barcadia 3.2.0__tar.gz

barcadia-3.2.0/LICENSE

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barcadia-3.2.0/PKG-INFO

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+Metadata-Version: 2.4
+Name: barcadia
+Version: 3.2.0
+Summary: A high-performance, memory-efficient toolkit for fast generation and validation of large-scale NGS barcodes
+Author: Danting Jiang
+License-Expression: Apache-2.0
+Project-URL: Repository, https://github.com/danting/barcadia
+Project-URL: PyPI, https://pypi.org/project/barcadia/
+Keywords: bioinformatics,NGS,barcodes
+Classifier: Development Status :: 5 - Production/Stable
+Classifier: Intended Audience :: Science/Research
+Classifier: Topic :: Scientific/Engineering :: Bio-Informatics
+Classifier: Environment :: Console
+Classifier: Programming Language :: Python :: 3 :: Only
+Classifier: Programming Language :: Python :: 3.12
+Classifier: Operating System :: POSIX :: Linux
+Classifier: Operating System :: MacOS :: MacOS X
+Classifier: Natural Language :: English
+Requires-Python: >=3.12
+Description-Content-Type: text/markdown
+License-File: LICENSE
+Requires-Dist: numpy==2.2.6
+Requires-Dist: numba==0.61.2
+Requires-Dist: llvmlite==0.44.0
+Requires-Dist: psutil==7.0.0
+Dynamic: license-file
+
+# Barcadia (v3.2.0)  
+*Best-in-class toolkit for large-scale NGS barcode generation and validation* 
+
+![version](https://img.shields.io/badge/version-3.2.0-blue)  
+![license](https://img.shields.io/badge/license-Apache%202.0-brightgreen)  
+![platform](https://img.shields.io/badge/platform-Linux%20%7C%20macOS-lightgrey) 
+
+---
+
+Barcadia is a **fast, memory-efficient, open-source toolkit** for generating and validating massive DNA barcode libraries for next-generation sequencing (NGS) applications. Designed for speed and scalability, it **outperforms existing tools** for both small- and large-scale operations.
+
+- **High performance & scalability** – Generates 100K barcodes in under 3 minutes and 1M in 40 minutes, largely outperforming existing tools limited to ~100K barcodes and requiring hours of compute. 
+- **Memory & compute efficient** – Runs on standard laptops with minimal resources (under 1 GB RAM used for generating 1M barcodes); no multi-core processing required.  
+- **Extended functionality** – Supports paired barcode generation for dual-indexing, extension from seed lists, validation of existing barcode sets, and estimation of library size limits — features not found in other tools.  
+
+Barcadia makes it easy to design small or large NGS barcode sets that are optimized for **robust performance in high-throughput sequencing workflows**.   
+
+## Table of Contents
+- [Background](#background)
+  - [Problem Statement](#problem-statement)
+  - [Existing Methods and their Limitations](#existing-methods-and-their-limitations)
+  - [This Toolkit and its Advantages](#this-toolkit-and-its-advantages)
+- [Default Filter Parameters](#default-filter-parameters)
+- [Theoretical Bounds](#theoretical-bounds)
+- [Benchmarking Highlights](#benchmarking-highlights)
+- [Installation](#installation)
+  - [Setup](#setup)
+  - [Requirements](#requirements)
+- [Quick Start](#quick-start)
+- [Package Overview](#package-overview)
+  - [Project Structure](#project-structure)
+  - [Main Commands](#main-commands)
+  - [Shared Modules](#shared-modules)
+  - [Utility Scripts](#utility-scripts)
+- [Citation](#citation)
+- [Changelog](#changelog)
+
+## Background
+
+### Problem Statement
+
+**Next-generation sequencing (NGS) is a high-throughput method that enables millions of DNA fragments to be sequenced in parallel, serving as the core technology for decoding genomes across all living organisms**. In this process, researchers use DNA barcodes to uniquely label and track individual biomolecules. Common examples include multiplex sample indexes and unique molecular identifiers (UMIs). 
+
+The practical utility of a DNA barcode library depends on controlling key features: **GC content** (the percentage of G and C nucleotides), **homopolymer repeats** (the length of the longest stretch of identical nucleotides), and **edit distance** (the measure of dissimilarity among sequences). GC content and homopolymer repeats are **within-sequence** criteria that ensure molecular stability during sequencing and are computationally inexpensive to evaluate. **In contrast, edit distance is a between-sequence constraint that provides error tolerance during analysis, but is computationally demanding to assess for large datasets**.
