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Practical Exam 2
Bioinformatics
signature

2020.8.11.
Practical Exam 2, Bioinformatics
Use the IBO Challenge 2020 Bioinformatics (IBOC) application to address the following
questions. The IBOC application may be accessed using any Web Browser.

Find the URL of your country’s server at:

URL: https://bit.ly/IBO2020file

Or refer to the URL list at the end of this exam file/booklet.

Then, enter the server using the following username and password

Username: ibo2020
Password: ibo2020binagasaki

If you have a connection problem, try the following alternative servers;

Competitors in Asia, Oceania, or America: 18.181.30.53/ec2-user/ibo2020bi
Competitors in Europe: 18.184.254.217:3838/ec2-user/ibo2020bi/

IBOC applications should not be accessed except during exam hours. The above URL should not
be shared with others even after taking the test.

If you experience any problems using the IBOC application, reload the application using the
reload button on of your Web Browser.

The IBOC application consists of 14 tabs. After starting the test, check first to see if you can open
all tabs. Immediately after the application is loaded for the first time, it may take about 1 minute
for a tab to be loaded.

This exam consists of three parts: Part 1: 13 marks, Part 2: 66 marks, and Part 3: 21 marks. The
total score is 100 marks. You will have 90 minutes to answer all questions. These questions are
designed to be solved in order, from beginning to end.
Overview of the topics covered in the bioinformatics questions

Bioinformatics is a discipline that aims to elucidate how information is transmitted and

interpreted within living organisms. Research scientists use bioinformatics tools to convert
observations of natural phenomena into digital format, and subsequently to visualize, analyze, and
interpret this information using computers.

Digitization has benefited scientific progress in ways that are well beyond simply making the data
easier to handle. First, the development of more powerful computers enabled the simultaneous
analysis of amounts of data so large that the task would be impossible to perform manually. In
addition, online sharing of the data has made it possible for researchers around the world to share
their observations on various life phenomena more efficiently. In particular, research on genomic
DNA sequences and on the three-dimensional structure of proteins benefited very early on from
the advent of computing technology. Nowadays, the benefits of computers in the life sciences are
widespread.

Use the IBOC application to address the following questions. IBOC application may be accessed
using any Web Browser.
Note: These application tools may return an error string written in red letters like the following:
" Error: An error has occurred. Check your logs or contact the app author for clarification". You
will see this if the query data is formatted incorrectly.

Part 1: Genome databases

Read the following information about amino acid and nucleotide sequence databases, and answer
the questions below.

Genbank is known as one of the first DNA sequence databases and is maintained by NCBI
(National Center for Biotechnology Information, USA). In Genbank, information for each gene is
displayed in a format called the GenBank Format, as shown in Genes 1 tab. Read the following
section and answer the questions.

The Genes 1 tab includes information about the human HoxA5 gene as registered in the RefSeq
database with the accession number NM_019102, and displayed in GenBank Format. In
GenBank Format, each gene entry line begins with a LOCUS tag, and ends with two slashes (//).
Most of the information is described as a combination of tags and data corresponding to the tags.
A tag is a word shown at the beginning of a line, such as LOCUS or DEFINITION. For
example, the data corresponding to the LOCUS tag is "NM_019102 1670 bp mRNA linear PRI
31-DEC-2019".

SOURCE tag shows the species name (Homo sapiens (human)). FEATURES tag corresponds to
several sub-sections; source, gene, exon, and CDS. "/chromosome=7" in the source sub-section
shows the chromosome number of which the gene of the entry is located. “/gene=HOXA5 in the
gene sub-section shows the name of the gene of the entry. The CDS sub-section indicates that the
region for amino acids starts at the 75th base and ends at the 887th base. The "/translation=" part
of the CDS sub-section contains the amino acid sequence translated from the coding sequence
(CDS) of this gene. There are two exon sub-sections in the FEATURES tag, described as "exon
1..636" and "exon 637..1670". This means that the gene has two exons, and that the boundary
between the two exons is located in between the 636th and 637th bases, for a final transcript length
of 1670 bp. Finally, The ORIGIN tag contains the DNA sequence of the genomic region that is
transcribed as messenger (mRNA), written from the 5’- to the 3’-end.

