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🛠️ GTF File Operations (mkgtf) • 📄 BAM to FASTQ (bam2fastq) • 🧬 Chromosome Splitting (chromsplit) • 📝 FASTQ Subsetting (fqsubC4)

$ dnbc4tools tools mkgtf -h

optional arguments:
  -h, --help            show this help message and exit

Basic Settings:
  --action <STR>        Select action type: 'mkgtf'(filter), 'stat'(statistics) or 'check'(validation) [default: mkgtf]
  --ingtf <FILE>        Path to input GTF annotation file
  --output <FILE>       Path to output file

Filter Settings:
  GTF file format requirements:
                  RNA analysis requires "gene"/"transcript" and "exon" types, plus gene_id/name and transcript_id/name attributes.

  --include <STR>       Set filter parameters in 'mkgtf' mode, multiple filters separated by commas. Default includes: protein_coding, lncRNA, lincRNA, antisense, IG_*/TR_* genes
  --type <STR>          Set according to gene type tag in GTF attributes [default: gene_biotype]
  --feature <STR>       Select information from feature column. If no 'gene' rows, select 'transcript' [default: gene]

--ingtf (Required)

Specify the path to the input GTF gene annotation file.

• Format Requirement: Standard GTF format. GFF or GFF3 formats are not supported.

Default: None

Example:

--ingtf Homo_sapiens.GRCh38.108.gtf

--output (Required)

Specify the output file for the processing results.

• Function: Generates different types of output files depending on the operation mode.
• Auto-creation: The specified output directory will be created automatically if it does not exist.

Default: None

Examples:

# When action is 'mkgtf' (filter)
--output ./filtered_genes.gtf

# When action is 'stat' (statistics)
--output ./gene_statistics.txt

# When action is 'check' (validation)
--output ./corrected.gtf

--action (Optional)

Select the type of operation to perform.

• mkgtf: (Default) Filter the GTF file based on gene types.
• stat: Count the gene types in the GTF file.
• check: Validate and fix the GTF file format.

Default: mkgtf

Example:

--action stat

--include (Optional)

In mkgtf mode, specify the gene types to keep, separated by commas.

• Function: Used to precisely filter for the gene sets you are interested in.

Default: protein_coding,lncRNA,lincRNA,antisense,IG_*,TR_*

Example:

--include protein_coding,lncRNA

--type (Optional)

Specify the tag in the GTF attributes used to identify the gene type.

• Function: Adapts to the annotation style of GTF files from different sources.

Default: gene_biotype

Example:

--type gene_type

--feature (Optional)

Specify from which column (feature) of the GTF file to extract information.

• Function: Typically used to specify whether the operation is at the gene or transcript level.
• Alternative: If there are no `gene` rows in the GTF file, it is recommended to select `transcript`.

Default: gene

Example:

--feature transcript

$ bam2fastq --help
BAM to FASTQ Converter for C4 Single Cell RNA seq Data

Usage: bam2fastq [OPTIONS] <BAM> <OUTPUT>

Arguments:
  <BAM>     Path to the input BAM file
  <OUTPUT>  Directory where FASTQ files will be written

Options:
  -t, --threads <THREADS>        Number of CPU threads for parallel processing [default: 4]
  -r, --locus <REGION>           Process reads from a specific genomic region (format: chr1:1000-2000)
  -n, --reads-per-fastq <READS>  Maximum number of reads per FASTQ file. All reads go to a single file if not specified.
      --max-memory <MEMORY>      Maximum memory to use in MB. If not specified, will be automatically determined based on system resources.
      --no-compress              Disable gzip compression for output FASTQ files
  -h, --help                     Print help
  -V, --version                  Print version

<BAM> (Required)

Specify the path to the input BAM file.

• Format Requirement: Must be a valid C4 RNA BAM file, supporting both single-end and paired-end data.
• Index Requirement: The BAM file must be indexed (i.e., a corresponding .bai file must exist alongside it).
• Paired-end Note: If it is paired-end data, you need to sort it by read name using samtools sort -n before processing.

Default: None

Example:

/path/to/your.bam

<OUTPUT> (Required)

Specify the directory for the output FASTQ files.

• Function: All converted FASTQ files will be saved in this directory.
• Auto-creation: The directory will be created automatically if it does not exist.

Default: None

Example:

/path/to/output_dir

-t, --threads (Optional)

Set the number of CPU threads for parallel processing.

• Performance Note: Since the tool needs to ensure the output order is consistent with the input, increasing the number of threads does not significantly improve the overall analysis speed.
• Recommendation: It is recommended to use the default 4 threads for analysis.

Default: 4

Example:

-t 8

-r, --locus (Optional)

Process only reads from a specific genomic region.

• Format: Standard genomic coordinate format (chromosome:start-end).
• Application: For targeted analysis of specific genes or chromosomal regions.

Default: None

Example:

-r chr1:1000-2000

-n, --reads-per-fastq (Optional)

Set the maximum number of reads per output FASTQ file.

• Splitting Strategy: Automatically splits large files into smaller ones for easier downstream processing.
• Default Behavior: If not specified, all reads will be written to a single file.

Default: None

Example:

-n 10000000

--max-memory <MEMORY> (Optional)

Set the maximum memory the tool can use (in MB).

• Function: Controls the tool's memory consumption to prevent failures due to insufficient memory.
• Auto-determination: If not specified, the tool will automatically allocate memory based on available system resources.