+
+### Existing Methods and their Limitations
+
+For a set of n sequences, the number of pairwise distance comparisons grows quadratically:
+
+$$\binom{n}{2} = \frac{n(n-1)}{2}$$
+
+**This makes brute-force approaches computationally prohibitive for large datasets**. Apart from the approach described by [Lyons et al. (2017)](https://www.nature.com/articles/s41598-017-12825-2), existing tools are not built to accommodate large-scale barcode library design (≥100K sequences). Lyons et al. circumvented computational limitations using a probabilistic Markov chain model; however, their resulting barcode sets are no longer accessible (invalid URLs in the paper), and the underlying code was not released (likely due to proprietary restrictions). 
+
+### This Toolkit and its Advantages
+
+**Here, I introduce Barcadia, a toolkit for efficient large-scale NGS barcode generation that integrates modern computational optimization with novel distance-constrained algorithms I developed to deliver best-in-class scalability and speed**. The software is openly available to promote reproducibility and is designed to run efficiently on minimal computing resources (e.g., standard laptops), ensuring broad accessibility.
+
+In comparison with [TagGD](https://doi.org/10.1371/journal.pone.0057521)—the only other open-source software reported to support barcode generation at the scale of up to 100,000 sequences—Barcadia generated 20,000 18-bp barcodes in just **1 minute** using only 100 MB of RAM on a comparable 8-core laptop, as opposed to the **5 minutes** highlighted in the TagGD abstract. For 100,000 18-bp barcodes, Barcadia completed the process in **under 5 minutes** with 180 MB of RAM, which is significantly faster than the **1.5 hours** reported in Table 1 of the TagGD paper. 
+
+**Notably, Barcadia can handle the generation of million-scale barcodes within reasonable time (i.e., 40 minutes) on minimal compute setups (e.g., standard laptops; <1 GB RAM and no multi-core processing required), far exceeding the capacity of TagGD and other existing tools**. More detailed benchmarking results are presented in a later section of this document. 
+
+**Additionally, it offers unique features not found in other tools**: paired barcode generation for dual-indexing applications, extension from user-provided seed sequences, and a comprehensive validation script for assessing the quality of existing barcode sets (which together enable the estimation of realistic bounds for achievable library sizes under all specified constraints).
+
+## Default Filter Parameters
+
+Barcadia uses carefully chosen default parameters that **optimize synthetic stability and sequencing reliability** in NGS workflows. Users can configure these based on their specific needs.
+
+### GC Content: 40-60%
+- **Rationale**: Sequences with extreme GC content (very low or very high) can form secondary structures and exhibit poor amplification efficiency during PCR
+- **Impact**: Moderate GC content ensures reliable sequencing performance across different platforms
+
+### Maximum Homopolymer Length: 2
+- **Rationale**: Long homopolymer runs (e.g., AAAA, TTTT) cause sequencing errors on most NGS platforms, particularly Illumina and Ion Torrent
+- **Impact**: Short homopolymers prevent read quality degradation and reduce sequencing artifacts
+
+### Minimum Hamming Distance: 3
+- **Rationale**: Distance ≥3 allows correction of single-base sequencing errors while maintaining sequence distinguishability
+- **Impact**: Provides error tolerance for typical NGS error rates (~0.1-1% per base)
+
+## Theoretical Bounds
+
+Leveraging established results from coding theory, **one can calculate lower and upper bounds on the number of valid barcode sequences under specified edit distance constraints**, assuming equal-length barcodes and applying the Hamming distance metric, which counts the base-pair mismatches.
