smftools 0.1.1__tar.gz → 0.1.3__tar.gz

{smftools-0.1.1 → smftools-0.1.3}/.gitignore

@@ -18,6 +18,10 @@ build/
 venv/
 /environment.yml
 
+# Tests
+/tests/_test_inputs/
+/tests/_test_outputs/
+
 # OS
 .DS_Store
 .LSOverride

{smftools-0.1.1 → smftools-0.1.3}/PKG-INFO

@@ -1,8 +1,9 @@
 Metadata-Version: 2.3
 Name: smftools
-Version: 0.1.1
+Version: 0.1.3
 Summary: Single Molecule Footprinting Analysis in Python.
 Project-URL: Source, https://github.com/jkmckenna/smftools
+Project-URL: Documentation, https://smftools.readthedocs.io/
 Author: Joseph McKenna
 Maintainer-email: Joseph McKenna <jkmckenna@berkeley.edu>
 License-Expression: MIT
@@ -31,6 +32,7 @@ Requires-Dist: numpy<2,>=1.22.0
 Requires-Dist: pandas>=1.4.2
 Requires-Dist: pod5>=0.1.21
 Requires-Dist: pomegranate>1.0.0
+Requires-Dist: pyfaidx>=0.8.0
 Requires-Dist: pysam>=0.19.1
 Requires-Dist: scanpy>=1.9
 Requires-Dist: scikit-learn>=1.0.2
@@ -38,9 +40,6 @@ Requires-Dist: scipy>=1.7.3
 Requires-Dist: seaborn>=0.11
 Requires-Dist: torch>=1.9.0
 Requires-Dist: tqdm
-Provides-Extra: base-tests
-Requires-Dist: pytest; extra == 'base-tests'
-Requires-Dist: pytest-cov; extra == 'base-tests'
 Provides-Extra: docs
 Requires-Dist: ipython>=7.20; extra == 'docs'
 Requires-Dist: matplotlib!=3.6.1; extra == 'docs'
@@ -56,13 +55,16 @@ Requires-Dist: sphinx-design; extra == 'docs'
 Requires-Dist: sphinx>=7; extra == 'docs'
 Requires-Dist: sphinxcontrib-bibtex; extra == 'docs'
 Requires-Dist: sphinxext-opengraph; extra == 'docs'
+Provides-Extra: tests
+Requires-Dist: pytest; extra == 'tests'
+Requires-Dist: pytest-cov; extra == 'tests'
 Description-Content-Type: text/markdown
 
 [![PyPI](https://img.shields.io/pypi/v/smftools.svg)](https://pypi.org/project/smftools)
 [![Docs](https://readthedocs.org/projects/smftools/badge/?version=latest)](https://smftools.readthedocs.io/en/latest/?badge=latest)
 
 # smftools
-A Python tool for processing raw sequencing data derived from single molecule footprinting experiments into [anndata](https://anndata.readthedocs.io/en/latest/) objects. Additional functionality for preprocessing, analysis, and visualization. Data structures are compatible with analyses developed within the [scverse](https://github.com/scverse) project, including [scanpy](https://github.com/scverse/scanpy) and [scvi-tools](https://github.com/scverse/scvi-tools).
+A Python tool for processing raw sequencing data derived from single molecule footprinting experiments into [anndata](https://anndata.readthedocs.io/en/latest/) objects. Additional functionality for preprocessing, analysis, and visualization.
 
