F Element Project
Quick Start Guide
This Quick Start Guide provides a minimum set of presentations, walkthroughs, and exercises that are designed to prepare students to participate in the F Element annotation projects. The Quick Start Guide is organized around five major concepts and tools. The curriculum materials listed under the first three concepts/tools can be used for other GEP scientific projects. The curriculum listed under concepts/tools 4 and 5 are specific to the F Element project. These materials assume that students have a basic background in molecular genetics.
- Exercises: require students to go through the process answering questions (can be graded)
- Walkthrough: highly scripted "click the button" tours that guide students through a protocol
- Lecture: provides an annotated slide set
- Workflows: one-page handouts to remind students of the steps for a protocol, including the logic for decision making
- Resource: project-specific materials (e.g., annotation protocols, strategy guides, and reports)
Concepts/Tools
Minimum Recommended Materials
Optional/Additional Materials
Concepts/Tools 1, 2, & 3
Beginning curriculum common to all GEP Research Projects.
- 1. Gene structure, Introduction to a genome browser, Introduction to gene model construction
- The recommended materials provide an introduction to gene structure by covering transcription, mRNA processing, and translation. The introduction to gene model covers topics including a codon consists of three nucleotides, that a coding exon can begin or end in the middle of a codon (i.e., phases of the splice acceptor and donor sites), and the need to maintain an open reading frame after splicing.
- Starting Points: Freshmen/Sophomores just learning about eukaryotic gene structure (exons/introns, etc.) may be best served by taking the time to work through all six “Understanding Eukaryotic Genes” (UEG) modules. Upper level students with a good grounding in molecular genetics need only review this material, which also introduces the nomenclature and tools of a genome browser (UCSC Genome Browser Mirror). UEG is highly scripted and can be given as an “at home” assignment before the start of the semester.
- 2. Homology/BLAST
- BLAST reports regions of sequence similarity; what is it looking for and how do we interpret the report?
- Using BLAST for Genomic Sequence Annotation
- Detecting and Interpreting Genetic Homology: Lecture Notes on Alignment accompanying notes for above PPT
- 3. RNA-Seq (Optional)
- We make extensive use of RNA-Seq data; this is primarily derived from processed transcripts, so we can identify splice sites based on spliced RNA-Seq reads.
Concepts/Tools 4
Prepares students to work on their own annotation projects; submitting completed projects to GEP allows them to become co-authors (if they read/critique/approve the resulting group manuscript or participate in microPublication).
Prepares students to work on their own annotation projects; submitting completed projects to GEP allows them to become co-authors (if they read/critique/approve the resulting group manuscript or participate in microPublication).
- 4. Annotation
- How to use multiple lines of evidence to establish the presence of a gene, determine its D. melanogaster ortholog, and construct the best gene model, as defined by the available evidence. In most cases our evidence includes:
- 1. sequence homology = evolutionary conservation
- 2. ab initio gene models = rules of ORF, start and stop codons
- 3. RNA-Seq data = local transcription, position of exons, splice site borders
- Annotation of Drosophila Primer describes annotation goals and strategies; accompanies the walkthrough below
- Annotation of a Drosophila Gene
- Simple Annotation Problem (optional)
- F Element Project: Annotation Report detailed Word document; students must complete this form providing evidence for their annotation to contribute to the research and be eligible to be a co-author
- GEP Annotation Workflow
- Annotating Splice Sites Workflow
- Identify the D. melanogaster Ortholog
- The following PPTs provide alternatives to the “Annotation of Drosophila Primer” (mix and match slides to fulfill pedagogic needs)1
- Annotation of Drosophila more detailed version of the “Annotation for D. virilis” PPT; provides additional information regarding the RNA-Seq data, interpretation of the BLAST search results, and phases of splice donor and splice acceptor sites; some faculty adapt the curriculum by combining a subset of slides from the “Annotation of Drosophila” presentation with the “Annotation for D. virilis” presentation
- Annotation for D. virilis uses mav gene which has two coding exons; provides quickest way to illustrate an overview of the annotation protocol
- Other Optional Materials:
- Annotation Instruction Sheet more in-depth description of the GEP approach; complements the "Annotation for D. virilis" PPT
- Annotation Strategy Guide how GEP strategies can be applied to more challenging cases
- Browser-based Annotation and RNA Seq-data provides more practice on comparative annotation, using BLAST and RNA-Seq
- GEP Digital Laboratory Notebook example of a digital notebook to guide students
- 1 typically used in workshops where the participants work on the “Annotation of a Drosophila Gene” walkthrough during the same training session; uses same gene as the walkthrough (i.e., CG31997) to illustrate the key steps of the annotation protocol (e.g., the group attending the Summer 2022 ABLE meeting used a modified version of the “Annotation of Drosophila Primer” and the walkthrough in their presentation
Concepts/Tools 5
If time permits, the project will benefit by students checking the Transcription Start Sites annotation for each gene as well.
If time permits, the project will benefit by students checking the Transcription Start Sites annotation for each gene as well.
- 5. Annotating the Transcription Start Sites (TSS) (Optional)
- Introduces concepts of promoter architecture and the experimental techniques for characterizing promoters (e.g., CAGE and RAMPAGE; RNA-Seq; ChIP-Seq data for RNA Polymerase II and transcription factors; DNase I Hypersensitive Sites in chromatin; 9-state chromatin models based on histone modifications).
- Note: We now have RAMPAGE data for the four species used here. The draft TSS annotation protocols and curriculum materials which use the new RAMPAGE data are available on Box.
- Searching for Transcription Start Sites in Drosophila
- Annotation of Transcription Start Sites in Drosophila
- TSS Annotation Workflow
- TSS Module Primer: Review of Transcription, Promoter Structure, and Chromatin Packaging provides an overview of promoter structure in eukaryotic genomes that might be helpful to students before they work on the TSS Modules
- TSS Modules (1-4)
- Page Last Updated: January 10, 2024