Question 1. According to the amino acid sequence shown in the /translation= part of the above
GenBank entry, the protein encoded by this gene starts with an M (methionine) at the first
position, followed by an S (serine) at the second position, and an another S (serine) at the third
position. Assuming the nucleotide sequence is exactly the same as the data shown in the above
GenBank entry, select the correct nucleotide sequences coding for the second and third position
serines. [ 3 marks ] [No. 1]

2nd position serine, 3rd position serine

1. AAT, TGT
2. AAT, AAT
3. GAC, GCA
4. GAC, GAC
5. TCT, TCT
6. AGC, AGC
7. AGC, TCT
8. GCC, CGG

In the GenBank entry, not only the amino acid sequence encoded by the target mRNA can be
found in /translation=, this information is linked to another database (GenPept) as
/protein_id="NP_061975.2".
NP_061975.2 is the accession ID of the GenPept database, also operated by NCBI. The GenPept
data for NP_061975.2 is shown Proteins tab.

Proteins consist of several regions with different characteristics functions (protein domains),
which confer both structure and function. In the above entry, the protein encoded by the HOXA5
gene has a total length of 270 amino acids, and the Region sub-section indicates that amino acids
176 to 181 constitute a protein domain called the "Antp-type hexapeptide". Another protein
domain called "Homeobox domain" spans amino acids 199 to 251. Similarly, you can find an
entry for the HOXA6 gene in the Proteins tab.

One of the tools for discovering functional domains in amino acid sequences is hmmscan in
Hmmer3 (Mistry et al., 2013). It enables the discovery of common domains (eg.
“Homeodomain”) in a given family of genes. In the HMMSCAN tab of the IBOC application,
you can use hmmscan to examine the functional domains contained in the amino acid sequence of
your protein of interest. This requires an existing protein domain database, and IBOC applications
are designed to search the Pfam-A database. Let's check the position of the Homeobox domain of
HOXA5 examined in Question 1 using hmmscan.

In the Protein Sequences 1 tab, several amino acid sequences are shown in FASTA format
which is one of the most commonly used formats to describe nucleotide and amino acid
sequences. A sequence in FASTA format begins with a single-line description, followed by lines
of sequence data. The description line (defline) is distinguished from the sequence data by a
greater-than (">") symbol at the beginning.

From the Protein sequences 1 tab, copy the amino acid sequence of HOXA5 protein in FASTA
format and paste it in the "Input sequence" box of the HMMSCAN tab. An E-value is calculated
to estimate the probability of the result happening by chance, and the recommended threshold is
1e-5 (=0.00001). Clicking on "Exec hmmscan" will display the regions containing functional
domains for HOXA5 (if present). If done correctly, the following should be displayed (Figure 1.):

Figure 1. An example of Hmmscan search result. This figure shows the output of the
hmmscan.

The search results display the domain name and Accession ID in the "target name:" and "target
accession:" columns, respectively. This information depends on the database provided, and in the
case of the IBOC application, the domain name and Accession ID from the Pfam-A database are
displayed. "tlen" is the total length of that particular domain in amino acids. Here, the
homeodomain registered in Pfam-A is 57 amino acids.

The name of the query amino acid sequence (the sequence you copied in, eg. NP_061975.2
homeobox protein Hox-A5 [Homo sapiens] in Figure 1.)" is displayed in the "query name:"
column, and its length in the "qlen:" column. If the same domain is found more than once, the
total number and serial number are displayed in the "of:" and "#:" columns, respectively. The
"from (hmm coord):" column and the "to (hmm coord):" column indicate which part of the
domain matched to the database. The "from (ali coord):" and "to (ali coord):" columns show the
start and end positions of the domain of interest inside the query sequence. This time, the results
show amino acids 1 to 57 of the PF00046 Homeodomain, which is exactly 57 amino acids long,
meaning that the entire length of this domain was included in the query sequence.
Question 2. Find a Homeodomain (PF00046.30) in HOXA6 (NP_076919.1 homeobox protein
Hox-A6 [Homo sapiens]). Use E-value: 1e-5 as threshold. [ 4 marks ]

