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  • Q1:B. Briefly describe Ensembl: what it is, what information it holds, and which other entities are like it See Answer
  • Q2:Recombinant DNA You have received a PCR product from an unknown organism that caused fever and a strange rash in young child. The family of the child has two kittens, and the physician treating the child suspects an infection with Bartonella. Strangely, the growth characteristics do not match those of known Bartonella species. The clinical lab has amplified an 825 base pair fragment of the unknown organism's genome and has asked you to use the PCR product to investigate the identity of the unknown bacteria. Once you received the DNA you decided to have the PCR product sent out for sequencing. Bioinformatics 1) Using a Blastn search determine the top 3 most similar DNA sequences and using Blastx determine the top 3 most similar proteins. What gene did the clinical lab PCR amplify? • What is the function of the protein? • Based on the results from this gene what organism did you isolated? And what species is your organism most similar to?See Answer
  • Q3:PCR/Primer Design and Cloning Now that you have learned a little bit about the genetics of the unknown organism you have been asked to create a real-time PCR assay to detect the unknown in other patient's blood samples using the gene that was amplified by the clinical lab. To create your standards for your real-time PCR assay you need to clone your DNA fragment into a plasmid (pUC19) using standard PCR techniques. 2) Using the 825 bp sequence for your unknown fragment, please create a primer pair that will amplify a 500 base pair fragment of the gene. Additionally, please add restriction sites to the ends of your primers. Please provide the following: • Sequences of the primers • Primer Tm • The location of the primers in the sequence • The restriction sites you used. Remember: Check to make sure that the restriction sites will only cut the primers and are not found anywhere else in the DNA sequence but are found in pUC19.See Answer
  • Q4:Part 1: Smith-Waterman Algorithm Instructions: 1. Copy the "STS protein query sequence" from the week 3 links page (This is the one letter code for the protein encoded by the Resveratrol synthase gene, the gene that catalyzes the final step in the resveratrol synthesis pathway). Be sure to include the ">STS query" on the first line, or the program won't accept it. 2. Go to https://www.ebi.ac.uk/Tools/sss/ and choose SSEARCH, then "protein". This will do a Smith-Waterman local alignment on your protein sequence. 3. Choose the UniProtKB/TrEMBL database (under Step 1 on the page) 4. Paste the STS protein query sequence into the paste window 5. Choose SSEARCH under step 3, then click "More options". Here you will find a number of parameters including the substitution matrix. Search UniProt using three different substitution matrices: • BLOSUM 50 • PAM 120 • PAM 250 Be patient, the calculation will take a while. Questions: 1. What is the name and score of the best hit for each matrix? 2. What is the e-value of the best hit for each matrix? 3. Why do you think the results may have changed? 4. What do e-values mean and how do we interpret them?See Answer
  • Q5:Part II: Needleman-Wunsch Algorithm Instructions: 1. Go to https://www.ebi.ac.uk/Tools/sss/ and select GGSEARCH then protein. This will do a Needleman-Wunsch global alignment on your protein sequence. 2. Choose UniProtKB/TrEMBL as your database (step 1) 3. Paste in your STS protein query sequence (step 2) 4. Click More options... on step 3 and choose BLOSUM50 as your scoring matrix and then click Submit. Questions: 1. What is the name and score of your top hit? 2. How do these results differ from what you got with the Smith-Waterman algorithm (SSEARCH)? 3. Why do you think the results differed?See Answer
  • Q6:Part III: FASTA Algorithm Instructions: 1. Go to https://www.ebi.ac.uk/Tools/sss/ and select FASTA, then protein. 2. Choose UniProtKB/TrEMBL as your database (step 1) 3. Paste in your STS protein query sequence (step 2) 4. Click More options... on step 3 and run your queries for the following choices of parameters: Scoring Matrix BLOSUM 50 BLOSUM 80 BLOSUM 80 BLOSUM 80 Gap Open -10 -10 0 -64 Gap Extend -2 -2 0 -16/nQuestions: 1. Did these calculations take as long as the Smith-Waterman search? If so, why? 2. Were the results different from the Smith-Waterman search? Why do you think this happens? 3. What is the effect of changing to a higher cutoff BLOSUM matrix (i.e. from 50 to 80) and what does it mean? 4. What is the effect of changing the Gap Open and Gap Extend parameters? Why do you think you observed what you did?See Answer
  • Q7:Part IV: using BLAST Instructions: 1. Go to http://blast.ncbi.nlm.nih.gov/Blast.cgi 2. Click on nucleotide BLAST. 3. Copy STS mRNA query sequence from the week 3 links page then paste it into the Query sequence window (note that this is a nucleotide sequence). 4. Under Database, choose Standard databases 5. Choose optimize for somewhat similar sequences (blastn) under Program selection, (note this is not the default) then click BLAST. 6. Now, run the same query against the expressed sequence tags database (est). (Database "Others", then scroll to "expressed sequence tags") Questions: 1. What are the names, scores, and e-values of your best hits for the two different searches you just performed? 2. Why are these results so different?See Answer
