CSB352H Bioinformatic Methods

Summer 2024 – Lab Report 1

CSB352: Lab Report 1

This assignment asks you to investigate a gene of unknown function from Arabidopsis thaliana, by identifying orthologous sequences from other organisms and performing a multiple sequence alignment of your discovered orthologs. In a nutshell, your lab report will introduce your gene (see arabidopsis.org to as described in the Gene Assignment section of the Lab Reports part of Portal; details on the gene/protein are also available from this database), give explicit details of the analysis you performed (BLAST and MSA), describe the particulars of what you found and comment on what you think it means in a biological context – what could the function of this gene be? You will also perform. a phylogenetic analysis on a subset of BLAST, generating the best-resolved (i.e. greatest number of nodes that are well supported based on their bootstrap scores) phylogenetic tree you can. Is there evidence of a gene duplication event in a recent ancestor of Arabidopsis thaliana and the other species whose sequences are homologous? The report should be no longer than 6 pages, double-spaced (excluding figures and references)! You should cite all primary sources plus all tools/websites/data sources used, using the journal Nature’s reference format.

Things you need to keep in mind

• about your query:

- are you going to BLAST a nucleotide or protein sequence (or both)? Genomic DNA or mRNA? Is your sequence long enough?

• about your search database:

- do you want an all-encompassing database or a well-curated one?

• about your BLAST parameters:

- which BLAST search algorithm is best for finding orthologs in other species?

- are you going to use blastn, blastp, blastx? Why?

- which scoring matrix should you use?

• about your alignment parameters:

- which alignment program are you going to use?

- how rigorous are your gap penalty parameters?

- does your alignment “make sense”?

• about your phylogenetic analysis:

- which method(s) are you going to use?

- include the closest A. thaliana paralog in the analysis (if there is one)

- note that duplicated genes are typically not identical and that the “nonredundant” database does actually have redundancies in it.

Your report should consist of the following sections:

Introduction: (4 marks)

A short, two-paragraph description of your gene and the analyses performed.

Be sure to include the following information:

- organism

- sequence source (i.e GI or AGI ID number, sequence type)

- physical location (if available)

- functional product (if known) – pI, MW, and other parameters as appropriate

Summarize the genes and taxa included in your analysis. You can make note of any pertinent alignment patterns you detected in your MSA (ie: conserved regions, range of sequence identity, etc.), and state what you are trying to ascertain with your phylogenetic analyses (do not copy the above objective!).

Methods: (8 marks)

A concise explanation of the parameters, data sources, web sites used and their citations, and settings for each in silico experiment performed in the final analysis you’re presenting in this report. Note: The marker must be able to replicate your results solely from the information in this section!

Be sure to detail the BLAST search(es) you performed along with any parameter adjustments you made and justify them. Include the Blast version and databases version. Describe which alignment program you chose as well as all parameters used in the analysis. Explain the design of your phylogenetic analysis in prose form, including the software used, the alignment and phylogenetic tree-building algorithm(s) used (justify your choices), the parameters you chose for each algorithm. DO NOT INCLUDE RESULTS IN THIS SECTION (see below.)

Tip: The literature accepted E-value cutoff for accepting a sequence as orthologous to the query sequence is 1e-20 as per Lecture 2. If your assigned gene returns few or no orthologs in the search, then contact [email protected] request a new gene.

Results: (12 marks)

Your results will include a short discussion on what was found and anything you did that was “out of the ordinary” and why. Also it will include 3 figures, complete with proper captions. Each figure should include a caption that describes the figure. The caption should be detailed enough that the readers do not need to read the text in order to understand the figure:

1) a table of your orthologous sequences (and the most similar A. thaliana paralog if there is one) and their associated BLAST statistical values. Be sure to justify your choice of sequences you selected for alignment (are all sequences   truly “nonredundant”?), and include only the sequence retrieval (do not include pairwise alignments, please).

2) your best multiple sequence alignment of the top 15 orthologs from your BLAST search – include only one other A. thaliana sequence that is not the same as your assigned gene, that is, include the closest Arabidopsis thaliana paralog, if one exists. Make sure the MSA is formatted in such away that conserved regions, residues, or nucleotides are highlighted. Trim it accordingly (i.e.: don’t include uneven tail-ends, as they are uninformative.)

3) A write-up in which the results of your phylogenetic analysis are displayed and described (but not analyzed). Include within this page your preferred phylogenetic tree with branch lengths and bootstrap values. In the text, explain any pertinent trends, divergences, or similarities seen in your tree.

Ifyour alignment or tree viewer program doesn’t generate imagefiles to import into your report document, use screenshots with the PrintScrn button then paste followed by cropping.

Ensure that the gene name and organism name (abbreviated if need be) is readable for each sequence in the alignment. Your query sequence should be at the top, followed the closest paralog and orthologs sorted from most to least similar.

Discussion: (6 marks)

Interpret your results in a biological context. Can you suggest a possible function of your gene? Do its relationships with other taxa make sense to you? Is this gene widespread in  the tree of life (or at least in plants)? Can you thus infer whether it is relatively old or new? Is it highly conserved across taxa? Are some regions highly conserved and others not? What might this tell you about its function? Do any species have duplicated versions of this gene? When did the duplication occur?