The homeodomain (PF00046.30) region in HOXA6 gene starts (“from (ali coord):”) at
[No.2][No.3][No.4], and ends (“to (ali coord):”) at [No.5][No.6][No.7].

eg) If you would like to answer "starts at 25” and “ends at 143”, the example of how to answer is
as follows;

No. 2 : 0
No. 3 : 2
No. 4 : 5

No. 5 : 1
No. 6 : 4
No. 7 : 3

Next, let's find where the two exons of HOXA5 are encoded in the DNA genome. It is convenient
to use the NCBI-BLAST+ tool developed by NCBI to search for sequence similarity. To do this,
you can choose between the following three programs. When blastp is selected, an amino acid
sequence query is searched for in an amino acid sequence database. If blastn is selected, a
nucleotide sequence query is searched against a nucleotide sequence database. Finally, when
tblastn is selected, an amino acid query may be searched against a nucleotide sequence database.

The first three tabs of the IBOC application show the following nucleotide sequences.
The "Human Genome DNA 1" tab shows the nucleotide sequence of human chromosome 7
from positions 27,140,701 to 27,150,700.
The "Human Genome DNA 2" tab shows the nucleotide sequence of human chromosome 7
from positions 26,188,001 to 26,218,000.
The "B. burgdorferi B31 Genome DNA 1" tab is the sequence of the entire cyclic genomic DNA
of a bacterium (Borrelia burgdorferi strain B31).

In the following questions we will refer to base positions within the analyzed sequence, not
positions along the chromosome. For example, we will refer to the first base of the Human
Genome DNA 1 sequence as 1, not 27,140,701. The genomic DNA of B. burgdorferi strain B31
is circular. Therefore, the first base was arbitrarily defined.

After copying the mRNA sequence of HOXA5 (NM_019102.4) from the Predicted mRNA
Sequences tab, paste it into the "Input sequence:" window of the BLAST tab. (Note: By
convention, RNA sequences are shown as a complementary DNA sequence. This means a coding
DNA strand and the transcribed RNA would be shown as identical sequences). Next, by selecting
"Human Genome DNA 1" from the "Reference Sequence:" options, a similarity search is
performed against “Human Genome DNA 1”. This step requires blastn. Clicking "Exec" gives
the predicted locus of HOXA5. In the search results, "sseqid" displays the IDs of the hit sequence.

Question 3. Choose the location which is including the first exon of HOXA5 from the following
options. Use “E-value:1e-5” as threshold for blastn. Hint: Use Predicted mRNA sequences tab,
and BLAST tab. [ 2 marks ] [No.8]

1. 1 bp ~ 1,000 bp
2. 1001 bp ~ 2000 bp
3. 2001 bp ~ 3000 bp
4. 3001 bp ~ 4000 bp
5. 4001 bp ~ 5000 bp
6. 5001 bp ~ 6000 bp
7. 6001 bp ~ 7000 bp
8. 7001 bp ~ 8000 bp
9. 8001 bp ~ 9000 bp
0. 9001 bp ~ 10,000 bp

Question 4. Next, find proteins with a similar amino acid sequence to HOXA6. Using HOXA6 as
a query, perform a blastp search on a human protein dataset (“human proteins” in the Reference
sequence field). Use “E-value:1e-5” as threshold for blastp. Choose the entry with the highest
sequence similarity from the following options, but not HOXA6 itself. Use “E-value:1e-5” as
threshold for blastp. Hint: Use Protein sequences 1 tab, and BLAST tab. [ 4 marks ] [No.9]

1. 1_06639
2. 1_05172
3. 1_07981
4. 1_09188
5. 1_12205
6. 1_12968
7. 1_15255
8. 1_15496
9. 1_17260
0. 1_18575

In order to obtain the protein sequence, you should go to the Entry Database tab within the IBOC
application (you would need in questions 18 and 19). The amino acid sequence of the protein will
be displayed upon entering the protein’s ID (eg. 1_05121) in the Entry_ID input field and choose
an organism name from the Sequence selection. Note: This feature may be useful for
answering Question 18 and 19.
Part 2. Analysis of sequence features and motif discovery

The question below introduces methods to investigate the GC composition of nucleic acid
sequences as a general property of genomic DNA. Read the following information about
chromosomes and the GC composition of DNA, and answer the questions below.

