BS2040-Using BLAST to Identify Genes – Engineering Assignment Help

Assignment Task

Task 

1.1 Resources required
This practical requires access only to the internet, and installation of the NoMachine Remote Desktop (Windows / iOS / Linux) or SSH Chrome plugin (Chrome OS) software. This will be used to access the SPECTRE2 (Special Computational Teaching and Research Environment) Linux Cluster (using your University IT username and password). You will need to use the command line to run BLAST searches on SPECTRE2, but full instructions are provided below.

1.2 Entry skills
To take part in this practical you will need to be able to carry out the following tasks:
1) Logging onto a UoL client PC
2) Using Chrome to access websites (including the Blackboard VLE) with multiple windows or tabs
3) Retrieval and recording of analysis results (by copying and pasting)
4) Installation of NoMachine Remote Desktop client on your personal computer
5) Accessing the SPECTRE2 Linux Cluster using NoMachine Enterprise Client
6) Manipulating files and running BLAST on the command line
7) Use of Microsoft Word to produce a practical report
8) Electronic submission of your report as an assignment, using the Blackboard VLE

2.0 Introduction
During this practical class, you will explore the use of the BLAST (Basic Local Alignment Search Tool) (Altschul et al., 1990) program to identify a mystery gene sequence. This is one of the simplest and most often used applications in sequence database searching and seeks to answer the question “what gene does my novel sequence come from?” Each student will have an individual sequence to analyse. Once you have answered this basic question you will need to do some research to answer a series of short questions about “your” gene. You will also need to make a blog post about the human disease(s) caused by sequence variants (mutations) in your gene. A description of your analysis and the answers to the questions should be included in your practical report, which should be submitted using Blackboard. To give you insight into how bioinformaticians really use BLAST, we will be using BLAST at the command line interface (CLI) on the University of Leicester Teaching Linux Cluster, a computer called SPECTRE2. You already have an account on SPECTRE and the username and   password are the same as your University IT services account. Using the CLI can seem strange to start with but it is how most bioinformaticians (and the programs they write) interact with the computers used for their analyses. Below is a brief guide to logging onto and manipulating files in the Linux environment, written by Professor Raymond Dalgleish (and adapted for this practical).

Questions to answer in the results section of your report Section 4.1 

1) Which organism does your query DNA sequence come from? In other words, from what species does the sequence that most closely matches your query come? You should find the common English name (if one exists) and the binomial Linnean (Latin) name of the species. If there are two (or more) 100% identical sequences you should make clear which (first, second, third, etc.) you are referring to. [2 marks]

2) a. What is the next most closely related nucleotide sequence, from a different organism, which shows significant similarity to your query DNA sequence? If there are two (or more) sequences showing the same level of identity you should make clear which(first, second, third, etc.) you are referring to.

b. What percentage of your query sequence does this other sequence cover (calculated as the length of the alignment, divided by the length of the query) 
c. What is the E-value associated with this match? 
d. How similar (in terms of % identity) are these sequences? 

3) Using your PROTEIN query sequence, find out which HUMAN protein sequence is MOST similar to your mystery gene?
a. You should not only name the protein that satisfies this criterion.
b. but also describe how you found it.
c. the percentage identity to your query,
d. and the E-value of the alignment.

4) What is the human reference messenger RNA sequence (NCBI RefSeq) corresponding to the MOST similar HUMAN protein sequence identified in question 3?
HINT: the simplest way to locate the reference mRNA sequence is to perform an NCBI Gene search using the protein name from question 3: paste it into the search box at use the dropdown box to select the “Gene” database. For some genes, there are a number of search results – you should select the result (usually the top one) that corresponds to the human gene name that you searched for. Clicking on the link for this result takes you to the Gene report, where data on the gene are organised into a number of sections. You should scroll down to the “Genomic Regions Transcripts and Products” section. If there are multiple NM_ mRNA sequences (likely due to alternative transcripts) simply select the longest isoform (the one with the largest
“Aligned Length” in the dropdown box that appears when you hover over the mRNA) –: this is an arbitrary choice – it may correspond exactly to your protein query sequence, but to be sure you would need to align the protein sequence to the selected mRNA using the software tool exonerate, but this is outside the scope of this practical. For your selected mRNA you should:
a) give the accession number of the reference mRNA (starts NM_…).
b) give the version number.
c) and describe how you found it.

(5) What is the protein sequence, from another organism with the lowest alignment score, which nevertheless shows significant similarity to your query protein sequence? Refer to the note below for a definition of what “significant similarity” means. In your answer you should not only name the protein that satisfies this criterion (a)but also describe how you found it (b) the percentage identity to your query (c)and the E-value of the alignment.

Section 4.2 
NOTE: You should answer the questions below by referring to the reference mRNA sequence (RefSeq) which you located in Section 4.1, question 4. If you are not sure that you have the correct sequence, check with a demonstrator.

1) What is the RefSeqGene entry (starts NG_…) for the human gene that corresponds to the RefSeq mRNA sequence you have identified [(a), 1 mark]. You should give the HGNC (Hugo Gene Nomenclature Committee) approved symbol for the gene (b) and the HGNC approved name (c) HINT: the “Summary” section of the NCBI Gene entry contains a link to this information, under “Primary Source”.

2) Is this gene the human orthologue of the gene from which your query sequences are derived? In your answer, you should define what an orthologue is (a) and explain how your BLAST results support this conclusion (b). 

3) What human disease(s) is / are associated with mutations in the gene you have identified? You should name the disease (or one of the diseases, if there are more than one) (a) and cite an original primary research paper (NOT a review paper) that supports this association HINT: OMIM might be useful here. 

4) What is meant by the term a “conserved protein domain”? Does your gene encode any such domains? You can check by copying and pasting your protein sequence into the search box at In your answer, you should define what a conserved protein domain is (a) describe any conserved domains present in your protein OR how you know there are NO conserved domains.

5) What is the “function” of the protein product of your gene, if known? Your answer should describe the protein’s function [(a), 5 marks] and how this is related to the disease caused when it is mutated. 

OR if the function of the protein product of your gene is not known you should summarise any information on the predicted function that can be inferred from any conserved domains of the protein (a) and what is known about how the protein might cause the disease phenotype, based on your research.