basename(annotation) == "vuoto.txt"
+    # load annotation bed files
+    annotation = None if empty else load_annotation_bed(annotation)
+    # annotate offtargets using input bed files
+    annotate_offtargets(offtargets_fname, annotation, offtargets_out_fname)
+    sys.stdout.write(f"Annotation completed in {time() - start:.2f}s\n")
+
+
+if __name__ == "__main__":
+    main()

PostProcess/annotation.py

@@ -31,6 +31,4 @@ else
     criteria=$sorting_criteria
 fi
 
-# python remove_contiguous_samples_cfd.py $fileIn.sorted $fileOut $thresh $chrom $position $total $true_guide $snp_info $cfd
-python remove_contiguous_samples_cfd.py $fileIn $fileOut $thresh $chrom $position $total $true_guide $snp_info $cfd $sort_criteria $criteria
-# rm $fileIn.sorted
+python remove_contiguous_samples.py $fileIn $fileOut $thresh $chrom $position $total $true_guide $snp_info $cfd $sort_criteria $criteria