+
+### Gilbert-Varshamov Bound (Lower Bound)
+
+The Gilbert-Varshamov bound provides a lower bound guarantee that a code of at least this size exists. For DNA sequences (i.e., alphabet of 4 characters: A, T, G, C), it states:
+
+$$M \geq \frac{4^n}{V(n,d-1)}$$
+
+where `V(n,d-1)` is the volume of a Hamming sphere of radius `d-1`:
+
+$$V(n,d-1) = \sum_{i=0}^{d-1} \binom{n}{i} \cdot 3^i$$
+
+### Hamming Bound (Upper Bound)
+
+The Hamming bound provides the theoretical maximum number of codewords that can exist for a given sequence length and minimum distance constraint. For DNA sequences of length `n` with minimum Hamming distance `d`, the bound is:
+
+$$M \leq \frac{4^n}{V(n,t)}$$
+
+where `t = ⌊(d-1)/2⌋` and `V(n,t)` is the volume of a Hamming sphere of radius `t`:
+
+$$V(n,t) = \sum_{i=0}^{t} \binom{n}{i} \cdot 3^i$$
+
+**As the sequence space is exhausted in search of valid barcodes, approaching the theoretical upper bound causes the search to slow down progressively**. In particular, a significant—often exponential—slowdown can be expected once the target size surpasses the Gilbert-Varshamov (GV) bound.
+
+For typical barcode applications (6-16 bp, as longer sequences may introduce molecular complexity) using the default minimum distance of 3, the theoretical bounds are summarized below:
+
+<div align="center">
+
+| Length (bp) | GV Bound (Lower) | Hamming Bound (Upper) |
+|:-----------:|:----------------:|:---------------------:|
+| 6           | 26               | 215                   |
+| 7           | 77               | 744                   |
+| 8           | 236              | 2.6K                  |
+| 9           | 744              | 9.4K                  |
+| 10          | 2.4K             | 34K                   |
+| 11          | 7.9K             | 123K                  |
+| 12          | 27K              | 453K                  |
+| 13          | 90K              | 1.7M                  |
+| 14          | 311K             | 6.2M                  |
+| 15          | 1.1M             | 23M                   |
+| 16          | 3.8M             | 88M                   |
+
+</div>
+
+When no seeds are provided—or when all provided seeds are the same length as the new barcodes to generate—**Barcadia will automatically calculate and display the theoretical bounds**. For the specified barcode length and minimum distance, it issues a warning if the requested count exceeds the GV lower bound (performance slowdown expected), or raises an error if the request is beyond the Hamming upper bound (not achievable).
+
+However, these theoretical bounds only capture the minimum distance constraints. **In practice, within-sequence biological filters (GC content and homopolymer restrictions) will further reduce the achievable library size**. Notably, Barcadia can be used to estimate more realistic bounds that account for all three constraints, following the procedures outlined below:
+
+1. **Generate sequences with only distance constraints** (no biological filters):
+   ```bash
+   barcadia generate --count <large_number> --length <your_length> --gc-min 0 --gc-max 1 --homopolymer-max <your_length> --output-dir <your_output_dir>
+   ```
+   *Note: Use a count near the GV bound for your parameters, and set homopolymer-max to your barcode length. This overrides the default biological filters (gc-min=0.4, gc-max=0.6, homopolymer-max=2).*
+
+2. **Check how many pass biological filters** (skipping distance validation):
+   ```bash
+   barcadia validate --input <your_output_dir>/barcodes.txt --skip-distance --output-dir <your_output_dir>
+   ```
+   *Note: By default, this validation step uses the biological filters (gc-min=0.4, gc-max=0.6, homopolymer-max=2) to check how many sequences pass. You can also set custom filter values if desired.*
+
+3. **Calculate empirical bounds**: Multiply the theoretical bounds by the observed pass rate from step 2 to get realistic achievable library sizes under all constraints.