As their name suggests, chromosomes can be dyed using one of several staining methods. Among
these methods, Giemsa staining results in a striped pattern of bands along the chromosome. The
staining is usually performed during the [ No. 10 ] of cell division, when the chromosomes are
visible because of chromosome condensation. The coloration is darker in regions that have a [ No.
11 ] AT content, and lighter and brighter in regions that have a [ No. 12 ] GC content.
Furthermore, regions with a [ No. 12 ] GC content were thought to have more genes. Such a
correlation between GC content and gene density was confirmed when the nearly complete
sequence of human nuclear DNA (draft genome) was determined by the Human Genome Project
in 2000. In addition, DNA tend to undergo denaturation (separation of the double helix into two
single strands) at [No. 13 ] temperatures. GC pairs are denatured at a [No. 14 ] temperature than
AT pairs. As described above, the GC content is a value that is both easy to obtain and very useful
in various aspects of bioscience and biotechnology research.

Question 5. Pick the answer most appropriate for [ No. 10 ]. [ 4 marks ]

1. G0 phase
2. S phase
3. Metaphase

Question 6. Pick the answer most appropriate for [ No. 11 ], [ No. 12 ], [No. 13], and [No. 14 ].
[ 8 marks ]

1. higher
2. lower

From here, let us focus on the GC content of DNA sequences. The GC content (GC%) is
represented by the following formula.

GC% = 100 × ( ( [G] + [C] ) / ( [A] + [T] + [G] + [C] ) )

In this formula, [A] represents the number of adenosines (A), and similarly, [T], [G], and [C] each
stand for the number of thymines (T), guanosines (G), and cytosines (C), respectively.

Question 7. For the DNA sequence shown in "Human Genome DNA 1" tab, calculate the GC
content in the ten bases from the 1st to the 10th position included. [ 2 marks ]
[ No. 15 ][ No. 16 ][ No. 17 ] %

eg) If you would like to answer "32%”, the example of how to answer is as following,

No. 15 : 0
No. 16 : 3
No. 17 : 2

The usage of the “Count nucleotide” tab and the “Window search” tab is shown below.

DNA sequences from the region of your choice can be extracted from "Human Genome DNA 1",
"Human Genome DNA 2", and "B. burgdorferi B31 Genome DNA 1" by using the Get
Subsequence function on the left side of the "Count Nucleotide" tab.
Select Human Genome DNA 1 from the Target Sequence selection list, then enter 1 and 10 in
the "Start" and "End" fields, respectively. This will display the first 10 bases from the first to the
tenth base of "Human Genome DNA 1". Note: This feature may be useful for answering
Question 15 and 16.

Window search is a method used to examine the distribution of DNA features over the entire
nucleotide sequence by shifting a window of fixed length by a fixed unit. The length of the
window is called Window Size. The length of the window shift is called Step Size.

Next, let's examine the sequence characteristics of "Human Genome DNA 1" and "Human
Genome DNA 2" using the "Window Search" tab of the IBOC application.

Question 8. Let's check the GC content for the first 10,000 nucleotides of "Human Genome
DNA 1" using window search.