  • Q8:Part V: Comparing FASTA and BLAST and SSEARCH Instructions: 1. Go to https://www.ebi.ac.uk/Tools/sss/ 2. You will do a nucleotide search using the STS mRNA from the previous step using FASTA, BLAST, and SSEARCH. 3. Leave the database as the default setting. 4. Select FASTA as your program, select more options, and set the following parameters: 5. Match/Mismatch: +3/-2 6. Gap Open: -5 7. Gap Extend: -2 8. Run the search and note the name, score, and e-value of the best hit. 9. Repeat this for BLAST and SSEARCH. Questions: 1. What were your best hits for each of the different search algorithms? 2. Were they different and if so why? 3. Why was it necessary to set the parameters as described above before running the searches?See Answer
  • Q9:Part VII: Using VAST+ to find structural homologs Instructions: 1. We will use the VAST+ program to find homologs of the SARS-CoV2 Spike Protein (PDB ID 6VXX). Go to https://www.ncbi.nlm.nih.gov/Structure/vastplus/vastplus.cgi and enter 6VXX for the PDB ID. The NCBI structural citation for this PDB file will come up. For one of these hits, click the little "+" box near the PDB ID. Click Visualize 3D Structure Superposition and then iCn3D. 2. 3. Take a look at some of the tools for analysis and visualization. You can learn quite a bit about the relationship between the aligned structures using these tools. 4. Go back to your search and use the filters to find a match around 80% identity, and one with around 30% identity. 5. Save a picture of each of your superimposed alignments. To do this go to Style then set the background to white. Then click the little camera (or go File to Save File to iCn3D PNG image). Paste the image inline into this homework. Questions: 1. What were the name, sequence identity, and RMSD of each of the hits you chose? 2. How did they differ both in terms of sequence and in terms of structure? 3. Is there anything special about the regions of the protein that have weaker structural alignment? 4. How might a tool like this be used to learn something about the coronavirus? 5. Why is structural alignment different from sequence alignment and why might it be useful? 6. Starting with this page: https://www.ncbi.nlm.nih.gov/Structure/vastplus/docs/vastplus_help.html, do some research and explain how does VAST+ works (in your own words, do not copy and paste anything!).See Answer
  • Q10:Part I: Learning about molecular phylogenies 1. What is the basic assumption underlying a molecular phylogeny? Why must we distinguish between gene trees and species trees? 2. 3. Why don't genes always evolve by a series of bifurcations (i.e., by a series of single base changes)? 4. What are the four steps to constructing a molecular phylogeny? 5. What is an orthologous sequence? 6. What is a paralogous sequence? 7. What is a xenologous sequence? 8. Which type of sequences should you use for a species phylogeny? 9. What is the difference between multiple sequence alignments to discover motifs, etc., vs for constructing phylogenies? 10. Why is Clustal W not a very good choice for constructing species phylogenies?/n10. Why is Clustal W not a very good choice for constructing species phylogenies? 11. Please use the supplemental material on the links page to answer the following questions. What is a phylogenetic tree composed of? What is the difference between rooted and unrooted phylogenetic trees? What are the two major groups of analyses used to examine phylogenetic relationships? 0 0 0 What is a paraphyletic grouping? What happens if a multiple alignment is poor? What is the best way to deal with parts of an alignment that are uncertain due to gaps? What sorts of phylogenies are best constructed using DNA sequence alignments? What sorts of phylogenies are best constructed using protein alignments? What sorts of phylogenies are best constructed using ribosomal RNA sequence alignments? 0 0 0 0 0 What is a homoplasy? 0 Why can't we simply construct all possible trees, score each one, then pick the one with the best score? 0See Answer
  • Q11:Part II: Distance matrix methods 1. Answer the following questions: What is the general approach used by distance matrix methods to construct a phylogeny? a. b. 2. 3. 4. a. 5. a. b. 6. What are the main differences between UPGMA and neighbor-joining methods? Take your protein sequences from the links page and import them into Mega. Align them via Clustal W and save the alignment. Use your alignment to construct UGMA and Neighbor-Joining Trees. What are the differences and similarities between the trees? Repeat the above with an alignment based on MUSCLE instead of ClustalW. How does this change the results? Why do you think the results are different? Include labeled screen shots of your different trees.See Answer
