RNA-Seq Analysis Pipeline: Quality Control

This pipeline provides a series of steps for performing RNA-Seq data quality control (QC) analysis using FastQC. It includes steps to download data, organize files, run FastQC, and generate QC reports for each sample.

Requirements

Before running the pipeline, ensure you have the following dependencies installed:

FastQC for quality control analysis

wget for downloading files

tar for extracting compressed files

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Step 1: Create a working directory

Create a new directory to organize your data and results. This directory will hold the raw data, FastQC results, and final reports.

mkdir RNAseq_analysis
cd RNAseq_analysis

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Step 2: Download data

wget ftp://ftp.ccb.jhu.edu/pub/RNAseq_protocol/chrX_data.tar.gz

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Step 3: Extract data

tar -xzf chrX_data.tar.gz

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Step 4: Activate your conda env

Step 5: Quality control with FastQC

Step 5a : Create an output folder for FastQC reports:

mkdir qc_output

Repeat the process for all the files

Step 5b: Run FastQC on your raw FASTQ files

fastqc chrX_data/samples/ERR188044_chrX_1.fastq.gz chrX_data/samples/ERR188044_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188104_chrX_1.fastq.gz chrX_data/samples/ERR188104_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188234_chrX_1.fastq.gz chrX_data/samples/ERR188234_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188245_chrX_1.fastq.gz chrX_data/samples/ERR188245_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188257_chrX_1.fastq.gz chrX_data/samples/ERR188257_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188273_chrX_1.fastq.gz chrX_data/samples/ERR188273_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188337_chrX_1.fastq.gz chrX_data/samples/ERR188337_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188383_chrX_1.fastq.gz chrX_data/samples/ERR188383_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188401_chrX_1.fastq.gz chrX_data/samples/ERR188401_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188428_chrX_1.fastq.gz chrX_data/samples/ERR188428_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR188454_chrX_1.fastq.gz chrX_data/samples/ERR188454_chrX_2.fastq.gz -o qc_output

fastqc chrX_data/samples/ERR204916_chrX_1.fastq.gz chrX_data/samples/ERR204916_chrX_2.fastq.gz -o qc_output

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Step 6: Run Trimmomatic for paired-end data

Trimmomatic processes paired-end FASTQ files and outputs the cleaned, trimmed sequences into paired and unpaired files. The following command demonstrates how to run Trimmomatic with a specific set of parameters to trim the sequences. Note: This step is optional. Based on the quality control results from FastQC, you may find that trimming is unnecessary for your dataset. If you find that trimming is required, you can follow the steps below.

trimmomatic PE chrX_data/samples/ERR188044_chrX_1.fastq.gz \
chrX_data/samples/ERR188044_chrX_2.fastq.gz \
paired/ERR188044_chrX_1_paired.fq.gz unpaired/ERR188044_chrX_1_unpaired.fq.gz \
paired/ERR188044_chrX_2_paired.fq.gz unpaired/ERR188044_chrX_2_unpaired.fq.gz \
ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 \
SLIDINGWINDOW:4:15 MINLEN:36

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