1. Open the "Window Search” tab and select "Human Genome DNA 1" from the
Reference Sequence list.
2. To check the total number of G’s and C’s combined, select "G+C" from the Nuc.
selection list.
3. Enter 1 in the Start field and enter 10,000 in the End field.
4. Set the “Window Size” to 100 bp, the “Step Size” to 100 bp, and the “Bin Size” to 10 %.
5. With the above settings, the GC content will be calculated for every 100 bp window from
the 1st base to the 10,000th base of "Human Genome DNA 1".
6. Finally, clicking on "Show Chart 1", displays the result as a histogram representing the
frequency of GC content values in intervals of 10 % (corresponding to the histogram’s
Bin Size) in "Histogram for the selected nucleotide(s)”.
7. Additionally, clicking on "Show Chart 2", displays the result as a plot representing the
frequency of GC content values along the region above in "Frequency for the selected
nucleotide(s)".
Note: X-axis in Chart-2: nucleotide position (bp). Y-axis in Chart-2: frequency for the selected
nucleotide(s).
Note: In Chart 2, Chart 3 and Chart4, the x-axis position may be displayed as “1e-4” instead of
“10,000”.
Note: If you have changed the parameters, click on "Show Chart 1( or 2, 3, 4)" again to reflect the
change.

Select the most appropriate item for [ No. 18 ] in the following sentence. [ 4 marks ]

Using the above settings, 100 bp windows with a GC content of [ No. 18 ] % appear most
frequently in "Human Genome DNA 1”.

1. 10-20
2. 20-30
3. 30-40
4. 40-50
5. 50-60
6. 60-70
7. 70-80
8. 80-90

Question 9. As we saw in Question 7. ~ 8. the GC% of DNA varies from region to region. Find
out where the 200 bp window with the highest GC content is located on "B. burgdorferi B31
Genome DNA", and choose the corresponding location from the following options. Note that the
genome DNA of this organism is circular and 910.724 bp long. Note that this calculation may
take some time (about one minute per calculation) in IBOC applications. [ 5 marks ] [ No. 19 ]

1. 50,000 bp ~ 100,000 bp
2. 100,000 bp ~ 200,000 bp
3. 200,000 bp ~ 300,000 bp
4. 300,000 bp ~ 400,000 bp
5. 400,000 bp ~ 500,000 bp
6. 500,000 bp ~ 600,000 bp
7. 600,000 bp ~ 700,000 bp
8. 800,000 bp ~ 900,000 bp
9. The region including 900,000 bp ~ 910,724 bp and 1 bp ~ 50,000 bp

In the following section, we will investigate bacterial DNA replication using the methods for
analysis such as Window Search, which we learned from the previous question.
Question 10. The replication of circular DNA starts at a single position and propagates in both
directions (Figure 2).

Figure 2. Schematic diagram of DNA replication in a bacterial circular genome. X

is the start point of replication (Ori) and Y is the end point of replication. There are two
replication forks, shown as Z1 and Z2.

If the whole genome sequence of the bacteria is available, to estimate the start and end points of
replication, it is useful to examine the GC-skew. GC-skew is an indication of the bias in the
balance between G’s and C’s on one strand of the DNA. It is expressed as the difference between
the number of C’s and the number of G’s divided by the total number of G’s and C’s over a given
sequence length.

GC-skew = ([C]-[G]) / ([C]+[G])

Using the Window Search, look up the GC-skew of the B. burgdorferi B31 Genome DNA. In
the "Window Search" tab, select "B. burgdorferi B31 Genome DNA" in the "Reference
Sequence", fill the first base and the last base in the Start and End fields, respectively, and then
choose ([C]-[G])/([C]+ [G]) for Skew field. Finally, press Show Chart 3, the GC-skew plot
appears in the Chart 3 area. Note that this calculation may take some time (about one minute
per calculation) in IBOC applications.

There are two switching points between high GC-skew and low GC-skew in the DNA. Choose
the corresponding locations among the following options. [ 8 marks ]

1. 50,000 bp ~ 100,000 bp
2. 100,000 bp ~ 200,000 bp
3. 200,000 bp ~ 300,000 bp
4. 300,000 bp ~ 400,000 bp
5. 400,000 bp ~ 500,000 bp
6. 500,000 bp ~ 600,000 bp
7. 600,000 bp ~ 700,000 bp
8. 800,000 bp ~ 900,000 bp
9. The region including 900,000 bp ~ 910,724 bp and 1 bp ~ 50,000 bp
The two switching points are located in [ No. 20 ] and in [ No. 21 ].

