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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.

Sequence Similarity Introduction

This lesson and exercise defines similarity in a non-biological and biological sense, quantifies the similarity between two sequences, explains how a substitution matrix is used to quantify similarity, calculates amino acid similarity scores using the BLOSUM 62 substitution matrix, explains how BLAST detects similarity between two sequences and how to use BLAST and interpret the alignments.

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.

Synteny Introduction Slides

This resource is a slide deck offering an expanded introduction to synteny. Instructors are encouraged to use and modify to fit the needs of specific courses and students.

Introduction to R and RStudio

This series of modules introduces students to the statistical platform R using an integrated development environment of RStudio. Both softwares can be downloaded for free. Once downloaded and installed according to Module 0, students should watch the accompanying video for an introduction to the new environment. Module 1 presents an exercise where students work with genomic sequence alignment data to learn how to construct R commands while performing basic summary statistics and making basic plots.

Genomic Neighborhood Check For Understanding

This was created in response to a member mentioning their students really struggled with the genomic neighborhood and the member didn’t realize until they were already too far into the annotation to correct their misconceptions. This is meant to be a quick in-class and/or homework assignment.

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:

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

Pathways Project: Annotation Form

This “Annotation Form” merged the “Annotation Report” and “Annotation Notebook” into a single document and the latter two items were archived.

The “Annotation Form” kept many of the Checks for Understanding type questions that were previously in the “Annotation Notebook.” However, given that not all faculty want their students to answer those questions, we marked any question that is NOT required for submission to the GEP as “OPTIONAL.” Currently the optional questions are organized as letters rather than numbers, which we hope will make it easy to quickly select and delete the OPTIONAL questions that faculty don’t wish to include. Again, all numbered questions are required for submission to the GEP for reconciliation.

Items that were previously asked for in the “Project Details” table of the “Annotation Report” are now available on the Genome Browser Gateway page for each species. Therefore, the “Project Details Table” instructions document is no longer needed.