Fernanda Rodríguez Reyes

2173035902
Actividad BLAST
Bioinformática
Part 1: Your first BLAST search
Below is the mRNA sequence for insulin from a South American rodent,
the Degu (Octodon degus).
>gi|202471|gb|M57671.1|OCOINS Octodon degus insulin mRNA, complete cds
GCATTCTGAGGCATTCTCTAACAGGTTCTCGACCCTCCGCCATGGCCCCGTGGATGCATCTCCTCACCGT
GCTGGCCCTGCTGGCCCTCTGGGGACCCAACTCTGTTCAGGCCTATTCCAGCCAGCACCTGTGCGGCTCC
AACCTAGTGGAGGCACTGTACATGACATGTGGACGGAGTGGCTTCTATAGACCCCACGACCGCCGAGAGC
TGGAGGACCTCCAGGTGGAGCAGGCAGAACTGGGTCTGGAGGCAGGCGGCCTGCAGCCTTCGGCCCTGGA
GATGATTCTGCAGAAGCGCGGCATTGTGGATCAGTGCTGTAATAACATTTGCACATTTAACCAGCTGCAG
AACTACTGCAATGTCCCTTAGACACCTGCCTTGGGCCTGGCCTGCTGCTCTGCCCTGGCAACCAATAAAC
CCCTTGAATGAG

We will now use a BLASTN search at NCBI to determine whether this sequence looks like the
human mRNA for insulin. There are two ways we can do this:

▪ Search the entire database and look for human hits in the results,
▪ specifically search the human part of the database.

We will try both of these possibilities.

Search against NR

1. Follow the "nucleotide blast" link from the main BLAST page.
2. In the section "Program Selection" select the option "Somewhat similar sequences
(blastn)"
3. Choose "Nucleotide collection (nr/nt)" as the search database. NR is the "Non
Redundant" database, which contains all non-redundant (non-identical) sequences from
GenBank and the full genome databases.
4. Click the BLAST button to launch the search.

After the search has completed, make yourself familiar with the BLAST output page. After a
header with some information about the search, there are three main parts:

▪ Graphic Summary
 ▪ Each hit is represented by a line showing which part of the query sequence the
alignment covers. The lines are coloured according to alignment score.
▪ Descriptions
▪ a table with a one-line description of each hit with some alignment statistics.
▪ Alignments
▪ the actual alignments between the query and the database hits.

Note that you can toggle between hiding and showing each part by clicking on the part
title (try it!).

The columns in the Descriptions table are:

▪ Description — the description line from the database

▪ Max score — the alignment score of the best match (local alignment) between the
query and the database hit
▪ Total score — the sum of alignment scores for all matches (alignments) between the
query and the database hit (if there is only one match per hit, these two scores are
identical)
▪ Query cover — the percentage of the query sequence that is covered by the
alignment(s)
▪ E value — the Expected value calculated from the Max score (i.e. the number
of unrelated hits with that score or better you would expect to find for random
reasons)
▪ Ident — the percent identity in the alignment(s)
▪ Accession — the accession number of the database hit.

First, take a look at the best hit. Since our search sequence (the query) was taken from
GenBank which is part of NR, we should find an identical sequence in the search. Make sure
this is the case!
 QUESTION 1.1:
Answer the following questions about the best hit:

▪ What is the identifier (Accession)? NM_001185098.2

▪ What is the alignment score ("max score")? 205

▪ What is the percent identity and query coverage? 74.49 %
▪ What is the E-value? 6e-48
▪ Are there any gaps in the alignment? 15/341 = 4 %
Then, find the best hit from human (Homo sapiens) that is not a
synthetic construct. (Tip: you can press Ctrl-F in most browsers
to search on the page).

Search against Human G+T

Note: In this context, G+T does not mean Gin and Tonic.

Open a new window/tab with the BLAST homepage.

Make a new BLASTN search with the same query sequence, this time
with Database set to Human genomic + transcript (Human G+T).
>gi|202471|gb|M57671.1|OCOINS Octodon degus insulin mRNA, complete cds
GCATTCTGAGGCATTCTCTAACAGGTTCTCGACCCTCCGCCATGGCCCCGTGGATGCATCTCCTCACCGT
GCTGGCCCTGCTGGCCCTCTGGGGACCCAACTCTGTTCAGGCCTATTCCAGCCAGCACCTGTGCGGCTCC
AACCTAGTGGAGGCACTGTACATGACATGTGGACGGAGTGGCTTCTATAGACCCCACGACCGCCGAGAGC
TGGAGGACCTCCAGGTGGAGCAGGCAGAACTGGGTCTGGAGGCAGGCGGCCTGCAGCCTTCGGCCCTGGA
GATGATTCTGCAGAAGCGCGGCATTGTGGATCAGTGCTGTAATAACATTTGCACATTTAACCAGCTGCAG
AACTACTGCAATGTCCCTTAGACACCTGCCTTGGGCCTGGCCTGCTGCTCTGCCCTGGCAACCAATAAAC
CCCTTGAATGAG

Remember again to select Somewhat similar sequences (blastn) under Program Selection.