 ## Philosophy
 While most genomic data structures handle low-coverage data (<100X) along large references, smftools prioritizes high-coverage data (scalable to at least 1 million X coverage) of a few genomic loci at a time. This enables efficient data storage, rapid data operations, hierarchical metadata handling, seamless integration with various machine-learning packages, and ease of visualization. Furthermore, functionality is modularized, enabling analysis sessions to be saved, reloaded, and easily shared with collaborators. Analyses are centered around the [anndata](https://anndata.readthedocs.io/en/latest/) object, and are heavily inspired by the work conducted within the single-cell genomics community.
@@ -73,10 +75,14 @@ The following CLI tools need to be installed and configured before using the inf
 2) [Samtools](https://github.com/samtools/samtools) -> For working with SAM/BAM files
 3) [Minimap2](https://github.com/lh3/minimap2) -> The aligner used by Dorado
 4) [Modkit](https://github.com/nanoporetech/modkit) -> Extracting summary statistics and read level methylation calls from modified BAM files
+5) [Bedtools](https://github.com/arq5x/bedtools2) -> For generating Bedgraphs from BAM alignment files.
+6) [BedGraphToBigWig](https://genome.ucsc.edu/goldenPath/help/bigWig.html) -> For converting BedGraphs to BigWig files for IGV sessions.
 
 ## Modules
-- Informatics: Processes raw SMF data coming from Nanopore POD5 files, BAM files, or FASTQ files and organizes it into an AnnData object.
-- Preprocessing: Filters the AnnData object on read length, total methylation, and a variety of QC metrics.
+### Informatics: Processes raw Nanopore/Illumina data from SMF experiments into an AnnData object.
+![](docs/source/_static/smftools_informatics_diagram.png)
+### Preprocessing: Appends QC metrics to the AnnData object and perfroms filtering.
+![](docs/source/_static/smftools_preprocessing_diagram.png)
 - Tools: Appends various analyses to the AnnData object.
 - Plotting: Visualization of analyses stored within the AnnData object.
 

{smftools-0.1.1 → smftools-0.1.3}/README.md

@@ -2,7 +2,7 @@
 [![Docs](https://readthedocs.org/projects/smftools/badge/?version=latest)](https://smftools.readthedocs.io/en/latest/?badge=latest)
 
 # smftools
-A Python tool for processing raw sequencing data derived from single molecule footprinting experiments into [anndata](https://anndata.readthedocs.io/en/latest/) objects. Additional functionality for preprocessing, analysis, and visualization. Data structures are compatible with analyses developed within the [scverse](https://github.com/scverse) project, including [scanpy](https://github.com/scverse/scanpy) and [scvi-tools](https://github.com/scverse/scvi-tools).
+A Python tool for processing raw sequencing data derived from single molecule footprinting experiments into [anndata](https://anndata.readthedocs.io/en/latest/) objects. Additional functionality for preprocessing, analysis, and visualization.
 
 ## Philosophy
 While most genomic data structures handle low-coverage data (<100X) along large references, smftools prioritizes high-coverage data (scalable to at least 1 million X coverage) of a few genomic loci at a time. This enables efficient data storage, rapid data operations, hierarchical metadata handling, seamless integration with various machine-learning packages, and ease of visualization. Furthermore, functionality is modularized, enabling analysis sessions to be saved, reloaded, and easily shared with collaborators. Analyses are centered around the [anndata](https://anndata.readthedocs.io/en/latest/) object, and are heavily inspired by the work conducted within the single-cell genomics community.
@@ -13,10 +13,14 @@ The following CLI tools need to be installed and configured before using the inf
 2) [Samtools](https://github.com/samtools/samtools) -> For working with SAM/BAM files
 3) [Minimap2](https://github.com/lh3/minimap2) -> The aligner used by Dorado
 4) [Modkit](https://github.com/nanoporetech/modkit) -> Extracting summary statistics and read level methylation calls from modified BAM files
+5) [Bedtools](https://github.com/arq5x/bedtools2) -> For generating Bedgraphs from BAM alignment files.
+6) [BedGraphToBigWig](https://genome.ucsc.edu/goldenPath/help/bigWig.html) -> For converting BedGraphs to BigWig files for IGV sessions.
 
