Overview

This document provides an overview of the methods used for RNA sequencing and subsequent data analysis. This pipeline was designed for studying Patient-Derived Xenograft (PDX) tumor tissue. The workflow includes sample collection, RNA extraction, RNA sequencing, and comprehensive data analysis.

Method

Sample Collection:

• Material Used: Snap frozen PDX tumor tissue.

RNA Extraction:

Kit Used: Allprep DNA/RNA Mini Kit (Cat. 80204 QIAGEN).

RNA Sequencing Kit/Platform

• Sequencing Kit: Illumina TruSeq Standard Total RNA Kit with RiboZero Plus.
• Sequencing Goal: Achieving approximately 50 million reads per sample to ensure depth and coverage for robust downstream analysis.
• Sequencing Platform: Novaseq, known for its high-throughput sequencing capability.

Analysis

Applied Tools and Methodology

• Xengsort Package:
  • Function: Used on FASTQ files to differentiate and split raw FASTQ files into human and mouse reads.
  • Data Organization: Data saved in separate folders for host and graft analysis.
    • Host Folder: "20210927 Host Analysis"
    • Graft Folder: "20210927 Host Analysis"

Patient sample 21A1 was established orthotopically, excluded for LRT DESeq2 analysis due to different model systems.

Host Analysis (Folder: 20220109analysis; ignore other folders):

• DESeq2 LRT Analysis: Differential expression analysis using the Likelihood Ratio Test. Basically it’s a method to compare differential expression gene across different “levels”. Here we use patient as factor to make the comparison. More details can be found here.
• PCA Plot: Principal Component Analysis for visualizing the genetic variation.

Gene Set Enrichment Analysis (LRT Ranked Gene)

Differential expression cluster analysis

• WGCNA Analysis
• Result Sheet Format:
  • Patient Meta Data
  • Raw Counts
  • Normalized Counts
  • Variance Stabilized Counts
  • LRT Ranked Genes with Differential Expression Statistic
  • Top 1000 LRT Ranked Gene Hallmark Gene Set Enrichment Analysis

Graft Analysis (Folder: 20220109analysis):

• DESeq2 LRT Analysis: Differential expression analysis for graft samples. Same pipeline as Host analysis.
• PCA Plot: Conducting PCA for graft-derived samples

Gene Set Enrichment Analysis

Gene Set Variance Analysis

• Result Sheet Format:
  • Patient Meta Data
  • Raw Counts
  • Normalized Counts
  • Variance Stabilized Counts
  • LRT Ranked Genes with Differential Expression Statistic
  • Top 1000 LRT Ranked Gene Hallmark Gene Set Enrichment Analysis