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Sequence Comparison and
Genome Alignment in the
Human Genome
Jian Ma
Powerpoint: Casey Hanson
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Introduction
This goals of the lab are as follows:
1.
Gain experience using BLAST and Genome Browsers by looking at
repeat families in the VHL gene.
2.
Become familiar with BLAT and the UCSC website by discovering the
identity of a mystery sequence.
3.
Visualize pairwise multi-genome alignment and chromosomal
rearrangements.
4.
View phylogeny based multi-genome alignment.
5.
Use UCSC tools and Galaxy to intersect annotated functional regions
between human and other placental animals.
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Step 0: Shared Desktop Directory
For viewing and manipulating files on the classroom
computers, we provide a shared directory in the
following folder on the desktop:
classes/mayo
In today’s lab, we will be using the following folder in
the shared directory:
classes/mayo/ma
Bacterial Genome Assembly v9 | C. Victor Jongeneel
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BLAST & Genome Browser
In this exercise, we will use BLAST (Basic Local Alignment Search Tool) to search for
significant occurrences of a class of transposable elements (TEs) called Short INterspersed
Elements (SINEs), specifically of the ALU family, in the well-known VHL tumor suppressor
gene.
The goal of this exercise is to gain experience using BLAST, particularly blastN, and the
UCSC genome browser to answer biologically relevant questions.
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Step 1A: BLAST VLH in ALU Database
Go to the following web page: http://blast.ncbi.nlm.nih.gov/Blast.cgi
Click nucleotide_blast
In the Enter Query Sequence box, paste the accession # for VHL:
AF010238
In the Database drop-down list, select the following:
Human ALU repeat elements (alu_repeats)
Click the BLAST button.
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Step 1B: BLAST VLH in ALU Database
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Step 2A: Interpreting BLAST Results
Color Indicates Quality of Match
Coordinates of VHL gene
Very Good Matches
A match is a significant
similarity between a
region of the query and
a region of a database
sequence.
Good Matches
Lines between boxes
indicate ‘gaps’ between
matches in the query
sequence. (The next
slide has a legend for
interpretation)
Okay Matches
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Step 2B: Interpreting BLAST Results
Exonic regions less likely to have ALU repeats.
Matches like this are likely to be located in intronic regions.
Note the following legend for interpreting a match.
Intron
Excellent Match
Exon
Intron
Good Match
Jian Ma | Sequence Comparison and Genome Alignment
Exon
Intron
Okay Match
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Step 3A: Examine VHL in UCSC Browser
Let’s look at the structure of the VHL gene in a Genome Browser to
verify that ALU elements are confined to the introns.
Go to the following web page: http://genome.ucsc.edu/
Click Genome Browser
In the search term, type VHL
Click submit
Click the 2nd link: VHL (uc003bvd.3) at chr3:10183319-10195354
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Step 3B: Examine VHL in UCSC Browser
Enter chr3:10,177,301-10,201,372 into input box and click go.
Right click on tracks NOT shown below and hide them.
Right click on the RepeatMasker track and click full. It is dense by default.
Adjust the zoom until you get a view you are comfortable with.
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Step 3C: Examine VHL in UCSC Browser
Repeat tracks are 3’ to the gene, 5’ to the gene, or in the intronic region.
This validates our hypothesis.
ALUs are not the only family of SINEs located in the intronic regions. What
other SINE families does VHL have? What about other TE classes other than
SINE?
(Answers provided in separate pdf)
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BLAT
In this exercise, we will use BLAT (Basic Local Alignment Tool) to search for the identity of a
mystery gene annotated in the human genome.
The goal of this exercise is to gain experience using BLAST, particularly blastN, and the
UCSC genome browser to answer biologically relevant questions.
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BLAST v. BLAT
BLAST
BLAT
 Can find matches to a query in any set
of GenBank sequences.
× Limited to matches to a query in a
particular reference genome.
 Not limited to a given k-mer size.
× Limited to non-overlapping 11-mers for
DNA.
× Consumes a lot of memory.
 Can fit an entire genome in memory ( <
1GB) of RAM.
× Slow compared to BLAT.
 Fast compared to BLAST.
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Step 1A: BLAT the Mystery Sequence
Go to the following web page: http://genome.ucsc.edu/
Click BLAT
Open our mystery sequence, located below, in Notepad.
classes/mayo/ma/mystery_sequence.txt
Paste the sequence into the textarea
Click submit
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Step 1B: BLAT the Mystery Sequence
Screenshot of the web form for BLAT.
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Step 2A: Identify Mystery Sequence
BLAT will return a list of significant matches in the genome.
Investigate the matches in the list by clicking browser for each match
For example, click the first browser link here.
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Step 2B: Identify Mystery Sequence
The screenshot below shows UCSC and RefSeq genes aligned to the Mysterious Sequence.
In particular, CYP2A13.
Examine the other matches on the previous slide in the genome browser.