 ## Modules
-- Informatics: Processes raw SMF data coming from Nanopore POD5 files, BAM files, or FASTQ files and organizes it into an AnnData object.
-- Preprocessing: Filters the AnnData object on read length, total methylation, and a variety of QC metrics.
+### Informatics: Processes raw Nanopore/Illumina data from SMF experiments into an AnnData object.
+![](docs/source/_static/smftools_informatics_diagram.png)
+### Preprocessing: Appends QC metrics to the AnnData object and perfroms filtering.
+![](docs/source/_static/smftools_preprocessing_diagram.png)
 - Tools: Appends various analyses to the AnnData object.
 - Plotting: Visualization of analyses stored within the AnnData object.
 

smftools-0.1.3/docs/source/api/index.md

@@ -0,0 +1,26 @@
+# API
+
+Import smftools as:
+
+```
+import smftools as smf
+```
+
+```{toctree}
+:maxdepth: 2
+
+informatics
+preprocessing
+tools
+datasets
+```
+
+## Informatics module diagram
+```{image} ../_static/smftools_informatics_diagram.png
+:width: 800px
+```
+
+## Preprocessing module diagram
+```{image} ../_static/smftools_preprocessing_diagram.png
+:width: 800px
+```

smftools-0.1.3/docs/source/api/informatics.md

@@ -0,0 +1,27 @@
+## Informatics: `inform`
+
+## Informatics module diagram
+```{image} ../_static/smftools_informatics_diagram.png
+:width: 1000px
+```
+
+```{eval-rst}
+.. module:: smftools.inform
+```
+
+```{eval-rst}
+.. currentmodule:: smftools
+```
+
+Processes raw sequencing data to load an adata object.
+
+
+### Diagram of final steps of Direct SMF workflow
+```{image} ../_static/modkit_extract_to_adata.png
+:width: 1000px
+```
+
+### Diagram of final steps of Conversion SMF workflow
+```{image} ../_static/converted_BAM_to_adata.png
+:width: 1000px
+```

{smftools-0.1.1 → smftools-0.1.3}/docs/source/api/preprocessing.md

@@ -1,5 +1,10 @@
 ## Preprocessing: `pp`
 
+## Preprocessing module diagram
+```{image} ../_static/smftools_preprocessing_diagram.png
+:width: 1000px
+```
+
 ```{eval-rst}
 .. module:: smftools.pp
 ```

smftools-0.1.3/docs/source/basic_usage.md

@@ -0,0 +1,75 @@
+# Basic Usage
+
+Import SmfTools:
+
+```
+import smftools as smf
+```
+
+## Informatics Module Usage
+
+Many use cases for smftools begin here. For most users, the call below will be sufficient to convert any raw SMF dataset to an AnnData object:
+
+```
+config_path = "/Path_to_experiment_config.csv"
+smf.inform.load_adata(config_path)
+```
+
+## Loading AnnData objects created by the informatics module
+
+After creating an AnnData object holding your experiment's SMF data, you can load the AnnData object as so:
+
+```
+import anndata as ad
+input_adata = "/Path_to_experiment_AnnData.h5ad.gz"
+adata = ad.read_h5ad(input_file)
+adata.obs_names_make_unique()
+```
+
+If you don't have an AnnData object yet, but want to play with the downstream Preprocessing, Tools, and Plotting modules, you can load a pre-loaded SMF dataset.
+
+Currently, you can do this with our lab's in vitro dCas9 binding kinetics dataset generated from a Hia5 SMF dataset generated with direct m6A high accuracy basecalls:
+
+```
+adata = smf.datasets.dCas9_kinetics()
+adata.obs_names_make_unique()
+```
+
+Alternatively, you can do this with our lab's M.CviPI SMF test data in F1-hybrid natural killer cells generated by NEB EMseq conversion followed by canonical basecalling:
+
+```
+adata = smf.datasets.Kissiov_and_McKenna_2025()
+adata.obs_names_make_unique()
+```
+
+## Writing out AnnData objects to save analysis progress
+
+After preprocessing and downstream analysis of the AnnData object, you can save the AnnData object at any step as so:
+
+```
+import anndata as ad
+import os
+
+output_dir = '/Path_to_output_directory'
+output_adata = 'analyzed_adata.h5ad.gz'
+final_output = os.path.join(output_dir, output_adata)
+adata.write_h5ad(final_output, compression='gzip')
+```
+
+
+## Troubleshooting
+For more advanced usage and help troubleshooting, the API and tutorials for each of the modules is still being developed.
+However, you can currently learn about the functions contained within the module by calling:
+
+```
+smf.inform.__all__
+```
+
+This lists the functions within any given module. If you want to see the associated docstring for a given function, here is an example:
+
+```
+print(smf.inform.load_adata.__doc__)
+```
+
+These docstrings will provide a brief description of the function and also tell you the input parameters and what the function returns.
+In some cases, usage examples will also be provided in the docstring in the form of doctests.

