Drosophila Annotation Goals: Final Presentation and Written Reports

The abstract should provide a brief statement of the goals and a report of your findings. This should be similar to and about the same length as a typical abstract found at the beginning of a scientific paper (~300 words). Your abstract should be a summary of your results, NOT the process by which you achieved those results. For the D. willistoni annotation project, be sure to include the size of the project, number of Genscan predictions, protein-coding genes documented, and other verified features (e.g., novel repeats, non-coding RNAs) or unusual findings (e.g., changes in exon structure, changes in the number of isoforms), etc. Be specific in reporting what you have found and your resulting conclusions.

One to three paragraphs: Why are we studying this problem? Why is comparative genomics a powerful approach for analyzing the features of genes and chromosomes? What kinds of results are we generating right now, and what do we hope to derive in the future aggregate analysis? Try to keep this section concise and informative but give some context — what are the goals of the overall study, and what are your goals in particular. Include a figure covering the entire sequence of your project(s) that indicates the size and position of each putative feature you will investigate. A screenshot of the GEP UCSC Genome Browser for your project region may provide a good starting point for this figure. Box and number the features that you will investigate/report on for ease of communication.

This section will be significantly larger (reflecting the number of features in your project), and it should be subdivided with a sub-section for each putative feature. You should present here a more detailed analysis of any genes, pseudogenes or partial genes you find in your project. Use the Genscan predictions (or similar) as an organizer for your presentation. Remember that while in D. melanogaster pseudogenes are rare, this may not be the case in other Drosophila species so keep an eye out for evidence suggesting the presence of pseudogenes in your project. Because the projects are partitioned into sub-regions, you may find only part of a gene at one end or the other of your project. Also be aware that no published genome is perfect — sequencing errors may occur in the genome assembly. Note that Genscan and other gene finders may miss some candidate genes, so a complete analysis will not be limited to just gene predictions; look at the other evidence tracks (homology search with D. melanogaster, RNA-seq, etc.) and examine the orthologous region of the D. melanogaster genome as annotated by the curators at FlyBase. In particular, Genscan will not predict non-coding RNAs (ncRNAs); be sure to investigate those posted for D. melanogaster, looking for conserved sequences in your species (e.g., via a blastn search of the non-coding RNA against your project region). When investigating a feature, you should include a discussion of all the information available to you: gene predictions, results from BLAST searches, RNA-seq data (e.g., splice junction predictions and assembled transcripts), and conservation tracks for the other Drosophila species (see the walk-throughs and practice examples). Be sure to discuss all the important sources of evidence. If the results are negative, ambiguous, or absent, simply mention that and move on.

For each gene in your D. willistoni projects, start by confirming the D. melanogaster orthologue; include the FlyBase ID and the name of any D. melanogaster gene that you consider likely to be orthologous in your report. After making approximate assignments based on evidence of conservation as detected using the “Align two or more sequences” functionality in BLAST, determine the exact location of each coding exon using all available evidence. Construct a model for each putative isoform. Use the Gene Model Checker to confirm your basic decisions; be sure to check both the dot plot and the peptide sequence alignment comparison to D. melanogaster. Every difference between your model and the D. melanogaster orthologue represents an assumption of evolutionary change that underlies your gene model. Based on the GEP annotation protocol, the best gene model should minimize these assumptions; as such, you should be able to account for every difference and convince yourself that there is no other acceptable gene model with fewer differences. [If possible, these differences should be supported by biological evidence (e.g., RNA-Seq data) or sequence conservation with other Drosophila species.] The dot plot and peptide sequence alignment, with a discussion of important differences (for each isoform), should be part of your report. You do not need to show multiple dot plots and alignments for isoforms that are highly similar. You may wish to include these in a “Supplemental Materials” section at the end of your final report.

Note that information from high-throughput sequencing of RNA, the RNA-Seq track and its derivatives, will be very useful. RNA-Seq sequences were generated by producing many short reads (100-125 bases) from expressed RNAs using the Illumina sequencing technology. These reads have been mapped back to the genome sequences and the number of reads that overlap each base plotted in the “RNA-Seq Coverage” tracks. In addition to using the RNA-Seq results to help you with your annotations, you should also compare the RNA-Seq data with your final gene model. Relevant questions include: How well does the RNA-Seq coverage track correspond to the locations of the individual exons in your annotation? Can you reliably use RNA-Seq coverage to identify untranslated regions (5' and 3' UTRs) for each of your genes? For every intron in your gene model, how many were supported and how many were unsupported by results in the splice junction and transcript tracks? Once you have mapped all the genes in your project region, be sure to investigate all regions that have high RNA-Seq coverage, especially in regions that do not correspond to the genes you have annotated. (However, some of the regions with high RNA-Seq coverage could be spurious if they overlap with transposon remnants or other repeats, so they should be investigated but treated skeptically.) Note that genes that are expressed only at low levels or in a restricted set of tissues or developmental stages may not display significant RNA-Seq data for our species. You can assess the expression status of the orthologous gene in detail from the data available for D. melanogaster on FlyBase.