+
+## Benchmarking Highlights
+
+Below are performance benchmarks for **barcode generation** on a MacBook Pro 2019 (8-core, 16GB RAM) using default parameters with target sizes near the Gilbert-Varshamov bound (generating equal-length libraries from scratch without seed sequences provided):
+
+<div align="center">
+
+| Length (bp) | Library Size | Time (Peak Memory)    |
+|:-----------:|:------------:|:---------------------:|
+| 6           | 10           | 0.2 seconds (82 MB)   |
+| 8           | 100          | 0.2 seconds (82 MB)   |
+| 10          | 1,000        | 0.6 seconds (82 MB)   |
+| 12          | 10,000       | 12.3 seconds (88 MB)  |
+| 14          | 100,000      | 2.8 minutes (160 MB)  |
+| 16          | 1,000,000    | 40 minutes (950 MB)   |
+
+</div>
+
+Below are performance benchmarks for **barcode validation** on a MacBook Pro 2019 (8-core, 16GB RAM) using default parameters with pool sizes near the Gilbert-Varshamov bound (validating barcodes that pass all the within-sequence and between-sequence filters):
+
+<div align="center">
+
+| Length (bp) | Library Size | Time (Peak Memory)    |
+|:-----------:|:------------:|:---------------------:|
+| 6           | 10           | 0.2 seconds (81 MB)   |
+| 8           | 100          | 0.2 seconds (81 MB)   |
+| 10          | 1,000        | 0.5 seconds (81 MB)   |
+| 12          | 10,000       | 6.1 seconds (88 MB)   |
+| 14          | 100,000      | 1.5 minutes (160 MB)  |
+| 16          | 1,000,000    | 20 minutes (943 MB)   |
+
+</div>
+
+## Installation
+
+### Setup
+
+#### Method 1: Install from PyPI (Recommended)
+```bash
+pip install barcadia
+```
+
+#### Method 2: Install from Source (Development)
+```bash
+git clone https://github.com/danting/barcadia.git
+cd barcadia
+pip install -e .
+```
+
+### Requirements
+- Python 3.12+
+- Dependencies (automatically installed):
+  - numpy==2.2.6
+  - numba==0.61.2 (for JIT compilation acceleration)
+  - llvmlite==0.44.0
+  - psutil==7.0.0
+
+This installs Barcadia as a Python package with the `barcadia` command-line tool.
+
+## Quick Start
+
+```bash
+# Generate 1000 barcodes of length 12
+barcadia generate --count 1000 --length 12
+
+# Validate existing barcodes
+barcadia validate --input test/barcodes.txt
+
+# Check version
+barcadia --version
+
+# Get help for any command
+barcadia --help
+barcadia generate --help
+barcadia validate --help
+```
+
+## Package Overview
+
+### Project Structure
+
+```
+barcadia/                                       # Main package
+├── __init__.py                                 # Public API
+├── cli.py                                      # Command-line interface
+├── generate_barcodes.py                        # Barcode generation
+├── validate_barcodes.py                        # Barcode validation
+├── config_utils.py                             # Configuration utilities
+├── filter_utils.py                             # Core filtering utilities
+└── tools/                                      # Utility scripts
+    ├── generate_random_sequences.py            # Random sequence generator
+    └── memory_benchmark.py                     # Performance monitoring
+```
+
+### Main Commands
+
+#### 1. `barcadia generate` - Barcode Generation
+
+**Purpose**: Generate high-performance NGS barcodes efficiently using a novel iterative growth algorithm (extension from seeds and paired mode supported).
+
+**Algorithm Overview**:
+1. Load seed sequence files as existing pool and report length distribution (will generate from scratch if no seeds are provided)
+2. Generate a batch of unique random sequence candidates passing biological filters (GC content, homopolymer repeats)
+3. **Two-step distance filtering with adaptive method selection (neighbor enumeration or pairwise)**:
+   - Filter candidates against existing pool (if pairwise, parallelized for large sets when multiple CPUs available)
+   - Filter remaining candidates against each other within the current batch (always sequential)
+4. Add verified batch of passing candidates to existing pool and repeat until target count reached
+
+**Key Features**:
+- Uses Hamming distance for generation when no seeds are provided (always equal-length)
+- Supports **extension from seed sequence** file(s) (when `--seeds` is specified) and can accommodate: 
+  - Multiple seed files of equal or variable lengths (concatenated automatically as seed pool)