{smftools-0.1.1 → smftools-0.1.3}/docs/source/index.md

@@ -42,6 +42,7 @@ smftools GitHub link
 :maxdepth: 1
 
 installation
+basic_usage
 tutorials/index
 api/index
 release-notes/index

smftools-0.1.3/docs/source/installation.md

@@ -0,0 +1,60 @@
+# Installation
+
+## PyPi version
+
+Pull smftools from [PyPI](https://pypi.org/project/smftools):
+
+```shell
+pip install smftools
+```
+
+It is recommended to first create and activate a conda environment before installing smftools to ensure dependencies are managed smoothly:
+
+```shell
+conda create -n smftools
+conda activate smftools
+pip install smftools
+```
+
+Ensure that you can access dorado, samtools, modkit, bedtools, and BedGraphtoBigWig executables from the terminal in this environment. These are all necessary for the functionality within the Informatics module.
+You may need to add them to $PATH if they are not globally configured.
+For example, if you want to check if dorado is executable, simply run this in the terminal:
+
+```shell
+dorado
+```
+
+On Mac OSX, the following can be used to congigure bedtools (with brew) and BedGraphToBigWig (with wget). Change the BedGraphToBigWig link to include the correct architecture for your OS.
+
+```shell
+brew install bedtools
+wget http://hgdownload.soe.ucsc.edu/admin/exe/macOSX.x86_64/bedGraphToBigWig
+chmod +x bedGraphToBigWig
+sudo mv bedGraphToBigWig /usr/local/bin/
+```
+
+## Development Version
+
+Clone smftools from source and change into the smftools directory:
+
+```shell
+git clone https://github.com/jkmckenna/smftools.git
+cd smftools
+```
+
+A virtual environment can be created for the current version within the smftools directory:
+
+```shell
+python -m venv venv-smftools
+source venv-smftools/bin/activate
+pip install .
+```
+
+Subsequent use of the installed version of smftools can be run by changing to the smftools directory and activating the venv:
+
+```shell
+cd smftools
+source venv-smftools/bin/activate
+```
+
+You can now run smftools from the terminal, an IDE, or a notebook within the virtual environment.

{smftools-0.1.1 → smftools-0.1.3}/experiment_config.csv

@@ -1,8 +1,8 @@
 variable,value,help,options,type
 smf_modality,conversion,Modality of SMF. Can either be conversion or direct.,"conversion, direct",str 
-pod5_dir,/path_to_POD5_directory,Path to directory containing input POD5 files (If doing Nanopore SMF),,str 
-basecalled_path,/path_to_basecalled_HTS_file.bam,Path to directory containing input BAM file (if doing SMF from an already basecalled experiment). Can also be a path to a FASTQ for conversion SMF.,,str 
+input_data_path,/path_to_POD5_directory,Path to directory/file containing input sequencing data,,str 
 fasta,/path_to_fasta.fasta,Path to initial FASTA file,,str 
+fasta_regions_of_interest,/path_to_bed.bed,Path to a bed file to subsample the fasta on.,,str 
 output_directory,/outputs,Directory to act as root for all analysis outputs,,str 
 experiment_name,,An experiment name for the final h5ad file,,str 
 model,None,The dorado basecalling model to use,,str 

