Bioinformatics

Part 1: BLASTP search and multiple sequence alignment

1. Use the Unknown sequence in the sequence.fsa file as a query sequence.

2. Choose an appropriate BLAST tool to search against the NCBI nr (non-redundant) database using this unknown sequence as a query.
https://blast.ncbi.nlm.nih.gov/Blast.cgi

3. Based on the blast search result (usually the top 1 hit), answer the following questions:
(a) what is this query sequence most likely to be? From which species / organism it comes?
(b) what is its possible function?
(c) can you find a homologous sequence in Drosophila melanogaster? If not, how can you modify your blast search to get one?

4. Retrieve TEN sequences from the hit list (each species can only show once, skip sequences in the top list if from the same species) and save into a file:
Each species can only show once, skip sequences in the top list if from the same species (* hint: do not choose the sequences labelled as isoform)
Must include one homologous sequence from Drosophila melanogaster.
(a) file must be saved in FASTA format (same as the query sequence file). You can make a copy of this query file and edit it use text editor such as Notepad or Wordpad.
(b) name your file as BIO760MidtermSeq_your last name.fas (fas is the file extension). For example, my file will be named as MidtermSeq_Gu.fas

5. Visit the CLUSTAL webserver at https://www.ebi.ac.uk/Tools/msa/clustalo/
(a) Align all ten sequences by upload your saved sequence file (BIO760MidtermSeq_your last name.fas) in step 4.
(b) Align with default options. Run a complete alignment.
(c) Download the alignment file (CLUSTAL_NUM File) and saved it as MidtermAln_your last name.clustal_num

Part 2: Phylogenetic Analysis

Now you can use the following data from the Florida dentist case. In 1990 a young woman tested positive for HIV, but she had no known risk factors for getting the virus. Investigation showed that she had had an invasive dental procedure done by an HIV-positive dentist years earlier. It turned out that a few other patients of this dentist were also HIV-positive without identifiable risk factors. The CDC (Centers for Disease Control) took blood samples and isolated Env viral sequences from three HIV-positive patients (E, F and G), the same viral sequence from the dentist, and from two HIV-positive individuals from the area who had no contact with the dentist. The last two are controls: Local Control 3 and Local Control 22.

1. Viewing the multiple sequences in dentist.fas, how are these sequences similar or different?

2. Download and install software package MEGAX from the website: https://www.megasoftware.net/home

3. Download the input file: dentist.fas (in FASTA format)

4. Use MEGA to align the sequences: choose Align by ClustalW with default parameters. Save your alignment file in .meg format.

Use the sequence alignment in step 4 to complete the following:

5. Does the information in the multiple sequence alignment verify patterns you saw by looking at the raw sequence in step 1? Summarize what the alignment shows in your own words. Does the multiple sequence alignment show you additional patterns? If so, elaborate them.

6. Calculate a Pairwise distance matrix using p-distance model. Fill in the following table:

Dentist PatientE PatientF PatientG LocalControl3 LocalControl22
Dentist
PatientE
PatientF
PatientG
LocalControl3
LocalControl22

7. Using the p distance matrix, build a tree with the Neighbor Joining (NJ) distance method. Run bootstrap analysis of this tree with 1,000 replicates. Label your NJ tree with branch length and bootstrap values. Copy your NJ tree to clipboard and paste it here.

8. Looking at the NJ distance tree, make a summary of argument to judge and jury regarding the claims from patients F, G and E that they acquired HIV from the dentist. Be use to include data from local controls in your argument.

9. Build a maximum likelihood (ML) tree using Tamura-Nei model. Run bootstrap analysis of this tree with 500 replicates. Label your ML tree with branch length and bootstrap values. Copy your ML tree to clipboard and paste it here.

10. Draw a Maximum Parsimony (MP) tree.

11. Compare the topology of ML and MP tree with NJ tree, do they support your argument based on the NJ tree?