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Home » RNA-Seq

RNA-Seq

Long-Read Sequencing Technology

This lecture on Long-Read Sequencing Technology was created by Katie Sandlin for the 2024 National GEP Faculty Workshop. The lecture covers four major topics: 1) Review of RNA-Seq Short-read Sequencing, 2) Overview and benefits of Long-read Sequencing, 3) Oxford Nanopore Sequencing, and 4) Pacific Biosciences Sequencing. Each slide is scripted in the Notes section for reference.

Pathways Project Primer

This PowerPoint presentation provides a primer on the recommended annotation strategy for the Pathways Project. The presentation provides an overview of the goals of the Pathways Project annotations, an introduction to RNA-Seq, web databases, and a discussion on the phases of the splice donor and acceptor sites.

Pathways Project: Annotation Videos

The Pathways Project is focused on annotating genes found in well-characterized signaling and metabolic pathways across the Drosophila genus. The current focus is on the insulin signaling pathway which is well-conserved across animals and critical to growth and metabolic homeostasis. The long-term goal of the Pathways Project is to analyze how the regulatory regions of genes evolve in the context of their positions within a network. For a general project overview, see the following video by Dr. Laura K. Reed:

Pathways Project Walkthrough Series

This series of videos based on the Pathways Project Annotation Walkthrough is intended to help GEP students annotate a Pathways Project gene from start to finish. The full series is available on the GEP YouTube Channel.

Pathways Gene Annotation Walkthrough Videos

Introduction

Part 1: Examine genomic neighborhood surrounding target gene in D. melanogaster

Part 2.1: Retrieve protein sequence of target gene in D. melanogaster

Part 2.2: Perform a BLAST search of D. melanogaster protein against the target species' genome

Part 2.3: Summarize tblastn results for protein on target species' scaffold

Part 3.1: Examine evidence for a protein-coding gene in region surrounding the tblastn alignment in the target species

Part 3.2: Use synteny to gather additional evidence for the ortholog assignment

Part 4: Determine target gene’s structure in D. melanogaster

Part 5: Determine approximate location of coding exons (CDS's) in target species

Part 6.1: Verify start codon coordinates

Part 6.2: Verify stop codon coordinates

Part 6.3: Determine phases of donor and acceptor splice sites

Part 6.4: Use spliced RNA-Seq reads to verify coordinates for Intron-1

Part 6.5: Use splice junction predictions to verify coordinates for second intron

Part 7.1: Verify gene model of protein

Part 7.2: Download files required for project submission

Part 7.3: Merge project files

Appendix A: Combining (or Batching) BLAST Searches

Motif Discovery in Drosophila

This walkthrough uses FlyBase RNA-Seq Search and the MEME suite to discover motifs that are enriched in a collection of D. melanogaster Muller F element genes that show similar expression patterns.

Annotation of Other Genomic Features within the F Element Project

This presentation illustrates the unusual genomic features that GEP students have encountered as part of their annotation of Muller F Elements from Drosophila ananassae and D. bipectinata. The Muller F Elements in these two species have undergone substantial expansion compared to D. melanogaster. The presentation describes the basic strategy for identifying pseudogenes, retrogenes, partial gene duplications, pseudogene clusters, and nuclear mitochondrial DNA segments (NUMT) within these F Element annotation projects.

Annotation Strategy Guide

This document illustrates how the strategies outlined in the Annotation Instruction Sheet can be applied to more challenging annotation cases.

Annotation of Drosophila

This PowerPoint presentation describes the recommended annotation strategy for Drosophila projects. The presentation provides an overview of the goals of the GEP annotation project, an introduction to NCBI BLAST, web databases, and the issue of reading frames and phase.

Annotation of a Drosophila Gene

This walkthrough uses the annotation of a gene on the D. biarmipes Muller F element to illustrate the GEP comparative annotation strategy. This document shows how you can investigate a feature in an annotation project using FlyBase, the Gene Record Finder, and the gene prediction and RNA-Seq evidence tracks on the GEP UCSC Genome Browser. The walkthrough then shows how you can identify the coordinates of each coding exon using NCBI BLAST, and also includes a discussion on the phases of the donor and acceptor splice sites. The walkthrough concludes by verifying the proposed gene model using the Gene Model Checker; it also includes a sample GEP Annotation Report.

Annotation of Drosophila Primer

This PowerPoint presentation provides a brief primer on the recommended annotation strategy for Drosophila projects. The presentation provides an overview of the goals of the GEP annotation project, an introduction to RNA-Seq, web databases, and a discussion on the phases of the splice donor and acceptor sites.

Browser-Based Annotation and RNA-Seq Data

This exercise continues your introduction to practical issues in comparative annotation. You will be annotating genomic sequence from the dot chromosome of Drosophila mojavensis using your knowledge of BLAST and some improved visualization tools. You will also consider how best to integrate information from high-throughput sequencing of expressed RNA.