+  - Differences in lengths between seed pool and newly-generated sequences (NOTE: new sequences are always the same length as specified by `--length`)
+  - Uses Hamming distance for equal-length comparisons, Levenshtein distance for mixed lengths (compared to seed pool)
+  - Incompatible with `--paired` mode
+- Supports **paired barcode generation** (when `--paired` flag is on) by generating 2x the target count and splitting output into 2 files with suffixes `_paired1.txt` and `_paired2.txt`
+- Supports paired barcode generation with **paired seed** files (when`--paired-seed1` and `--paired-seed2` are specified), but is more restrictive than `--seeds` in unpaired mode:
+  - Only accepts one file each and both must be specified
+  - Both files must have the same number of sequences with all sequences being the same length within and across both files
+  - Similar to `--seeds`, can accommodate differences in lengths between seed pool and newly-generated sequences (Hamming for equal/Levenshtein for mixed)
+  - Incompatible with `--seeds`
+- **Adaptive method selection for distance filtering**:
+  - Small barcode sets (<10K sequences including seeds): Pairwise distance checking (fast for small sets)
+  - Large mixed-length (within seeds and/or between seeds and new barcodes): Pairwise distance checking (neighbor enumeration requires complex Levenshtein handling)
+  - Large equal-length (no seeds or everything equal-length): Choose between pairwise and neighbor enumeration based on min_distance
+    * Pairwise distance checking: when min_distance > 4 (large number of neighbors to check)
+    * Neighbor enumeration: when min_distance <= 4 (limited number of neighbors to check)
+
+**Basic Usage**:
+```bash
+# Generate 1000 barcodes of length 12 from scratch (no seeds)
+barcadia generate --count 1000 --length 12
+
+# Build from a seed sequence file
+barcadia generate --count 1000 --length 12 --seeds seed.txt
+
+# Build from multiple seed sequence files (concatenated automatically)
+barcadia generate --count 1000 --length 12 --seeds seed_file1.txt seed_file2.txt
+
+# Generate paired barcodes from scratch (no seeds)
+barcadia generate --count 1000 --length 12 --paired
+
+# Generate paired barcodes with paired seed files
+barcadia generate --count 1000 --length 12 --paired --paired-seed1 seed_paired1.txt --paired-seed2 seed_paired2.txt
+```
+
+**Required Arguments**:
+- `--count`: Number of barcodes or barcode pairs to generate
+- `--length`: Length of barcodes or barcode pairs to generate
+
+**Optional Arguments**:
+- `--gc-min`: Minimum GC content (default: 0.4)
+- `--gc-max`: Maximum GC content (default: 0.6)
+- `--homopolymer-max`: Maximum homopolymer repeat length (default: 2)
+- `--min-distance`: Minimum edit distance between sequences (default: 3)
+- `--cpus`: Number of CPU cores to use during the parallel filtering step (default: all available)
+- `--seeds`: Seed sequence files (any number of files, one sequence per line as .txt; if not provided, will generate from scratch; incompatible with --paired mode; default: None)
+- `--paired`: Generate paired barcodes (doubles target count, splits output randomly into two equal parts; incompatible with --seeds; default: off)
+- `--paired-seed1`: Paired seed sequence file 1 (used only with --paired and --paired-seed2, only one file is accepted, all sequences must be same length and match count/length of --paired-seed2; default: None)
+- `--paired-seed2`: Paired seed sequence file 2 (used only with --paired and --paired-seed1, only one file is accepted, all sequences must be same length and match count/length of --paired-seed1; default: None)
+
+- `--output-dir`: Output directory (default: test)
+- `--output-prefix`: Output filename prefix (default: barcodes)
+
+**Output Files**:
+- `{prefix}.txt` or `{prefix}_paired1.txt` & `{prefix}_paired2.txt`: Generated barcodes
+- `generate_barcodes_{timestamp}.log`: Detailed generation log
+
+**Important Notes**:
+- **Seed sequences are not validated**: If using seed files (paired or unpaired), run `barcadia validate` first to ensure they pass all filters
+- In paired mode with seeds, both `--paired-seed1` and `--paired-seed2` must be provided and have the same count/length
+- Seeds are preserved in the output files (paired or unpaired)
+
+---
+
+#### 2. `barcadia validate` - Barcode Validation
+
+**Purpose**: Validate existing barcode lists against quality filters with support for variable-length sequences.