smftools-0.1.3/notebooks/Kissiov_and_McKenna_2025_example_notebook.ipynb

@@ -0,0 +1,85 @@
+{
+ "cells": [
+  {
+   "cell_type": "code",
+   "execution_count": null,
+   "metadata": {},
+   "outputs": [],
+   "source": [
+    "import anndata as ad\n",
+    "import pandas as pd\n",
+    "import numpy as np\n",
+    "import matplotlib.pyplot as plt\n",
+    "import smftools as smf\n",
+    "import os"
+   ]
+  },
+  {
+   "cell_type": "code",
+   "execution_count": null,
+   "metadata": {},
+   "outputs": [],
+   "source": [
+    "# Define file paths\n",
+    "adata_path = '/Path_to_input_adata.h5ad.gz'\n",
+    "output_directory = '/Path_to_output_directory'\n",
+    "output_adata = 'analyzed_adata.h5ad.gz'\n",
+    "final_output = os.path.join(output_directory, output_adata)\n",
+    "\n",
+    "# Load adata\n",
+    "adata = ad.read_h5ad(adata_path)\n",
+    "adata.obs_names_make_unique()"
+   ]
+  },
+  {
+   "cell_type": "code",
+   "execution_count": null,
+   "metadata": {},
+   "outputs": [],
+   "source": [
+    "# Define path to sample sheet and run first part of preprocessing.\n",
+    "sample_sheet_path = '/path_to_sample_sheet.csv'\n",
+    "variables = smf.pp.recipe_1_Kissiov_and_McKenna_2025(adata, sample_sheet_path, output_directory)\n",
+    "# Update global variables\n",
+    "globals().update(variables)"
+   ]
+  },
+  {
+   "cell_type": "code",
+   "execution_count": null,
+   "metadata": {},
+   "outputs": [],
+   "source": [
+    "# Filter adata based on defined read length statistics, using the plots from preprocessing part 1 to direct the input parameters here.\n",
+    "smf.pp.filter_reads_on_length(adata, filter_on_coordinates=[lower_bound, upper_bound], min_read_length=2700)"
+   ]
+  },
+  {
+   "cell_type": "code",
+   "execution_count": null,
+   "metadata": {},
+   "outputs": [],
+   "source": [
+    "# Filter adata on defined read methylation statistics\n",
+    "smf.pp.filter_converted_reads_on_methylation(adata, valid_SMF_site_threshold=0.8, min_SMF_threshold=0.025)"
+   ]
+  },
+  {
+   "cell_type": "code",
+   "execution_count": null,
+   "metadata": {},
+   "outputs": [],
+   "source": [
+    "# Run second part of preprocessing\n",
+    "duplicates = smf.pp.recipe_2_Kissiov_and_McKenna_2025(adata, output_directory, binary_layers)"
+   ]
+  }
+ ],
+ "metadata": {
+  "language_info": {
+   "name": "python"
+  }
+ },
+ "nbformat": 4,
+ "nbformat_minor": 2
+}

smftools-0.1.3/notebooks/Kissiov_and_McKenna_2025_sample_sheet.csv

@@ -0,0 +1,11 @@
+Sample,Sample_names,MTase,Time (sec),Group
+0,,Hia5,0,0
+1,,Hia5,15,0
+2,,Hia5,30,0
+3,,Hia5,120,0
+4,,Hia5,300,0
+5,,Hia5,0,1
+6,,Hia5,15,1
+7,,Hia5,30,1
+8,,Hia5,120,1
+9,,Hia5,300,1

{smftools-0.1.1 → smftools-0.1.3}/pyproject.toml

@@ -48,6 +48,7 @@ dependencies = [
     "pandas>=1.4.2",
     "pod5>=0.1.21",
     "pomegranate>1.0.0",
+    "pyfaidx>=0.8.0",
     "pysam>=0.19.1",
     "scanpy>=1.9",
     "scikit-learn>=1.0.2",
@@ -60,9 +61,10 @@ dynamic = ["version"]
 
