QuBi/module/bio203-lab12—2018 - Revision history

imported>Weigang: /* Exercise 1. Homology searching using BLAST */

2019-05-01T19:40:54Z

Exercise 1. Homology searching using BLAST

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	<li>Sequence identity, number of matched bases, and number of gaps between the matched sequences		<li>Sequence identity, number of matched bases, and number of gaps between the matched sequences
	</ol>		</ol>
	<li>Click "Genome Data Viewer" at top right will bring you to a genome browser~~</ol>~~		<li>Click "Genome Data Viewer" at top right will bring you to a genome browser
	<li>Mouse-over the central segment and click the link "GenBank View". A standard GenBank file of this gene will load. Locate the 1st "mRNA" feature block and write down the following structural information about this gene in your lab report file:		<li>Mouse-over the central segment and click the link "GenBank View". A standard GenBank file of this gene will load. Locate the 1st "mRNA" feature block and write down the following structural information about this gene in your lab report file:
	<ol>		<ol>

imported>Weigang: /* Exercise 1. Homology searching using BLAST */

2019-05-01T19:40:01Z

Exercise 1. Homology searching using BLAST

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	<li>Sequence identity, number of matched bases, and number of gaps between the matched sequences		<li>Sequence identity, number of matched bases, and number of gaps between the matched sequences
	</ol>		</ol>
	<li>Click ~~the link for~~ "~~5' side~~" ~~(next to '''Features''')~~ will bring you a standard GenBank file of this gene. Locate and write down the		<li>Click "Genome Data Viewer" at top right will bring you to a genome browser</ol>
	following structural information about this gene in your lab report file:		<li>Mouse-over the central segment and click the link "GenBank View". A standard GenBank file of this gene will load. Locate the 1st "mRNA" feature block and write down the following structural information about this gene in your lab report file:
	<ol>		<ol>
	<li>Gene ~~accession (~~ID ~~number)~~		<li>Gene ID
	<li>Total length of the gene		<li>Total length of the gene
	<li>Number of introns		<li>Number of introns

imported>Weigang at 03:22, 4 May 2018

2018-05-04T03:22:52Z

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	<span style="color: Seagreen;font-weight:bold;font-size:large;">Lab 12. Bioinformatics Exercises: BLAST & Genomes>		<span style="color: Seagreen;font-weight:bold;font-size:large;">Lab 12. Bioinformatics Exercises: BLAST & Genomes
	==Expected Learning Outcomes==		==Expected Learning Outcomes==
	* Be able to perform NCBI BLAST search for homologous sequences in GenBank.		* Be able to perform NCBI BLAST search for homologous sequences in GenBank.

imported>Lab at 18:08, 2 May 2018

2018-05-02T18:08:50Z

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	# Click on "CDS" and notice that coding sequences are now highlighted		# Click on "CDS" and notice that coding sequences are now highlighted
	# Fill in '''Table 3''' in your lab report file for each coding sequence you could identify:		# Fill in '''Table 3''' in your lab report file for each coding sequence you could identify:
	# ~~DRAW a diagram of this gene based on~~ the ~~above~~ exon and ~~intron coordinates~~. ~~Use the provided graph paper for~~ this~~. MAKE SURE~~ to ~~indicate your names+ section # and RETURN IT TO YOUR TA at the end of~~ the ~~session, otherwise you will not get credit~~ for ~~this exercise.~~		# Obtain the intron/exon gene structure and copy into your lab report file. To do this:
	##~~Label the top of~~ the ~~diagram with basic information, such as the gene's name and species information.~~		##go back to the Genbank page for AF527840 (as instructed above)
	##~~Label coordinates for introns~~, ~~exons~~, ~~3'/5' UTRs, start-codon~~, and ~~stop-codon coordinates.~~		##click on the "Graphics" link
	##~~Draw the~~ diagram ~~mostly to scale. It does NOT have to be perfect~~, ~~but make a reasonable effort. Put a scale bar~~ and ~~length markers on~~ your ~~drawing.~~		##you will see a window with a diagram, showing the genomic sequence in green, the primary transcript in purple, and the coding sequence in red
			##copy and paste this diagram into your lab report (or create a desktop picture, crop as needed and paste into your lab report file)
	# Answer the following questions, in your lab report file:		# Answer the following questions, in your lab report file:
	## What is the total length of exons, introns, and coding sequences of this gene?		## What is the total length of exons, introns, and coding sequences of this gene?