Default: Auto-determined

Example:

--max-memory 8192

--no-compress (Flag)

Disable gzip compression for the output FASTQ files to significantly increase analysis speed.

• Performance Bottleneck: The main speed bottleneck of the program is writing compressed files.
• Strongly Recommended: Using this parameter disables compression, thereby significantly improving the overall analysis speed.
• Trade-off: The resulting uncompressed files will occupy more disk space, so ensure you have sufficient storage.

Default: Not set

$ chromsplit --help

Usage: chromsplit [OPTIONS] --fasta <FA> --prefix <PREFIX>

Options:
  -f, --fasta <FA>           Input genome sequence file in FASTA format
  -g, --gtf <GTF>            Optional GTF/GFF annotation file for the genome
  -o, --prefix <PREFIX>      Prefix for output files
  --min_length <MIN_LENGTH>  Minimum length of output scaffold fragments [default: 300000000]
  --max_length <MAX_LENGTH>  Maximum length of output scaffold fragments [default: 500000000]
  --cut_site <CUT_SITE>      Optional cut site file containing predefined split positions
  -h, --help                 Print help
  -V, --version              Print version

-f, --fasta <FA> (Required)

Specify the input genome sequence file.

• Format Requirement: Standard FASTA format (.fa, .fasta, .fna).
• Content: Must contain complete chromosome or scaffold sequences.

Default: None

Example:

--fasta genome.fasta

-o, --prefix <PREFIX> (Required)

Specify the prefix for the output files.

• Output Files: The tool will automatically generate files like <prefix>.fa, <prefix>.cutsite.tsv, etc.
• File Management: Facilitates batch processing and result tracking.

Default: None

Example:

--prefix split_genome

-g, --gtf <GTF> (Optional)

Specify the gene annotation file (GTF/GFF format).

• Intelligent Splitting: Providing an annotation file ensures that split points are located in intergenic regions, protecting gene integrity.
• Annotation Sync: The tool automatically adjusts and outputs a new annotation file with synchronized coordinates.

Default: None

Example:

--gtf annotation.gtf

--min_length <MIN_LENGTH> (Optional)

Set the minimum length of the output fragments (unit: bp).

• Function: Ensures that the split fragments are not too small, which could affect subsequent analysis.

Default: 300000000

Example:

--min_length 300000000

--max_length <MAX_LENGTH> (Optional)

Set the maximum length of the output fragments (unit: bp).

• Technical Limitation: Primarily used to ensure fragment length meets requirements for downstream analyses like ATAC library construction (usually < 2^29-1 bp).

Default: 500000000

Example:

--max_length 500000000

--cut_site <CUT_SITE> (Optional)

Provide a text file containing predefined split positions.

• Precise Control: Prioritizes splitting at the specified positions in the file, allowing for precise control over split locations.

Default: None

Example:

--cut_site predefined_cuts.txt

$ fqsubC4 --help

Usage: fqsubC4 [OPTIONS] --input <FILE> --output <FILE> --regions <REGIONS>

Options:
  -i, --input <FILE>           Path to input FASTQ file
  -o, --output <FILE>          Path to output FASTQ file
  -r, --regions <REGIONS>      Comma-separated regions in format start:end (e.g., 7:16,23:32,38:47)
  -b, --batch-size <BATCH_SIZE>  Batch size for processing [default: 100000]
  --buffer-size <BUFFER_SIZE>  Buffer size for channel between reader and writer [default: 500]
  -h, --help                   Print help
  -V, --version                Print version

-i, --input <FILE> (Required)

Specify the path to the input FASTQ file.

• Format Support: Supports both uncompressed (.fq, .fastq) and gzipped (.fq.gz, .fastq.gz) formats.
• Auto-detection: The tool automatically determines the compression format based on the file extension.

Default: None

Example:

--input sample_R1.fastq.gz

-o, --output <FILE> (Required)

Specify the path for the output FASTQ file.

• Auto-compression: The output file will be automatically compressed if the filename ends with .gz.
• Performance Note: GZIP compression will significantly slow down the processing speed.

Default: None

Example:

--output extracted_R1.fastq.gz

-r, --regions <REGIONS> (Required)

Specify the regions to be extracted from the sequences.

• Format Specification: Use start:end format, with multiple regions separated by commas.
• Coordinate System: Coordinates are 1-based (the first base of the sequence is position 1).
• Application: Used for extracting Barcodes, UMIs, or for trimming sequences.

Default: None

Example:

--regions 7:16,23:32,38:47

-b, --batch-size <BATCH_SIZE> (Optional)

Set the number of records per batch for processing (i.e., the number of FASTQ records read into memory at one time).

• Performance Impact: Higher values use more memory but may improve processing performance.
• Balancing Act: A balance needs to be found between memory usage and processing efficiency.

Default: 100000

Example:

--batch-size 200000

--buffer-size <BUFFER_SIZE> (Optional)

Set the buffer size for the channel between the reader and writer.

• Throughput Optimization: Adjust this parameter for better throughput when processing large files.

Default: 500

Example:

--buffer-size 1000

💡 Tip

This document is continuously updated. If you find any errors or have information to add, your feedback is welcome.

📝 Document Version: 3.0 beta | Last Updated: 2025

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🛠️ DNBelab C Series HT Tool-based Analysis Parameters
A parameter configuration guide for high-performance single-cell data analysis tools