Question 11. It is known that the replication start point, OriC, is in the vicinity of the region
encoding the DnaA protein. Examine the region encoding the DnaA protein on the "B.
burgdorferi B31 Genome DNA" and select the region that contains it.
Think about which tools you need to use to answer this question. You can use the DnaA protein
sequence from the Protein Sequences 2 tab. [ 2 marks ] [ No. 22 ]

1. 50,000 bp ~ 100,000 bp
2. 100,000 bp ~ 200,000 bp
3. 200,000 bp ~ 300,000 bp
4. 300,000 bp ~ 400,000 bp
5. 400,000 bp ~ 500,000 bp
6. 500,000 bp ~ 600,000 bp
7. 600,000 bp ~ 700,000 bp
8. 800,000 bp ~ 900,000 bp
9. The region including 900,000 bp ~ 910,724 bp and 1 bp ~ 50,000 bp

Question 12. It is known that GC-skew values change significantly at both the replication start
and end points of replication. Considering the answers to the previous questions, select the region
that is most likely to contain the replication start point (OriC) for this organism from the
following. [ 4 marks ] [ No. 23 ]

1. 50,000 bp ~ 100,000 bp
2. 100,000 bp ~ 200,000 bp
3. 200,000 bp ~ 300,000 bp
4. 300,000 bp ~ 400,000 bp
5. 400,000 bp ~ 500,000 bp
6. 500,000 bp ~ 600,000 bp
7. 600,000 bp ~ 700,000 bp
8. 800,000 bp ~ 900,000 bp
9. The region including 900,000 bp ~ 910,724 bp and 1 bp ~ 50,000 bp
From here, we will examine the DNA motifs involved in the regulation of gene expression in
eukaryotes.
Gene expression can be regulated by the chemical addition of methyl groups to specific bases on
DNA (Figure 3).

Figure 3. Structural formula of 5-methylcytosine, a methylated cytosine. The

dotted area is the added methyl group.

In vertebrates, the cytosines of CpG dinucleotides are often subject to DNA methylation. A CpG
dinucleotide, as the name indicates, consists of a consecutive C and G in the 5’ to the 3’ direction
(Figure 4). 'p' between C and G, which stands for the phosphodiester bond so that CpG is
distinguished from CG which is a nucleotide pair in a double-stranded DNA.

Figure 4. A CpG dinucleotide consists of a consecutive C and G in the 5’ to the 3’

direction. The dotted area is an example of a CpG dinucleotide.

A DNA region that is rich in CpG dinucleotides is known as a CpG island. CpG islands are
regularly found in the genomic DNA of vertebrates, and may be located either around the exons
of protein-coding genes, or a few hundred bp upstream of their transcription start sites. Not all
CpG islands are methylated, but these methylation events are correlated with inactivation of the
downstream gene.
Several methods have been proposed to look for CpG islands. Among them, the CpG-score is
expressed by the following formula, and can be calculated over a shifting window as
demonstrated previously

CpG-score = ( [CpG] / ( [C] x [G] ) ) x Window-Size

In this formula, [CpG] represents the number of CpG in the window, and similarly, [G], and [C]
each stand for the number of guanosines (G), and cytosines (C) in the window, respectively.
To obtain the CpG score, “Window-Size = 100” and “Step-Size = 1” are generally the standard
parameters used. Subsequently, areas with a CpG score of 0.6 or higher are often considered as
candidate CpG islands.

Question 13. As mentioned above, "Human Genome DNA 1" is the sequence from positions
27,140,701 to 27,150,700 on human chromosome 7, for a total of 10,000 bases.
In the "Window Search" tab, select “Human Genome DNA 1” from the Reference Sequence
field, and examine the distribution of the CpG-score. Enter the appropriate values for the Start
and End fields. Use “Window-Size = 800” and “Step-Size = 1” as the parameters.
Select "CpG-score" from the CpG-score field shown in the lower right corner of the screen, and
click Show Chart 4. This will display the distribution of CpG-scores for the target sequence
region. Estimate the length of the longest candidate CpG island region found in this sequence, and
choose the closest value from the following options. In this question, regions with a CpG score
greater than 0.6 are considered to be candidate CpG islands. Note that this calculation may take
some time (about one minute per calculation) in IBOC applications. [ 6 marks ] [ No. 24 ]