+
+**Algorithm Overview**:
+1. Load and parse input file(s) and report lengths distribution
+2. Check if sequences fail both biological filters (GC content, homopolymer repeats)
+3. **Computational complexity-optimized distance validation**:
+   - **Method selection**: Automatically chooses optimal validation approach based on barcode set and distance filter characteristics
+   - **Early stopping**: Terminates on first violation found (prevents unnecessary computation)
+4. Generate detailed validation report with comprehensive violation details
+
+**Key Features**:
+- Supports multiple input files (automatically concatenated) with variable lengths
+- Uses Hamming distance for equal-length sequences, Levenshtein for mixed lengths (for sequences passing biological filters)
+- **Adaptive method selection for distance validation**:
+  - Small barcode sets (<10K sequences): Sequential pairwise validation
+  - Large barcode sets (≥10K sequences) with mixed lengths and/or min_distance > 4: Parallel pairwise validation (when multiple CPUs available)
+  - Large equal-length barcode sets (≥10K sequences) with min_distance ≤ 4: Neighbor enumeration
+  - Early stopping on first violation
+  - Can be skipped when `--skip-distance` flag is on
+- Comprehensive reporting with violation cases (for both biological and distance constraints)
+
+**Basic Usage**:
+```bash
+# Validate a single file
+barcadia validate --input test/barcodes.txt
+
+# Validate multiple files (automatically concatenated)
+barcadia validate --input file1.txt file2.txt file3.txt
+
+# Skip distance validation entirely (biological filters only)
+barcadia validate --input test/barcodes.txt --skip-distance
+```
+
+**Required Arguments**:
+- `--input`: Input file(s) containing DNA barcodes (one per line)
+
+**Optional Arguments**:
+- `--gc-min`: Minimum GC content (default: 0.4)
+- `--gc-max`: Maximum GC content (default: 0.6)
+- `--homopolymer-max`: Maximum homopolymer repeat length (default: 2)
+- `--min-distance`: Minimum edit distance between sequences (default: 3)
+- `--skip-distance`: Skip distance validation entirely (default: off)
+- `--cpus`: Number of CPUs for pairwise parallel validation (default: all available)
+- `--output-dir`: Output directory for logs and reports (default: test)
+
+**Output Files**:
+- `validation_report_{timestamp}.txt`: Detailed validation report
+- `validate_barcodes_{timestamp}.log`: Validation process log
+
+---
+
+### Shared Modules
+
+#### `config_utils.py`
+Configuration utilities and DNA encoding/decoding:
+- DNA bases encoded as 0-3 integers (A=0, T=1, G=2, C=3) for enhanced efficiency
+- Convert between DNA strings and integer arrays for optimized processing
+- Logging setup and file reading utilities (ExistingSequenceSet class)
+
+#### `filter_utils.py`
+High-performance filtering algorithms with Numba JIT compilation:
+- Simple validation on filter arguments (GC content, homopolymer, min-distance)
+- GC content and homopolymer repeat filtering with early stopping
+- Hamming distance for equal-length sequences with early stopping 
+- Levenshtein distance for variable-length sequences with early stopping
+- Adaptive method selection between pairwise and neighbor enumeration approaches
+- Neighbor enumeration for efficient distance constraint checking
+
+### Utility Scripts
+
+#### 1. `generate_random_sequences.py` - Test Data Generation
+
+**Purpose**: Generate random DNA sequences for testing validation scripts.
+
+**Usage**:
+```bash
+python -m barcadia.tools.generate_random_sequences --count <num> --lengths <length1> [length2...] [--output <file>]
+```
+
+**Output**: Auto-generated filename in `test/` directory based on `count` and `length` if `--output` not specified.
+
+---
+
+#### 2. `memory_benchmark.py` - Performance Monitoring
+
+**Purpose**: Monitor memory usage and performance of the main scripts.
+
+**Usage**:
+```bash
+# General usage
+python -m barcadia.tools.memory_benchmark [--mem-output-dir <dir>] <command> [args...]
+
+# Examples
+python -m barcadia.tools.memory_benchmark barcadia generate --args
+python -m barcadia.tools.memory_benchmark barcadia validate --args
+```
+
+**Output**: Memory usage report with peak memory consumption and execution time. Log saved to specified directory (default: `test/`).
+
+## Citation
+
+If you use Barcadia in your research, please cite:
+
+```bibtex
+@software{barcadia2025,
+  title={Barcadia: a high-performance, memory-efficient toolkit for fast generation and validation of large-scale NGS barcodes},
+  author={Jiang, Danting},
+  year={2025},
+  date={2025-09-12},
+  url={https://pypi.org/project/barcadia/},
+  note={Code repository: https://github.com/danting/barcadia},
+  version={3.2.0}
+}
+```
+
+A preprint will be posted soon. Citation information will be updated with a DOI once available.
+
+## Changelog
+
+See [CHANGELOG.md](CHANGELOG.md) for detailed version history.