As described above, you should provide a detailed description of your general approach with the description of the first gene discussed in the paper. Be sure to include screenshots documenting the following:

Include the final model in a table, showing the exact exon coordinates, size, percent identity with D. melanogaster, reading frame, phase of the donor and acceptor sites. While you should include a dot plot and protein alignment from Gene Model Checker, a screenshot of the Gene Model Checker checklist (i.e., green check marks) should not be shown if every test passes. Figures like this do not hold informational content that cannot simply be described in the text (i.e., “all tests passed in Gene Model Checker”) and is evident from the dot plot and protein alignments. However, you should include a discussion if the Gene Model Checker checklist contains any warnings or errors (e.g., non-canonical splice donor), along with the evidence you used to support these exceptions in your final model. Be sure to include a detailed justification in your report if the proposed gene model for your species differs substantially from the D. melanogaster orthologue. There will be differences — evolution happens! But you should be able to explain any substantial gaps in the dot plot, particularly large vertical or horizontal gaps (which correspond to large insertions or deletions compared to the D. melanogaster orthologue, respectively) and discuss why your model is better than any alternatives.

Once you have established the general approach, you can summarize your results for subsequent genes. Focus on anything unusual or different in the results of subsequent annotations. Each gene summary should include a summary table as described above and a final dot plot and protein alignment. Do not include multiple dot plots and alignments if the coding exons are identical, just describe which isoforms are identical and mention that the dot plot and alignment figures represent the results for all of them.

As we have discussed, we would like you to use the TSS Analysis Protocol to define, given the data, the smallest possible TSS search region(s) that is likely to contain the TSS. Do this for all the genes in your project region. You should give details for those promoters you have analyzed, and provide a short summary of the TSS’s for the other genes in your region (simply refer to the other reports from the group for more details). If there are multiple TSS’s annotated in D. melanogaster, you should analyze your results with the assumption that the same structure exist in your orthologous genes. If your final results indicate a change in the number of promoters, your analysis should include your best evidence to support this conclusion.

For any promoter, begin your TSS analysis by characterizing the number and shape of the promoter(s) for the orthologous gene in D. melanogaster based on the TSRchitect results. Then, use all available evidence to define the broad genomic region to search for the TSS’s in your project based on the gene structure of the ortholog in D. melanogaster, the organization of neighboring genes, the blastn search result, and any available RNA-Seq data. Remember the goal in the written report is not to give a historical record of the analysis, but to write the results in a way that best describe the evidence that pertains to your TSS annotations and logically flows from observations to your conclusion. Negative results should, at a minimum, be discussed briefly in your report to assure the reader that all sources of evidence were examined in your analysis.

Following the Basic Instructions for Repeat Analysis, report on the repeat landscape in your individual analysis region. Outline the steps you took and the results you found in a way that convinces the reader to trust your results. Discuss the overall data for both the D. melanogaster F Element and the F Element region in D. willistoni. Look over the data for patterns or differences between the D. melanogaster F Element and the portion of D. willistoni chromosome 3 that contains most of the F Element genes.

Start with your local synteny analysis of the genes in your project region. Generate a simplified map of your D. willistoni project region, including genes and possibly any other features you have found. Analyze the genes from your project, and assess synteny with D. melanogaster. Using one of the D. willistoni genes in your project region, identify the location of the corresponding ortholog on the D. melanogaster F element. Generate a map of the D. melanogaster genome centered around this gene which covers approximately the same number of protein-coding genes as your D. willistoni project region. Note which Muller element each gene is on in D. melanogaster.

Using these two maps, compare the relative gene order and orientation in D. willistoni and D. melanogaster. Remember that the orientation of your D. willistoni project is arbitrary (based on computer generated files), so you should focus on the relative orientations of the genes when analyzing synteny. (The project region is considered to be syntenic with D. melanogaster if the reverse complement of the D. willistoni project has the same gene order and orientation as D. melanogaster.)

If synteny has been preserved, the genes in your project will all be from the same region of the D. melanogaster genome, and your comparison will show a one-to-one map of the orthologs from one genome to the other (two horizontal lines). If the regions are completely syntenic, the genes will be in the same relative order and orientation in the two species. If synteny has not been preserved, please include a comparison based on each gene in your project to the same gene and flanking regions in D. melanogaster (several horizontal lines, stacked up). Look for evidence of events (such as inversions, transpositions, etc.) that could have easily occurred during evolution that could explain the changes in synteny. If there is no set of simple events, then consider the changes as “complex” and report as such.

The second part will delve into the synteny “Block” analysis. Write up the analysis of your specific region in detail — summarizing the results as you proceed through the protocol to the end results for your project region. Then, examine all the data in the shared Excel Online workbook to develop observations that you think are relevant to the initial question: "how likely is it that the F Element syntenic block would stay intact for over 15 million years?". Big questions that are related and can be addressed in the discussion section of your final report are: how relevant are the observations of these syntenic blocks? What are the assumptions behind this experimental design? How could you change the protocol to improve quality and reliability of the results?

As time permits, we encourage you to explore one of the genes or features in your project in more detail. What is the presumed function(s) of one of the genes? Can you generate evidence in support of these functions? Are key regions of the predicted protein conserved? Looking at the “Gene Ontology” and “Protein Domains” sections of the FlyBase report, and the Clustal Omega analysis results may be informative. Is this gene part of a known biological pathway in D. melanogaster (see the “Pathways” section of the FlyBase Gene Report)? Anything is possible here but not required.

One to three paragraphs. Start with a “final map” in this section. Use custom tracks to show the entire project region with all the final annotations of all CDS-based gene models for all members of the group. Point out the genes and TSS’s you were directly involved in annotating. Summarize the accomplishments of the group. In subsequent paragraphs, put your work in context. How do your findings support or differ from prior analyses? Refer in particular to the results of Leung et al., 2017 and questions raised in the synteny block analysis.