1. 200 bp
2. 800 bp
3. 1,400 bp
4. 2,000 bp
5. 2,600 bp
6. 3,200 bp
7. 3,800 bp
8. 4,400 bp
9. 5,000 bp

Question 14. Then, predict the transcription start site of human HoxA6 gene in the Human
genome DNA 1. Consider the most 5’-end-most position among the search results (also called
“hits”) of this full-length sequence as the transcription start site, and also consider the most 3’-end
position among the hits of this full-length sequence as the end of transcript. Choose the closest
answer from the following options. For the HoxA6 sequence, use the sequence shown in the
"Predicted mRNA sequences" tab.

The transcription start site of HoxA6 is nearest to nucleotide position [ No. 25 ]. [ 4 marks ]

1. 1,000
2. 2,000
3. 3,000
4. 4,000
5. 5,000
6. 6,000
7. 7,000
8. 8,000
9. 9,000

Question 15. Referring to Questions 13 - 14 above, choose the incorrect statement from the
following options. [ 6 marks ] [ No. 26 ]

1. The CpG island, which overlaps with the first exon of HoxA5, is longer than another CpG
island which overlaps with the first exon of HoxA6.
2. The intron of HoxA6 is roughly consistent with the region containing the fewest CpG
dinucleotides.
3. In this sequence, the region with the lowest CpG-score is roughly consistent with the region
with the highest AT content.
4. Both HoxA5 and HoxA6 have a GC content of >60% in the first exon.
5. The intergenic region between HoxA5 and HoxA6 genes is approximately 1.7 kb in length.

In the following question, we will examine the characteristics of functional sequence motifs found
on genomic DNA.

DNA contains a variety of functional sequence motifs. A famous example is the polyadenylation
signal (AAUAAA sequence) encoded by the transcribed RNA in its 3' untranslated region (UTR).
On mRNA precursors, a group of protein complexes recognizes this polyadenylation signal and
truncates the 3’-end of RNA before adding a poly(A) tail. In other words, we can expect to find a
polyadenylation signal in the last exon at the 3'-end of a coding sequence.

If the location of the motif is known, an analysis method called “sequence logo” enables statistical
evaluation of the consensus sequence. As an example, let's use the sequence logo program to
visualize the polyadenylation signal motif. The 3'-end of genes are shown in FASTA format
below. The sequence of the polyadenylation motif is shown in capitals. Ten bases either side are
shown in lowercase. (Same sequences are shown on the bottom of the Sequence Logo tab).

>HoxA5_1638bp_1663bp
tggaacaaaaAATAAActttctattg
>CBX3_2027bp_2052bp
acagttgggaAATAAAagtttcatgt
>HNRNPA2B1_3591bp_3617bp
ggctgtccccAATAAAtgctgttcat
>Stk31_3238bp_3263bp
ggttgtgaaaAATAAAgatgtttggc
>Tra2a_1752bp_1777bp
agtagtctcaAATAAAagctaatttc
The figure below is a representation of the sequence alignment as a sequence logo (Figure 5).

Figure 5. An example of a result of the sequence logo.

Using the Sequence Logo tab, make sure that you actually get the same diagram as above. (Copy
the above sequences in the Sequence Logo tab and paste those into the "Input Sequence" window
in the Sequence Logo tab, and then click on the “Exec Sequence Logo”).

Thus, the AATAAA sequence, a common polyadenylation-signal motif in all five sequences, is
highlighted on the sequence log as you can see. In this example, only five gene sequences were
used, but in the actual discovery of motifs, many more sequences need to be analyzed. Otherwise,
you run the risk of highlighting sequences that just happen to be coincidental matches.

