BGGN-213: FOUNDATIONS OF BIOINFORMATICS

The find-a-gene project assignment

Overview:
The find-a-gene project is a required assignment for BGGN-213. You should prepare a written
report in PDF format that has responses to each question labeled [Q1] - [Q10] below. You may
wish to consult the scoring rubric at the end of this document and the example report provided
online.
The objective with this assignment is for you to demonstrate your grasp of database searching,
sequence analysis, structure analysis and the R environment that we have covered in class.

Due Date:
Your responses to questions Q1-Q4 are due at the beginning of class Thursday November
16th (11/16/17). Note that these answers can be obtained very quickly (at best within 10 or 15
minutes), so if you don’t succeed at first, just keep trying.
The complete assignment, including responses to all questions, is due at the beginning of class
Tuesday December 5th (12/5/17). Late responses will not be accepted under any
circumstances.

Submission instructions:
Email your PDF document as an attachment named BGGN213_F17_[yourUCSDname].pdf to
both Ileana (ileenamitra@[Link]) and me (bjgrant@[Link]). For example, my
document would be named BGGN213_F17_bjgrant.pdf
Be sure to include your UCSD email and PID number on the first page of your report.
Email your preliminary report with answers to Q1-Q4 by November 16th so we can determine if
you have found a novel gene. Submit this preliminary report as one document with screen shots
of the results inserted appropriately.
See the demonstration report linked to on the course website for an example of format. I will
email you my decision; proceed with subsequent questions only after we are sure you have
found a novel gene.
For the final report add your results for Q5-Q10 to the preliminary report and send a final
document containing the results for all questions. Please do not send only Q5-Q10 answers as
the final report.
Questions:
[Q1] Tell me the name of a protein you are interested in. Include the species and the accession
number. This can be a human protein or a protein from any other species as long as it's function
is known.
If you do not have a favorite protein, select human RBP4 or KIF11. Do not use beta globin as
this is in the worked example report that I provide you with online.

[Q2] Perform a BLAST search against a DNA database, such as a database consisting of
genomic DNA or ESTs. The BLAST server can be at NCBI or elsewhere. Include details of the
BLAST method used, database searched and any limits applied (e.g. Organism).

Also include the output of that BLAST search in your document. If appropriate, change the font
to Courier size 10 so that the results are displayed neatly. You can also screen capture a
BLAST output (e.g. alt print screen on a PC or on a MAC press ⌘-shift-4. The pointer becomes
a bulls eye. Select the area you wish to capture and release. The image is saved as a file called
Screen Shot [].png in your Desktop directory). It is not necessary to print out all of the
blast results if there are many pages.

On the BLAST results, clearly indicate a match that represents a protein sequence,
encoded from some DNA sequence, that is homologous to your query protein. I need to
be able to inspect the pairwise alignment you have selected, including the E value and
score. It should be labeled a "genomic clone" or "mRNA sequence", etc. - but include no
functional annotation.

In general, [Q2] is the most difficult for students because it requires you to have a “feel”
for how to interpret BLAST results. You need to distinguish between a perfect match to
your query (i.e. a sequence that is not “novel”), a near match (something that might be
“novel”, depending on the results of [Q4]), and a non-homologous result.
If you are having trouble finding a novel gene try restricting your search to an organism
that is poorly annotated.

[Q3] Gather information about this “novel” protein. At a minimum, show me the protein
sequence of the “novel” protein as displayed in your BLAST results from [Q2] as FASTA
format (you can copy and paste the aligned sequence subject lines from your BLAST
result page if necessary) or translate your novel DNA sequence using a tool called
EMBOSS Transeq at the EBI. Don’t forget to translate all six reading frames; the ORF
(open reading frame) is likely to be the longest sequence without a stop codon. It may
not start with a methionine if you don’t have the complete coding region. Make sure the
sequence you provide includes a header/subject line and is in traditional FASTA format.
Here, tell me the name of the novel protein, and the species from which it derives. It is
very unlikely (but still definitely possible) that you will find a novel gene from an
organism such as S. cerevisiae, human or mouse, because those genomes have
already been thoroughly annotated. It is more likely that you will discover a new gene in
a genome that is currently being sequenced, such as bacteria or plants or protozoa.