  • Q12:Part III: Maximum parsimony methods 1. 2. 3. 4. What is the key assumption of maximum parsimony methods? How does this differ from distance matrix methods? What are the advantages of maximum parsimony methods? What are the disadvantages of maximum parsimony methods?See Answer
  • Q13:Part IV: Maximum likelihood methods 1. Go to a. b. C. d. e. f. https://evolution.genetics.washington.edu/phylip/programs.html What are the assumptions of DNAML? What does DNAMLK do? How is DNAMLK related to DNAML? What are the assumptions of DNAMLK? What is PROTML? Why isn't DNAML suited to protein sequence data?/nWhy can the inclusion of the third base in a codon be misleading when constructing phylogenetic trees? h. What is the strategy taken by DNAML? i. How does Felstenstein recommend that you compensate for the dependence on the order of species inputs? j. What is "star decomposition?" 2. Use MEGA 10 to construct Maximum Likelihood and Maximum Parsimony Trees from your previous alignments. How do they compare to the other trees?See Answer
  • Q14:Part V: Tree evaluation 1. 2. 3. 4. 5. What are the three basic ways to resample the data for tree-building? What is jackknife resampling? What is bootstrap resampling? How does it differ from jackknife resampling? Recreate one of your ML trees except use Bootstrap Resampling as a method of tree evaluation. 6. How did your tree change?See Answer
  • Q15:Part I: Finding specific types of prokaryotic sequences 1. Why is it relatively easy to find genes in bacterial genomes? 2. Go to the week 7 folder, select E.coli, then copy the entire DNA sequence. These are the first 10,000 bp of the raw E.coli genomic DNA sequence that we are going to study using various DNA analysis programs. 3. Go to http://www.fruitfly.org/seq tools/promoter.html paste the sequence into the window, then select "Prokaryotic," "yes" for "include reverse strand?" and click "submit." • How many promoters does it find on the forward strand? • Where do the three highest-scoring promoters start and end? • How many promoters does it find on the reverse strand? • Where do the three highest-scoring promoters start and end? 5. Go to BPROM - Prediction of bacterial promoters (softberry.com) and click "help.” • How accurate is BPROM? • How far is it from most bacterial promoters to the protein coding sequences? 6. Return to BPROM - Prediction of bacterial promoters (softberry.com) paste the E. coli query into the window then click "process." 4. How many promoters does it find? 5. Where do the three highest-scoring promoters start and end? 6. How do these compare with the previous site?See Answer
  • Q16:Part II: Finding prokaryotic genes 1. Go to FGENESB - Bacterial Operon and Gene Prediction (softberry.com) 2. Select "Escherichia coli K-12" as closest organism, then paste the E. coli query into the window and click "process." • How many genes does it find? • How many are on the + strand? • How many are on the - strand? Open a new window and go to https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3 the GenBank flatfile for the Escherichia coli str. K-12 substr. MG1655 genomic sequence. You will need to select "Change region shown" to "selected region : "begin" to "10000" then click "update view" to see the annotation for this region. • Did fgenesB find any genes not listed on the GenBank Flatfile? • Do the starts and stops agree with those listed on the GenBank flatfile? • If not, which ones are different? 3. Now go to https://en.wikipedia.org/wiki/GeneMark • How does GeneMark find genes? 4. Now go to http://opal.biology.gatech.edu/GeneMark/gmhmmp.cgi •paste the E. coli sequence into the window, then select "species: Escherichia coli_K_12_substr_MG 1655, Output format should be "GFF." Output options should be "PDF" then click "Start GeneMark.HMM™ • When the output comes up click on the link to "coordinates of predicted genes" • How many genes does it find? How many are on the + strand? How many are on the - strand? • Which genes were not listed on the GenBank Flatfile? • Do the starts and stops of the genes they both found agree with those listed on the GenBank flatfile? If not, which ones are different?See Answer
  • Q17:1. What is the difference between energy and free energy and what does it have to do with RNA structure? What structural elements are used in MFE algorithms to compute the free energy of an RNA secondary structure?See Answer
  • Q18:2. An MFE algorithm tries to find the secondary structure with the lowest free energy. What is the point of doing this? What is the significance of the MFE structure?See Answer
  • Q19:3. RNAFold also uses partition-function methods (AKA thermodynamic ensemble methods). What is a partition function and how is it related to free energy? What are the advantages and disadvantages of calculating partition functions?See Answer
  • Q20:1. For BLAST/FASTA tell us how many significant results were found, and which sequences were most closely related and who they came from. Identify and try to explain any unexpected similarities and any differences between the searches using DNA versus amino acid sequences.See Answer
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