Tips for the First Time Annotator
The following information was cultivated for first-time annotators to gain their bearings in the eukaryotic annotation process for the GEP. Special thanks to GEP TA D’Andrew Harrington for creating the original version of this document. This page is a living document.
Genome annotation allows scientists to attach biological information to sequences of A’s, T’s, C’s, and G’s by identifying where the genes and other functional regions of the genome are located.
What do I need to do to succeed?
Success starts and ends with you. There isn’t any other method or secret it boils down to. Find a system that works for your time constraints and be curious. By asking questions and spending time studying the materials – you will exponentially increase your chances of success in your annotations and any other scientific endeavor you are looking to achieve.
What are recommendations for time management?
Are there materials that will make this course easier?
Who can I reach out to if I need help?
Breakdown of Annotation Tools
What tools do I have to help me in annotation?
The annotation process can be broken down into “stages” or “steps” that will create a more manageable timeframe for you to complete your assignments. Each tool is useful in its own way and shouldn’t be skipped or dodged.
Annotation Files Merger
The Annotation Files Merger allows for the merging of multiple files gathered during the annotation process. This is important because it allows consistency in data collection and a quick method to review everything from a top-down glance. The supported file types for the annotation files merger are: GFF (Genetic Feature Format), FASTA (FAST-ALL), PEP (Peptide File Format), and VCF (Variant Call Format).
The file types listed will be found throughout your annotations so it will also be important to recognize what files are which and why they are important too.
FlyBase is a bioinformatic database for all things Drosophila. This website can be a great place to study the who, what, where, when, and why behind a particular gene within D. melanogaster. You can best use FlyBase by searching for individual genes to uncover detailed reports that summarize genomic location, functionality, orthological pairing, and much, much more.
- If you are looking for a deeper understanding of your assigned genes – look no further than FlyBase.
- It can be incredibly dense within the FlyBase reports, so be sure to have a general idea of what you are looking for.
- When looking for a gene, remember that gene names are case-sensitive.
See the FlyBase Tools and Downloads Documentation for more information.
Gene Model Checker
The Gene Model Checker is a key “checkpoint” in the annotation process. This allows us to visualize our annotation as compared to that of the D. melanogaster gene. Here we can see a bigger picture as to what may or may not be missing so be sure to review your dot plot and protein alignment for possible errors or common mistakes.
Gene Record Finder
The Gene Record Finder can be used to break down the complex information from FlyBase into a quick and easy-to-read form. More importantly, the Gene Record Finder can provide transcription details, polypeptide details, and isoforms for the gene you are examining.
- If you are annotating the UnTranslated Regions (UTRs), be sure to use the Transcription Details.
- If you are looking for gene CoDing Sequences (CDSs), be sure to use the Polypeptide Details.
- Make sure you account for EACH isoform. Unique or identical, we need information on all isoforms!
See the Gene Record Finder User Guide for more information.
GEP UCSC Genome Browser
- Be sure to check if your gene is located in the reverse or forward position in relation to your scaffold. This error commonly occurs!
See the UEG | Genome Browser Video for a breakdown of how to use the GEP UCSC Genome Browser.
BLAST is vital for success in genomic annotation as a whole. As mentioned in the introductory video for navigating and interpreting BLAST results, there are numerous steps that need to be done in order to make sure BLAST is working for you and you aren’t working for BLAST.
- BLAST is heuristic and deterministic, which means it will not give you the same answer every single time, so it should only be used as a guide, along with other lines of evidence for your model.
- Evidence gathered from BLAST is not always exact or precise. Be sure to check your coordinates carefully!
- Within BLAST be sure to collect these key points from the top two hits:
(sequence to match)
|Database/Subject (searching for match)||Function||Use Cases|
|blastn (nucleotide)||nucleotide||nucleotide||searching with shorter queries, cross-species comparison||map mRNAs against genomic assemblies|
|blastp (protein)||protein||protein||general sequence identification and |
|search for proteins similar to predicted genes|
|blastx||nucleotide → protein||protein||identifying potential protein products encoded by a nucleotide query||map proteins/CDS against genomic sequence|
|tblastn||protein||nucleotide → protein||identifying database sequences encoding proteins similar to query||map proteins against genomic assemblies|
|tblastx||nucleotide → protein||nucleotide → protein||identifying nucleotide sequences similar to the query based on their coding potential ||identify genes in unannotated sequences|
Arrows indicate the BLAST program translates the nucleotide sequence before performing the search.
Pathways Project Genome Assemblies
The Sequence Updater creates a VCF (Variant Call Format) file, which can be used to update an existing assembly. This is used whenever a student may have sufficient evidence to suggest that an assembly has an error causing an incorrect alignment.
- Before suggesting a consensus error, make sure to contact your instructor and/or a TA to confirm that you have looked at all other lines of evidence; using the Sequence updater and proposing a consensus error should always be your last resort. Updating a sequence does not happen often and should not be used unless there is significant evidence to suggest it. More often than not, this tool will not be used.
- You must have multiple sequences that suggest an error. Much like verifying your work, it’s better to have numerous pieces pointing to the same potential error than to only have one. Is the change you are seeking valid and accurate?
- As outlined in the GEP Tools | Sequence Updater video tutorial, keep in mind once you apply these changes, that’s it. Be absolutely certain this is what you want.
Small Exons Finder
Do you have an exon that is too small to find with BLAST? Is the result, no matter what you change, coming up with “No Significant Matches Found?” If so, the Small Exons Finder can offer some insight in locating small exons that would otherwise be finding a needle in a very large haystack.
- When using the Small Exons Finder, you should use the whole scaffold from the GEP UCSC Genome Browser and not just the position of your predicted gene. You can do this by following the steps below:
Ideally, the annotation process for anyone who is just starting out will take time and that’s okay. The initial hardship you face is not because you don’t understand the science but are learning to use these specific tools, the use of which will become easier with time. Patience, time, and dedication to your work will not only offer you a better sense of why annotation can be an effective scientific process, it will also provide you with skills that can eventually be utilized in a scientific or research career. We are not here to create unneeded difficulty; we simply strive to offer a genuine look at an introduction to bioinformatics and genomic annotations. If at any time during your annotation you feel lost or unsure where to go, simply reach out the GEP TAs, and we will be more than happy to provide a nudge in the right direction.