[Q4] Prove that this gene, and its corresponding protein, are novel. For the purposes of
this project, “novel” is defined as follows. Take the protein sequence (your answer to
[Q3]), and use it as a query in a blastp search of the nr database at NCBI.
• If there is a match with 100% amino acid identity to a protein in the database, from the
same species, then your protein is NOT novel (even if the match is to a protein with a
name such as “unknown”). Someone has already found and annotated this sequence,
and assigned it an accession number.
• If the top match reported has less than 100% identity, then it is likely that your protein
is novel, and you have succeeded.
• If there is a match with 100% identity, but to a different species than the one you
started with, then you have likely succeeded in finding a novel gene.
• If there are no database matches to the original query from [Q1], this indicates that
you have partially succeeded: yes, you may have found a new gene, but no, it is not
actually homologous to the original query. You should probably start over.

[Q5] Generate a multiple sequence alignment with your novel protein, your original
query protein, and a group of other members of this family from different species. A
typical number of proteins to use in a multiple sequence alignment for this assignment
purpose is a minimum of 5 and a maximum of 20 - although the exact number is up to
you. Include the multiple sequence alignment in your report. Use Courier font with a size
appropriate to fit page width.
Side-note: Indicate your sequence in the alignment by choosing an appropriate name
for each sequence in the input unaligned sequence file (i.e. edit the sequence file so
that the species, or short common, names (rather than accession numbers) display in
the output alignment and in the subsequent answers below). The goal in this step is to
create an interesting an alignment for building a phylogenetic tree that illustrates
species divergence.
[Q6] Create a phylogenetic tree, using either a parsimony or distance-based approach.
Bootstrapping and tree rooting are optional. Use “simple phylogeny” online from the EBI
or any respected phylogeny program (such as MEGA, PAUP, or Phylip). Paste an image
of your Cladogram or tree output in your report.

[Q7] Generate a sequence identity based heatmap of your aligned sequences using R.
If necessary convert your sequence alignment to the ubiquitous FASTA format (Seaview
can read in clustal format and “Save as” FASTA format for example). Read this FASTA
format alignment into R with the help of functions in the Bio3D package. Calculate a
sequence identity matrix (again using a function within the Bio3D package). Then
generate a heatmap plot and add to your report. Do make sure your labels are visible
and not cut at the figure margins.

[Q8] Using R/Bio3D (or an online blast server if you prefer), search the main protein
structure database for the most similar atomic resolution structures to your aligned
sequences.
List the top 3 unique hits (i.e. not hits representing different chains from the same
structure) along with their Evalue and sequence identity to your query. Please also add
annotation details of these structures. For example include the annotation terms PDB
identifier (structureId), Method used to solve the structure (experimentalTechnique),
resolution (resolution), and source organism (source).

HINT: You can use a single sequence from your alignment or generate a consensus
sequence from your alignment using the Bio3D function consensus(). The Bio3D
functions [Link](), [Link]() and [Link]() are likely to be of most relevance
for completing this task. Note that the results of [Link]() contain the hits PDB
identifier (or [Link]) as well as Evalue and identity. The results of [Link]() contain
the other annotation terms noted above.
Note that if your consensus sequence has lots of gap positions then it will be better to
use an original sequence from the alignment for your search of the PDB. In this case
you could chose the sequence with the highest identity to all others in your alignment by
calculating the row-wise maximum from your sequence identity matrix.

[Q9] Generate a molecular figure of one of your identified PDB structures using VMD.
You can optionally highlight conserved residues that are likely to be functional. Please
use a white or transparent background for your figure (i.e. not the default black).
Based on sequence similarity. How likely is this structure to be similar to your “novel”
protein?

[Q10] Perform a “Target” search of ChEMBEL ( [Link] ) with

Scoring Rubric:
[45 total points available]

Q1 (4 points)
Protein name 1
Species 1
Accession number 1
Function known 1

Q2 (6 points)
Blast method 1
Database searched 1
Limits applied 1
Search output list (top hits) 1
Alignment of choice 1
Evalue and other alignment stats 1

Q3 (3 points)
Protein sequence of choice matches Subject above 1
Name in header 1
Species 1

Q4 (3 point)
Blastp output list with identities & Evalue 1
Top alignment shown with alignment statistics 1
Results indicates a “novel” gene found 1

Q5 (3 points)
MSA labeled with useful names 1
MSA trimmed appropriately (i.e. no gap overhangs) 1
Pasted MSA fits report page width (i.e. font, format) 1

Q6 (1 point)
Figure illustrates sequence clustering pattern 1

Q7 (10 points)
Heatmap figure included in report 5
Heatmap is legible (i.e. no labels obscured) 5

Q8 (10 points)
PDB identifiers from multiple species reported 5
Annotation of PDB source, resolution and technique 4
Annotation of Evalue and Sequence Identity 1

Q9 (4 points)
Structure figure provided 2
Uses white background for molecular figure 1
Figure of high resolution (i.e. not just snapshot) 1

Q10 (1 point)
Evidence of ChEMBEL searches 1