A versatile toolkit for FASTX file processing, including quality control, merging, demultiplexing, and sequence analysis.

Hammer_fastx is a Rust-based command-line tool designed for processing FASTA and FASTQ files. It provides a comprehensive set of subcommands for quality control, paired-end read merging, demultiplexing, sequence statistics, filtering, and alignment to references with ambiguous regions (N-regions). The tool leverages external dependencies like fastp and flash2 for specific tasks and includes parallelized workflows for efficient processing.

• Version: v1.2.0
• Author: CZH with the help of Gemini 2.5 pro
• License: MIT (or specify your preferred license)

• Workflows:
  • demux_all: Complete pipeline for quality control, merging, and demultiplexing.
  • mergePE: Quality control and merging of paired-end reads with customizable output formats.
• Single-Step Commands:
  • demux_only: Demultiplex merged FASTQ files based on barcodes.
  • fastp: Quality control for paired-end FASTQ files using fastp.
  • flash2: Merge paired-end reads using flash2.
  • stats: Generate sequence statistics for FASTA/FASTQ files.
  • filter: Filter FASTA/FASTQ files based on sequence length.
  • Ns_count: Align reads to a reference with N-regions using anchor-based logic.

• Rust: Ensure you have Rust installed (cargo is used for building).
• External Tools:
  • fastp: Required for quality control (fastp subcommand and workflows).
  • flash2: Required for paired-end read merging (flash2 subcommand and workflows).
• Ensure both fastp and flash2 are in your system's PATH.

1. Clone the repository:

   git clone https://github.com/your-username/hammer_fastx.git
   cd hammer_fastx

2. Build the project:

   cargo build --release

3. Install the binary:

   cargo install --path .

The executable will be available as Hammer_fastx.

Run Hammer_fastx --help to see all available subcommands and options.

Hammer_fastx v1.2.0
CZH with the help of Gemini
A versatile toolkit for FASTX file processing, including QC, merging, and demultiplexing.

USAGE:
    Hammer_fastx [SUBCOMMAND]

SUBCOMMANDS:
    demux_all    Run the complete pipeline from QC and merging to demultiplexing
    mergePE      Quality control and merge paired-end data, with optional output formats
    demux_only   Demultiplex a merged FASTQ file based on barcodes
    fastp        Quality control paired-end FASTQ files using fastp
    flash2       Merge paired-end reads using flash2
    stats        Get sequence statistics from one or more FASTA/FASTQ files
    filter       Filter one or more FASTA/FASTQ files based on sequence length
    Ns_count     Align reads to a reference with Ns using a strict anchor-based method

1. Run the full demultiplexing pipeline:

   Hammer_fastx demux_all -i read1.fastq.gz -I read2.fastq.gz --tags samples.csv -o output_dir --fastp-threads 8 --flash-threads 8 --demux-threads 8

2. Merge paired-end reads:

   Hammer_fastx mergePE -i read1.fastq.gz -I read2.fastq.gz -o merged.fastq --out-fasta --fastp-threads 4

3. Demultiplex a merged FASTQ file:

   Hammer_fastx demux_only --inputfile merged.fastq --output demux_out --tags samples.csv --threads 16 --trim

4. Generate sequence statistics:

   Hammer_fastx stats --inputfile sample1.fasta sample2.fastq

5. Filter sequences by length:

   Hammer_fastx filter --inputfile input.fasta --outfile filtered.fasta --min-len 100 --max-len 1000

6. Align reads to a reference with N-regions:

   Hammer_fastx Ns_count --reads reads.fasta --refSEQ reference.fasta --output results --threads 8 --mismatches 2

• Input: Supports FASTA and FASTQ files, including gzipped files.
• Output:
  • Workflows and demultiplexing: FASTQ (default) or FASTA (optional).
  • Statistics: Tabular summary printed to the console.
  • Filtering: FASTA/FASTQ output to file or stdout.
  • Ns_count: CSV files with combination counts and optional FASTA files for matching reads.

The tag file for demultiplexing must be a CSV with the following columns:

• SampleID: Unique identifier for the sample.
• F_tag: Forward tag sequence.
• R_tag: Reverse tag sequence.

Example (samples.csv):

SampleID,F_tag,R_tag
sample1,ACGTACGT,TGCACTGC
sample2,GGTTCCAA,CCATGGTT

• Rust Crates:
  • anyhow: Error handling.
  • clap: Command-line argument parsing.
  • bio: FASTA/FASTQ parsing and DNA sequence utilities.
  • flate2: Gzip file handling.
  • indicatif: Progress bars.
  • rayon: Parallel processing.
  • crossbeam-channel: Thread-safe communication.
  • csv: CSV file handling.
• External Tools:
  • fastp: For quality control.
  • flash2: For paired-end read merging.

Contributions are welcome! Please submit issues or pull requests to the GitHub repository. Ensure your code follows the project's coding style and includes appropriate tests.

This project is licensed under the MIT License. See the LICENSE file for details.