otkwnt_embryo

RNA-seq analysis of Otk1, Wnt4, Otk2 Drosophila embryos

➤ Table of Contents

• ➤ 1. About The Project
• ➤ 2. Introduction
• ➤ 3. Dataset
• ➤ 4. Preprocessing
• ➤ 5. Quality Control
• ➤ 6. Differential Expression Analysis
• ➤ 7. Clustering
• ➤ 8. KEGG pathview plots
• ➤ 8. Results
• ➤ 9. MD5 checksums
• ➤ 10. Version info
• ➤ 11. Acknowledgements

➤ 1. About The Project

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This project analyzes bulk RNA-seq data from various Otk1, Otk2 and Wnt4 mutants in Drosophila embryos.

Specifically, we are interested in the following questions:

1. Does Wnt4 bind to Otk1 and activate specific pathways?
2. What is the relationship between Otk1 and Otk2?

➤ 2. Introduction

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Otk1 (Off-track 1) is a transmembrane protein that is associated with colon cancer, but its mechanism of action is still unknown. Wnt4 has been shown to bind to Otk1, but whether this binding is functional and activates any particular pathway has not been studied.

In this study, we overexpress and downregulate various combinations of these three factors (Otk1, Otk2 and Wnt4) and analyze the resulting bulk RNA-seq data. The UAS system was used to upregulate genes, while RNAi was used to downregulate target gene expression. In particular, we are looking for specific pathways that are either activated/ repressed by the Wnt4-Otk1-Otk2 signaling axis.

➤ 3. Dataset

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Mutation details:

Label	Condition	Description
Ctrl	Control	Wild-type Drosophila embryos
W4_OE	UAS-wnt4	Drosophila embryos overexpressing Wnt4
O1_OE	UAS-otk1	Drosophila embryos that overexpress Otk1
O1W4_OE	UAS-otk1, UAS-wnt4	Drosophila embryos overexpressing both Otk1 and Wnt4
O2_KO	otk2RNAi	Drosophila embryos downregulating Otk2
W4_KO	wnt4RNAi	Drosophila embryos downregulating Wnt4
O1_KO	otk1RNAi	Drosophila embryos downregulating Otk1
O1W4_KO	otk1RNAi, wnt4RNAi	Drosophila embryos downregulating Otk1 and Wnt4
O2W4_KO	otk2RNAi, wnt4RNAi	Drosophila embryos downregulating Otk2 and Wnt4
O1O2_KO	otk1RNAi, otk2RNAi	Drosophila embryos downregulating Otk1 and Otk2

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➤ 4. Preprocessing

Initial quality control was performed with fastqc. Reads were then aligned with STAR, and counted with RSEM.

Data was of high quality overall, and no issues were flagged during the QC process.

MultiQC report:

• View on Github: data/02_aligned/03_multiqc/multiqc_report.pdf

• Download interactive copy: data/02_aligned/03_multiqc/multiqc_report_interactive.html

➤ 5. Quality Control

Folder: analysis/01_QC/

Contents: Read counts per sample, gene counts per sample, correlation heatmaps, PCA plots.

➤ 6. Differential Expression Analysis

Folder: analysis/02_DE/

Contents: Volcano plots, MA plots, log2FoldChanges compared to control. Also contains GO and KEGG for upregulated and downregulated genes.

➤ 7. Clustering

Folder: analysis/03_clust/

Contents: K-means clustering, GO and KEGG enrichments. Also contains silhouette plots and WSS plots.

➤ 8. KEGG pathview plots

Folder: analysis/03b_keggplot/

*Contents: KEGG pathview plots for selected pathways. *

➤ 8. Results

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• Master DEG list (LRT p-values, log2FoldChange, padj, cluster) analysis_all/output/04_compile/master_de_gene_list.xlsx

➤ 9. MD5 checksums

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• Drosophila_melanogaster.BDGP6.22.97.chr.gtf: cba22b0ac8e925a53f3e60f450d4888a
• dm6.fa: 5aadf7ccab5a6b674e76516bf75eaa09

➤ 10. Version info

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• FastQC: v0.11.8
• STAR: 2.7.1a
• RSEM: v1.3.1
• MultiQC: version 1.14 (931327d)
• Drosophila genome: dm6.fa, Drosophila_melanogaster.BDGP6.22.97.chr.gtf

➤ 11. Acknowledgements

This table of contents was built with https://github.com/andreasbm/readme