imported>Lab at 15:18, 2 May 2018

2018-05-02T15:18:45Z

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	#there, enter your input sequence format as 'ALN' and in the window below that, paste your aln file		#there, enter your input sequence format as 'ALN' and in the window below that, paste your aln file
	#click on 'run boxshade'		#click on 'run boxshade'
	#under results: click on '~~boxsgade~~ output 1"--- here's your alignment!		#under results: click on 'boxshade output 1"--- here's your alignment! (this should open with adobe acrobat and might take a bit of time)
	#the output is a pdf file-- save it and import into your word file (as page 2) by doing an "Insert--- picture from file" in MS Word- you will have two pages: page 1 with your MDM2 sequences, and page 2 with your alignment		#the output is a pdf file-- save it and import into your lab report word file (as page 2) by doing an "Insert--- picture from file" in MS Word- you will have two pages: page 1 with your MDM2 sequences, and page 2 with your alignment


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	# Explain the following BLAST terms: “Expect” (e-value) [http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=FAQ#expect Read this FAQ], “Identities”, “Gap”, “Strand”.		# Explain the following BLAST terms: “Expect” (e-value) [http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=FAQ#expect Read this FAQ], “Identities”, “Gap”, “Strand”.
	# Which is a statistically more significant match by BLAST, a match with an e-value=1e-5 or a match with an e-value of 1?		# Which is a statistically more significant match by BLAST, a match with an e-value=1e-5 or a match with an e-value of 1?
	~~# If you want your match to be biologically relevant (and not random, chance matches), should you use the default e-value cutoff of 10?~~
	# List and describe individual elements of a typical human gene based on mdm2.		# List and describe individual elements of a typical human gene based on mdm2.
			#what are two determinants that can lead to the production of isoforms for a specific locus?
	# What is the "GT-AG" rule? Explain how to read the sequence logos. Explain the significance of sequence conservation at exon-intron junctions.		# What is the "GT-AG" rule? Explain how to read the sequence logos. Explain the significance of sequence conservation at exon-intron junctions.
	# ~~Describe~~ biological significance of alternative splicing, using mdm2 gene as an example.		# Discuss biological significance of alternative splicing, using mdm2 gene as an example.
			#look at your alignment from part III: what are the black boxes- the grey boxes?
			#do you see many gaps/insertions?do you think there is a pattern?

	----		----

imported>Lab at 19:40, 1 May 2018

2018-05-01T19:40:22Z

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	==Exercise 3. MDM2 homologs in other species==		==Exercise 3. MDM2 homologs in other species==
	This exercise will consist in comparing the predicted protein sequences of MDM2 in three species: human (H. sapiens), mouse (M. musculus) and zebrafish (D. rerio).		This exercise will consist in comparing the predicted protein sequences of MDM2 in three species: human (H. sapiens), mouse (M. musculus) and zebrafish (D. rerio).
	You will need to download ~~each~~ sequence ~~from~~ the ~~appropriate database~~, and copy each sequence into a MS Word file using the following format:		You will need to download the human MDM2 sequence, find the mouse and fish homologs, and copy each sequence into a MS Word file using the following format:

	Names (First, Last)		Names (First, Last)

imported>Lab at 18:58, 1 May 2018

2018-05-01T18:58:27Z

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	==Exercise 3. MDM2 homologs in other species==		==Exercise 3. MDM2 homologs in other species==
	This exercise will consist in comparing the predicted protein sequences of MDM2 in three species: ~~humans~~ (H. sapiens), mouse (M. musculus) and ~~Zebrafish~~ (D. rerio).		This exercise will consist in comparing the predicted protein sequences of MDM2 in three species: human (H. sapiens), mouse (M. musculus) and zebrafish (D. rerio).
	You will need to download each sequence from the appropriate database, and copy each sequence into a MS Word file using the following format:		You will need to download each sequence from the appropriate database, and copy each sequence into a MS Word file using the following format:

imported>Lab at 18:56, 1 May 2018

2018-05-01T18:56:28Z

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	#find the protein sequence and paste into your page as above. Make sure you use the right program and database for a protein BLAST!		#find the protein sequence and paste into your page as above. Make sure you use the right program and database for a protein BLAST!