Question 16. In eukaryotes, protein-coding genes consist of several exonic and intronic
sequences on a genomic DNA. It is known that the boundary between exons and introns contains
a distinctive sequence that allows the spliceosome (the enzyme that cuts out the intron) to
recognize the intron sequences to be spliced. In addition to their commonality within eukaryotes,
they also have species-specific and taxonomic specificity. The HNRNPA2B1 gene has a typical
exon-intron boundary sequence motif in the human genome. Determine the correct motif of exon-
intron boundary and intron-exon boundary. You can use the information of the HNRNPA2B1
gene in the GenBank format in the Genes 1 tab, mRNA sequence in the Predicted mRNA
sequences tab. Then choose the most appropriate answer from the following options. [ 13 marks ]

Note that these represent the 4 bp motif (2 bp at the 3'-end of the exon and 2 bp at the 5'-end of
the intron) that make up the main exon-to-intron boundary in human ([No. 27 – No. 30]), and the
3 bp motif (2 bp at the 3'-end of intron and 1 bp at the 5'-end of exon) that makes up the major
intron-to-exon boundary in human ([No. 31 – No. 33]).

[ Exon-side bases ] - [ Intron-side bases ]

[ [No. 27], [No. 28] ] - [ [No. 29], [No.30] ]

[ Intron-side bases ] - [ Exon-side base ]

[ [No. 31], [No. 32] ] - [ [No. 33] ]
1. a
2. t
3. g
4. c

eg) If you would like to answer “ …exon… at - gg …intron... ca - t … exon …”, the answer
should be as follows:

No. 27 : 1
No. 28 : 2
No. 29 : 3
No. 30 : 3
No. 31 : 4
No. 32 : 1
No. 33 : 2

Part 3: Task

Answer the following questions using the IBOC application.

Using the BLAST tab and the Entry Database tab, a sequence-similarity search can be performed
against the protein data set of the following 7 organisms; 5 animals (human, mouse, fruit fly,
octopus, and starlet sea anemone) and 2 unicellular organisms (choanoflagellate. and fission
yeast). By examining some of the cadherin family genes in these organisms, answer the following
questions.

Question 17. E-Cadherin and N-Cadherin are known to be representative of the cadherin family
of genes. Information on these two genes are shown in the Genes tab, Proteins tab, Predicted
mRNA sequences tab, and Protein sequences 1 tab. Many of the cadherin family genes in the
above animals have a tandemly duplicated (=repeatedly lined up) domain. Both E-Cadherin and
N-Cadherin repeat this domain at least five times or more. The domain is given by the Accession
ID of Pfam-A: (i)[ PF ***** ]. Identify the domain, use the e-value of less than 1e-5 for the
hmmscan threshold. (Note: Ignore the numbers after the dot in Pfam Accession ID.) [ 4 marks ]

(i) PF [No. 34][No. 35][No. 36][No. 37][No. 38]

eg) If you would like to answer “PF00001.12”, the answer should be as follows:

No. 34 : 0
No. 35 : 0
No. 36 : 0
No. 37 : 0
No. 38 : 1

Question 18. A comparison of the domain structure of cadherin proteins in animals and non-
animals shows that, (ii)[ PF ***** . * ] is added to the (iii) [No.44]-terminus next to the repeat of
the (i) domain in animals. (Note: Ignore the numbers after the dot in Pfam Accession ID.)
[ 10 marks ]

(ii) PF [No. 39][No. 40][No. 41][No. 42][No. 43]

(iii) [No.44]

1. N
2. C

Question 19. From the set of protein entries for the choanoflagellate, find one gene that has
multiple repeats of the above domain ( i ) in Question 17. To identify the domain ( i ), use the e-
value of less than 1e-5 for the hmmscan threshold. [ 7 marks ]
(The more repeats of domain (i) that are in the gene that you find, the higher score you will get.)

Hint: You may use following functions in HMMSCAN-tab as needed." ‘Show [ number ]
entries’ function can be used to change the number of displayed lines. 'Search: [ word ]' can be
used to perform a string [ word ] search. The total number of rows in the resulting table is
displayed at the bottom left of the screen.

(iv) [No.45] _ [No. 46][No. 47][No. 48][No. 49][No. 50]

// End of Practical Exam 2.

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