 [project.urls]
 Source = "https://github.com/jkmckenna/smftools"
+Documentation = "https://smftools.readthedocs.io/"
 
 [project.optional-dependencies]
-base_tests = [
+tests = [
     "pytest",
     "pytest-cov"
 ]
@@ -91,16 +93,16 @@ packages = ["src/smftools"]
 path = "src/smftools/_version.py"
 
 [tool.pytest.ini_options]
+addopts = [
+    "--import-mode=importlib",
+    "--strict-markers",
+    "--doctest-modules",
+    "--pyargs",
+]
 testpaths = ["tests"]
 pythonpath = ["src"]
 xfail_strict = true
-markers = [
-    "internet: mark tests that requires internet access",
-    "optional: mark optional tests",
-    "private: mark tests that are private",
-]
 
 [tool.coverage.run]
-branch = true
 source = ["smftools"]
 omit = ["tests/*"]

{smftools-0.1.1 → smftools-0.1.3}/requirements.txt

@@ -7,6 +7,7 @@ numpy>=1.22.0,<2
 pandas>=1.4.2
 pomegranate>1.0.0
 pod5>=0.1.21
+pyfaidx>=0.8.0
 pysam>=0.19.1
 scanpy>=1.9
 scikit-learn>=1.0.2

smftools-0.1.3/sample_sheet.csv

@@ -0,0 +1,11 @@
+Sample,Sample_names,MTase,Time (sec),Group
+0,,Hia5,0,0
+1,,Hia5,15,0
+2,,Hia5,30,0
+3,,Hia5,120,0
+4,,Hia5,300,0
+5,,Hia5,0,1
+6,,Hia5,15,1
+7,,Hia5,30,1
+8,,Hia5,120,1
+9,,Hia5,300,1

{smftools-0.1.1 → smftools-0.1.3}/src/smftools/_settings.py

@@ -1,4 +1,5 @@
 from pathlib import Path
+from typing import Union
 
 class SMFConfig:
     """\
@@ -8,9 +9,9 @@ class SMFConfig:
     def __init__(
         self,
         *,
-        datasetdir: Path | str = "./datasets/"
+        datasetdir: Union[Path, str] = "./datasets/"
     ):
-         self._datasetdir = Path(datasetdir) if isinstance(datasetdir, str) else datasetdir
+        self._datasetdir = Path(datasetdir) if isinstance(datasetdir, str) else datasetdir
 
     @property
     def datasetdir(self) -> Path:

smftools-0.1.3/src/smftools/_version.py

@@ -0,0 +1 @@
+__version__ = "0.1.3"

smftools-0.1.3/src/smftools/datasets/F1_sample_sheet.csv

@@ -0,0 +1,5 @@
+Sample,Sample_names,MTase,Time (min),Notes
+barcode0001_sorted,Neither,M.CviPI,7.5,Cultured in IL2
+barcode0002_sorted,BALBC,M.CviPI,7.5,Cultured in IL2
+barcode0003_sorted,B6,M.CviPI,7.5,Cultured in IL2
+barcode0004_sorted,Both,M.CviPI,7.5,Cultured in IL2

{smftools-0.1.1 → smftools-0.1.3}/src/smftools/datasets/datasets.py

@@ -1,10 +1,9 @@
 ## datasets
 
-def import_deps():
+def import_HERE():
     """
-    
+    Imports HERE for loading datasets
     """
-    import anndata as ad
     from pathlib import Path
     from .._settings import settings
     HERE = Path(__file__).parent
@@ -12,16 +11,18 @@ def import_deps():
 
 def dCas9_kinetics():
     """
-    
+    in vitro Hia5 dCas9 kinetics SMF dataset. Nanopore HAC m6A modcalls.
     """
-    HERE = import_deps()
+    import anndata as ad
+    HERE = import_HERE()
     filepath = HERE / "dCas9_m6A_invitro_kinetics.h5ad.gz"
     return ad.read_h5ad(filepath)
 
