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tRNA-Seq Pipeline Specification

Pipeline Details

  • Name: tRNA-Seq Pipeline
  • Pipeline UUID: f931xrcq6go72tr44eeesouo9fdtjr
  • Version: 1.0.1
  • View Pipeline:

Overview

tRNA-Seq Pipeline is designed for automated tRNA expression and modification analysis. It automates data preprocessing, quality control, tRNA clustering, read alignment, coverage calculation, expression quantification, differential expression analysis, and comprehensive modification analysis to ensure reliable and reproducible results for tRNA sequencing data.

Key Use cases:

  • tRNA Expression Quantification: Quantify tRNA expression levels from tRNA-seq data with SNP-tolerant alignment using GSNAP.
  • tRNA Modification Analysis: Comprehensive quantification of tRNA modifications and misincorporation signature analysis.
  • Differential Expression Analysis: Calculate tRNA differential expression between conditions using DESeq2.
  • Functional tRNA Analysis: Analyze functional tRNA pools and tRNA completeness via 3'-CCA analysis.

Features

  • SNP-Tolerant Alignment: Uses GSNAP for robust read alignment that handles tRNA modifications and SNPs.
  • Comprehensive Modification Analysis: Quantifies tRNA modifications and analyzes misincorporation signatures.
  • tRNA Clustering: Clusters tRNAs and deconvolves cluster-aligned reads back to unique tRNA transcript-level reads.
  • UMI Support: Supports UMI extraction and duplicate removal for improved quantification accuracy.
  • Quality Control: Implements FastQC, adapter removal with Trimmomatic, and quality filtering steps.
  • Differential Expression: Built-in DESeq2 analysis for identifying differentially expressed tRNAs.
  • Coverage Analysis: Generates coverage information and plots for quality control assessment.
  • Modular Design: Optional preprocessing steps including quality filtering, trimming, and UMI processing.

Input/Output Specification

Inputs

Required

Reads

  • Description: FASTQ files containing raw tRNA sequencing reads
  • Format: .fastq.gz
  • Example File Path: /path/to/input/sample.fastq.gz

Adapter Sequence

  • Description: Adapter sequence(s) for removal during preprocessing
  • Format: Text string
  • Example: AGATCGGAAGAGC

Optional Inputs

UMI Pattern

  • Description: Pattern for UMI extraction from reads
  • Format: Regular expression pattern
  • Example: .+(?P<umi_1>(NNNCGANNNTACNNN)|(NNNATCNNNAGTNNN)){s<=1}.+

Sample Groups

  • Description: Sample group definitions for differential expression analysis
  • Required Columns: Sample ID, Condition
  • Format: Tab-separated values (.tsv)

Outputs

Reported Outputs

  • tRNA Expression Matrix:
  • Description: Quantified tRNA expression levels across samples
  • Format: .tsv
  • Example File Path: /output/experiment/expression_matrix.tsv

  • Location: mimseq output directory

  • Differential Expression Results:

  • Description: DESeq2 results for differentially expressed tRNAs
  • Format: .tsv
  • Example File Path: /output/experiment/differential_expression.tsv

  • Location: mimseq output directory

  • Modification Analysis:

  • Description: Comprehensive tRNA modification quantification results
  • Format: .tsv
  • Example File Path: /output/experiment/modification_analysis.tsv

  • Location: mimseq output directory

  • Coverage Plots:

  • Description: Coverage visualization plots for quality control
  • Format: .pdf, .png
  • Example File Path: /output/experiment_reports/coverage_plots.pdf
  • Visualization App: PDF viewer, image viewer
  • Location: Reports folder

Supporting Outputs

  • Quality Control Reports:
  • Description: FastQC reports for input and processed reads
  • Format: .html

  • Example File Path: /output/qc/sample_fastqc.html

  • Alignment Statistics:

  • Description: GSNAP alignment statistics and logs
  • Format: .log

  • Example File Path: /output/alignment/alignment.log

  • Processing Summary:

  • Description: Overall summary of read processing statistics
  • Format: .tsv
  • Example File Path: /output/overall_summary.tsv

Associated Processes

References & Additional Documentation