	you will now make an ~~alignement~~ of all three sequences to see potential identities or similarities between them		you will now make an alignment of all three sequences to see potential identities or similarities between them

	#this involves two steps: first, the production of an output file by a program called "Clustal W"		#this involves two steps: first, the production of an output file by a program called "Clustal W"
	# and second, the processing of this file to generate an ~~alignement~~ figure by a program called "Boxshade"		# and second, the processing of this file to generate an alignment figure by a program called "Boxshade"
			#go to this link: http://www.genome.jp/tools-bin/clustalw
			#in the top window, paste in your three sequence by selecting from your first ">" sign to the end of your file (do not take your header, with your names)
			# click on "multiple alignment"
			#you will see an 'aln' output file: select the file including the header on top "CLUSTAL 2.1 multiple sequence alignment" down to the bottom of the file (no extra spaces)- copy in the buffer
			#to do the alignment figure, go to : https://embnet.vital-it.ch/software/BOX_form.html
			#there, enter your input sequence format as 'ALN' and in the window below that, paste your aln file
			#click on 'run boxshade'
			#under results: click on 'boxsgade output 1"--- here's your alignment!
			#the output is a pdf file-- save it and import into your word file (as page 2) by doing an "Insert--- picture from file" in MS Word- you will have two pages: page 1 with your MDM2 sequences, and page 2 with your alignment

imported>Lab at 18:35, 1 May 2018

2018-05-01T18:35:12Z

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	# copy the protein sequence by highlighting all residues from the initial 'M' to the last residue- nothing else		# copy the protein sequence by highlighting all residues from the initial 'M' to the last residue- nothing else
	# paste the sequence into your word file as instructed above		# paste the sequence into your word file as instructed above
	#
	#		now let's find the mouse homolog using the human sequence as a query:
	#now let's find the mouse homolog using the human sequence as a query:
	#go to the main NCBI link: https://www.ncbi.nlm.nih.gov		#go to the main NCBI link: https://www.ncbi.nlm.nih.gov
	#on the right side, under "Popular resources", click on "Blast"		#on the right side, under "Popular resources", click on "Blast"
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	#you get a number of hits- scroll down to the best one (under "alignments") and click on "gene" on the right side, under "related information"		#you get a number of hits- scroll down to the best one (under "alignments") and click on "gene" on the right side, under "related information"
	#you are now on the mouse MDM2 locus page: find the protein sequence to the LONGEST isoform and paste into your page as above		#you are now on the mouse MDM2 locus page: find the protein sequence to the LONGEST isoform and paste into your page as above
	#
	#		now let us get the Zebrafish homolog:
	#now let us get the Zebrafish homolog: go to the genome portal : http://zfin.org
			#go to the genome portal : http://zfin.org
	#find the protein sequence and paste into your page as above. Make sure you use the right program and database for a protein BLAST!		#find the protein sequence and paste into your page as above. Make sure you use the right program and database for a protein BLAST!

	you will now make an alignement of all three sequences to see potential identities or similarities between them		you will now make an alignement of all three sequences to see potential identities or similarities between them

	#this involves two steps: first, the production of an output file by a program called "Clustal W"		#this involves two steps: first, the production of an output file by a program called "Clustal W"
	# and second, the processing of this file to generate an alignement figure by a program called "Boxshade"		# and second, the processing of this file to generate an alignement figure by a program called "Boxshade"

imported>Lab at 18:33, 1 May 2018

2018-05-01T18:33:49Z

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	#you get a number of hits- scroll down to the best one (under "alignments") and click on "gene" on the right side, under "related information"		#you get a number of hits- scroll down to the best one (under "alignments") and click on "gene" on the right side, under "related information"
	#you are now on the mouse MDM2 locus page: find the protein sequence to the LONGEST isoform and paste into your page as above		#you are now on the mouse MDM2 locus page: find the protein sequence to the LONGEST isoform and paste into your page as above
			#
			#
			#now let us get the Zebrafish homolog: go to the genome portal : http://zfin.org
			#find the protein sequence and paste into your page as above. Make sure you use the right program and database for a protein BLAST!

			you will now make an alignement of all three sequences to see potential identities or similarities between them
			#this involves two steps: first, the production of an output file by a program called "Clustal W"
			# and second, the processing of this file to generate an alignement figure by a program called "Boxshade"