Keep in mind 2 questions: (Answers provided at the end of the document)
A. How many potential genes does the mystery sequence come from?
B. What is the relationship among these genes?
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Pairwise Whole Genome
Alignments
In this exercise, we will utilize the UCSC Genome Browser to view whole genome
alignments computed by lastZ of the following genomes individually to human: organutan,
mouse, dog, and opossum. We will investigate these alignments to see if we can discover
chromosomal rearrangements.
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Step 1: Create a Custom UCSC Track
Go to the UCSC Genome Browser: http://genome.ucsc.edu/index.html
Under the My Data Tab, click Create Custom Tracks:
In the Paste URLs textbox paste the following and click submit: (no commas)
chr13 58481798 58486558
On the next page, click Go to Genome Browser
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Step 2A: Track Addition
The track should look similar to what is below:
’
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Step 2B: Track Addition and Removal
To get ‘Pairwise Alignments’ we need to turn a few tracks on and one track off.
Specifically, we need to select:
Primate Chain/Net
Placental Chain/Net
Vertebrate Chain/Net.
Underneath the Comparative Genomics Tab, turn these tracks to dense.
Additionally, set Conservation to hide and click refresh.
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Step 2C: Track Addition
The resulting view should look like the figure below.
There is one problem: our species of interest are not being displayed.
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Step 2D: Species Selection
To select the correct species, go back to the Comparative Genomics Tab.
Click on the Primate Chain/Net link.
In the resulting window, set Chains to hide and make sure only Orangutan is selected.
Click submit
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Step 2E: Species Selection Continued
Conduct Step 2D for the other two tracks:
Placental Chain/Net
Vertebrate Chain/Net
Make sure your configuration resembles the screenshots below:
Placental Chain/Net
Vertebrate Chain/Net
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Step 2F: Expand Tracks
On the tracks for each species, Right Click and select Full.
The resulting Genome Browser (after moving the tracks to the top) should look like the
following:
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Step 3: Whole Genome Alignment Analysis.
Investigate the tracks for each species and answer the
following questions.
A. Are the sequence counterparts co-linear with
respect to human? If not, is their evidence of
genomics rearrangements in this region? Which
kind?
B.
Can you infer when these rearrangements
happened evolutionarily on the diagram to the
right?
Answers provided in separate pdf.
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Phylogeny Based Whole
Genome Alignment
In this exercise, we will utilize the UCSC Genome Browser to view a refined whole genome
alignment of orangutan, mouse, dog, and opossum genomes to human. This alignment is
produced by Multiz, a program that utilizes pairwise whole genome alignments of many
species and, using a phylogenetic tree, improves the alignment.
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Step 1: Setup Multiz Visualization
Go to the UCSC Genome Browser: http://genome.ucsc.edu/index.html
Upload the following as a Custom Track and go to the genome
browser, as in the previous exercise: (no commas)
chr20 61733467 61733528
Under the Comparative Genomics tab in the genome browser, click on
Conservation.
Ensure the following settings are in place on the next 2 pages:
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Step 1B: Setup Multiz Visualization
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Step 1C: Setup Multiz Visualization
Once your configuration resembles the last 2 figures, click submit
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Step 2: Multiz Visualization Analysis
After rearranging tracks, the genome browser should resemble the
figure below:
Investigate the tracks for each species and answer the following questions:
A.
Is this region highly conserved in mammals?
B.
Look closely at the Multiz track. Do you see anything strange in the human
sequence compared to the other species? What could be the reason for this
discrepancy?
(Answers provided in separate pdf)
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Intersection of Annotated
Regulatory Regions in Human
and Placental Mammals
In this exercise, we will use Galaxy to intersect annotated regulatory regions in
human with annotated regions in other placental mammals.
We will then view the intersection in the UCSC genome browser
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Step 1A: Place Regulatory Data in Galaxy
Login to Galaxy : https://galaxy.illinois.edu/
Upload the sequence of predicted regulatory regions in h19 to Galaxy:
classes/mayo/ma/PRe_Mod_hg19.bed
Make sure to identify hg19 as your reference genome.
Acquire all conserved regions in placental mammals from the UCSC Main Table Browser in Galaxy:
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Step 1B: Place Regulatory Data in Galaxy
Select Comparative Genomics for Group
Select Mammal E1: phastConsElements45wayPlacental for table.
Select Genome for region.
Select Galaxy for send output to.
Click Get Output
On the next screen, click Send Query to Galaxy.
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Step 2: Intersect Datasets
Go to Operate on Genomic Intervals in Galaxy and select Interesect.
Select the parameters below and click Execute.
When finished, click display at UCSC in history pane.
UCSC Results
chr19 regulatory regions.
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Step 3: Predicted Modules Overlap with
PAX5 Regulators
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Exploratory Exercise
Pick a gene of interest. (VHL, CMYC, ETS1, TBP, USF2, GATA-1, …)
Visualize the intersected intervals in the UCSC Genome Browser.
See how this region correlates with results from ENCODE to assess
their functional roles.
We will come around to help.
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