GuideWithTarget 2.0 is an optimized bioinformatics pipeline designed to identify and validate PIWIL3-associated piRNA cleavage events. Significant upgrades from version 1.0 include high-throughput batch processing for biological replicates and enhanced handling of sequencing duplicates, moving beyond one-by-one execution to a robust, integrated workflow. It bridges small RNA-seq (IP vs. Input) and degradome-seq (WT vs. Mutant) to pinpoint authentic cleavage signatures across the transcriptome and transposable elements with higher statistical power.
To run the pipeline, ensure your environment is configured (e.g., Conda environment with bowtie, bedtools, and samtools) and execute the main bash script:
Configuration Parameters The following parameters are defined within the script to control the behavior of the batch analysis:
# Edit PATHWAY DECLARATIONS inside the script first
Bowtie 1.0.0 – Short read aligner for hierarchical mapping.
Samtools 1.5 – Utilities for manipulating alignments.
Bedtools 2.27.1 – Genome arithmetic for cleavage site extraction.
Blast 2.12.0 – BLAST+ suite for target binding analysis.
Cutadapt 2.6 – Adapter trimming and quality control.
Seqtk 1.3 – Tool for processing sequences in FASTA/Q formats.
Python 3.13.9 – Core scripting language (includes Biopython, Pandas).
R 4.2.0 – Statistical analysis (includes ggplot2, dplyr).
Perl 5.26.2 – Legacy script support and text processing.
# Parameters Declarations
cleavage / nocleavage – Group prefixes for experimental conditions (e.g., WT vs MUT).
num_of_rep_cleav – Number of biological replicates for the cleavage group.
logfc_thres – Minimum Log2 Fold Change to consider a site enriched.
pval_thres – P-value cutoff (e.g., 0.05) for statistical validation across replicates.
set_range – Nucleotide window size (e.g., 20) flanking the cleavage site.
keep_zeroMUT – Set to Y to focus on sites with zero background in control groups.