 def Kissiov_and_McKenna_2025():
     """
-    
+    F1 Hybrid M.CviPI natural killer cell SMF. Nanopore canonical calls of NEB EMseq converted SMF gDNA.
     """
-    HERE = import_deps()
+    import anndata as ad
+    HERE = import_HERE()
     filepath = HERE / "F1_hybrid_NKG2A_enhander_promoter_GpC_conversion_SMF.h5ad.gz"
     return ad.read_h5ad(filepath)

smftools-0.1.3/src/smftools/informatics/__init__.py

@@ -0,0 +1,14 @@
+from . import helpers
+from .load_adata import load_adata
+from .subsample_fasta_from_bed import subsample_fasta_from_bed
+from .subsample_pod5 import subsample_pod5
+from .fast5_to_pod5 import fast5_to_pod5
+
+
+__all__ = [
+    "load_adata",
+    "subsample_fasta_from_bed",
+    "subsample_pod5",
+    "fast5_to_pod5",
+    "helpers"
+]

{smftools-0.1.1/src/smftools/informatics → smftools-0.1.3/src/smftools/informatics/archived}/bam_conversion.py

@@ -18,7 +18,7 @@ def bam_conversion(fasta, output_directory, conversion_types, strands, basecalle
     Returns:
         None
     """
-    from .helpers import align_and_sort_BAM, converted_BAM_to_adata, generate_converted_FASTA, split_and_index_BAM
+    from .helpers import align_and_sort_BAM, converted_BAM_to_adata, generate_converted_FASTA, split_and_index_BAM, make_dirs
     import os
     input_basecalled_basename = os.path.basename(basecalled_path)
     bam_basename = input_basecalled_basename.split(".")[0]
@@ -32,16 +32,28 @@ def bam_conversion(fasta, output_directory, conversion_types, strands, basecalle
     fasta_basename = os.path.basename(fasta)
     converted_FASTA_basename = fasta_basename.split('.fa')[0]+'_converted.fasta'
     converted_FASTA = os.path.join(output_directory, converted_FASTA_basename)
-    if os.path.exists(converted_FASTA):
+    if 'converted.fa' in fasta:
+        print(fasta + ' is already converted. Using existing converted FASTA.')
+        converted_FASTA = fasta
+    elif os.path.exists(converted_FASTA):
         print(converted_FASTA + ' already exists. Using existing converted FASTA.')
     else:
         generate_converted_FASTA(fasta, conversion_types, strands, converted_FASTA)
 
     # 2) Align the basecalled file to the converted reference FASTA and sort the bam on positional coordinates. Also make an index and a bed file of mapped reads
-    align_and_sort_BAM(converted_FASTA, basecalled_path, bam_suffix, output_directory)
+    aligned_output = aligned_BAM + bam_suffix
+    sorted_output = aligned_sorted_BAM + bam_suffix
+    if os.path.exists(aligned_output) and os.path.exists(sorted_output):
+        print(sorted_output + ' already exists. Using existing aligned/sorted BAM.')
+    else:
+        align_and_sort_BAM(converted_FASTA, basecalled_path, bam_suffix, output_directory)
 
     ### 3) Split the aligned and sorted BAM files by barcode (BC Tag) into the split_BAM directory###
-    split_and_index_BAM(aligned_sorted_BAM, split_dir, bam_suffix)
+    if os.path.isdir(split_dir):
+        print(split_dir + ' already exists. Using existing aligned/sorted/split BAMs.')
+    else:
+        make_dirs([split_dir])
+        split_and_index_BAM(aligned_sorted_BAM, split_dir, bam_suffix, output_directory)
 
     # 4) Take the converted BAM and load it into an adata object. 
     converted_BAM_to_adata(converted_FASTA, split_dir, mapping_threshold, experiment_name, conversion_types, bam_suffix)