Consider the best hit.

Note: even though you may not have found exactly the same database entry in the two
searches, the alignment should be the same. Make sure this is the case by comparing the
actual alignments in the two windows where you made the searches.

QUESTION 1.3:
Answer the same questions as before about the best hit you found in this
search. Answer the following questions about the best hit:

▪ What is the identifier (Accession)? NM_001291897.2

▪ What is the alignment score ("max score")?205
▪ What is the percent identity and query coverage? 76 %
▪ What is the E-value? 3e-50
▪ Are there any gaps in the alignment? 15/341 = 4 %

Concerning database size and E-values

When answering the previous two questions, you may have noticed that the E-value
changed, while the alignment score did not. We will now investigate this further.
 QUESTION 1.4:
What are the sizes (in base pairs) of the databases we used
for the two BLAST searches? (Tip: Expand the "Search summary"
section near the top by clicking it). 567 y 705

QUESTION 1.5:

▪ What is the ratio between the database sizes in the two

BLAST searches?
▪ What is the ratio between the E-values (for the best
human hits) in the two BLAST searches?
▪ What is the relationship between database size and E
value for hits with identical alignment scores?
▪ In conclusion: if the database size is doubled, what will
happen to the E-value?

Part 2: Assessing the statistical significance of BLAST hits

As discussed in the lecture, there will be a risk of getting false positive results (hits to
sequences that are not related to our input sequence) by purely stochastic means. In this first
part of the exercise, we will be investigating this further, by examining what happens when we
submit randomly generated sequences to BLAST searches.

Rather than giving out a set of pre-generated DNA/Peptide sequences where you only have
our word for their randomness, you'll be generating your own random sequences with the
SeqGen server. We previously used d4/d20 dice to generate these sequences manually, but we
have decided to let the computer do the work for you to save some time. It is important to
understand that these computer-generated sequences are totally random, just as if you were
rolling a die to determine each nucleotide/amino acid in each sequence.

Random DNA sequences and BLASTN

▪ Generate three DNA sequences of length 25bp using

https://www.bioinformatics.org/sms2/random_dna.html
 QUESTION 2.1:
Report the three sequences in FASTA format.
1. cgcagagatgcaacaacatgtcggc

2. cgagcacttctcggtgccgtactcg

3. cagtcggccgggtcggagggtcaga

▪ We will now do a BLASTN search using these three random sequences as

queries.

▪ Follow the "nucleotide blast" link from the main BLAST page, and, as before,

▪ select the option "Somewhat similar sequences (blastn)" in the section "Program
Selection".

▪ Choose "Nucleotide Collection (nr/nt)" as the search database.

VERY IMPORTANT: For this special situation where we BLAST small artificial
sequences, we need to turn off some the automatics NCBI incorporate when short
sequences are detected. Otherwise, we'll not be able to see the intended results:

▪ Extend the "Algorithm parameters" section (see the screen shot below) in
order to gain access to fine-tuning the options.
1. Deselect the "Automatically adjust parameters for short input
sequences" option.
2. Set the E-value cut-off ("Expect threshold") to 50
Remember to adjust the BLAST settings

▪ Paste in your three sequences in FASTA format and start the BLAST search.

Browsing BLAST results: select which of your query sequences to inspect in the
drop-down box near the top of the page

▪ Inspect the results.

QUESTION 2.2:
Answer the following small questions, and document your findings by pasting in
examples of alignments / text snippets from the overview table:

▪ Do you find any sequences that look like your input sequences (paste in a
 few example alignments in your report

▪ What is the typical length of the hits (the alignment length)? Approximately 22
▪ What is the typical % identity? 100%
▪ In what range is the bit-scores ("max score")? 39.2
▪ Notice: This is conceptually the same as the "alignment score" we
have already met in the pairwise alignment exercise.
▪ What is the range of the E-values? 4.1

QUESTION 2.3:

▪ What is the biological significance of these hits / is there any biological

meaning? El valor esperado (E) es un parámetro que describe el
número de aciertos que uno puede "esperar" ver por casualidad al
buscar en una base de datos de un tamaño particular . Disminuye
exponencialmente a medida que aumenta la puntuación (S) del partido.
Random protein sequences and BLASTP
Now it's time to work with a set of protein sequences - generate three
 sequences of length 25 aa using

https://www.bioinformatics.org/sms2/random_protein.html

▪ Notice 1: The distribution of amino acids will be equal (5% prob)

and this is different from true biological sequences - however this
is not important for this first part of the exercise.
▪ Notice 2: Please recall from the lecture that the
way BLASTP selects candidate sequences for full Smith
Waterman alignment is different from BLASTN. (BLASTN - a
single short (11 bp +) perfect match hit is needed. BLASTP - a
pair of "near match" hits of 3 aa within a 40 aa window is needed).

QUESTION 2.4:
Report the sequences in FASTA format.
1. HVDGVAIPPDSAYFEVSDFSNHQWA

2. FHFCPHARVMFDLDPESIWKNDYWT

3. CWPACNFPEEGDVGNWKPHFVEHDL

Locate the "Protein BLAST" page at NCBI and choose blastp as the
algorithm to use.

Paste in your sequences in FASTA format, and choose the "NR"

database (this is the protein version, consisting of translated CDS'es,
UniProt etc).

VERY IMPORTANT: We also need to tweak the parameters this time

- in the "Algorithm Parameters" section select BLOSUM62 as the
alignment matrix to use and set the "Expect threshold" to 1000
(default: 10) - and DISABLE the "Short queries" parameters as we
did in the DNA search a moment ago - otherwise our carefully
tweaked parameters will be ignored.

▪ Perform the BLAST search.

▪ Inspect the results.
 QUESTION 2.5:
(Remember to document your answers in the same manner as Q2.2)

▪ What is the typical length of the alignment and do they contain gaps?
The typical length is 700 the gaps are in between 10 and 25%

▪ What is the range of E-values? 44

▪ Try to inspect a few of the alignments in detail ("+" means similar

sequences) - do you find any that look plausible, if we for a moment ignore
the length/E-value?

▪ If we had used the default E-value cut-off of 10 would any hits have been
found? No significant similarity found.

QUESTION 2.6:

▪ If we compare the result from BLASTing random DNA sequences to random

Peptide sequences - which kind of search has the higher risk of returning
 false positives (results that appear plausible, maybe even significant, but are
truly unrelated)? the peptide sequences because of the algorithm parameter
that we established
▪ Remember to take E-values into your consideration.

Part 3: using BLAST to transfer functional information by finding

homologs
Homo-, Ortho- and Paralogs
One of the most common ways to use BLAST as a tool is in the situation where you have a
sequence of unknown functions, and want to find out which function it has. Since a large
amount of sequence data has been gathered during the years, chances are that an
evolutionarily related sequence with a known function has already been identified. In
general, such a related sequence is known as a "homolog".

Homo-, Ortho- and Paralogs:

▪ A Homolog is a general term that describes a sequence that is related by any

evolutionary means.
▪ An Ortholog ("Ortho" = True) is a sequence that is "the same gene" in a different
organism: The sequences shared a single common ancestor sequence, and has now
diverged through speciation (e.g. the Alpha-globin gene in Human and Mouse).
▪ A Paralog arises due to a gene duplication within a species. For example Alpha
and Beta-globin are each other's paralogs.
▪
Image source: gwLee's blog

Notice that in both cases it's possible to transfer information, for example, information about
gene family/protein domains. We have already touched upon a comparison of (potentially)
evolutionarily related sequences in the pairwise alignment exercise. However, this time we do
not start out with two sequences we assume are related, but we rather start out with a single
sequence ("query sequence") which we will use to search the databases for homologs (we
often informally speak of "BLAST hits", when discussing the sequences found).

BLAST example 1
Let's start out with a sequence that will produce some good hits in the database. The
sequence below is a full-length transcript (mRNA) from a prokaryote. Let's find out what it
is.
 >Unknown_transcript01
CCACTTGAAACCGTTTTAATCAAAAACGAAGTTGAGAAGATTCAGTCAACTTAACGTTAATATTTGTTTC
CCAATAGGCAAATCTTTCTAACTTTGATACGTTTAAACTACCAGCTTGGACAAGTTGGTATAAAAATGAG
GAGGGAACCGAATGAAGAAACCGTTGGGGAAAATTGTCGCAAGCACCGCACTACTCATTTCTGTTGCTTT
TAGTTCATCGATCGCATCGGCTGCTGAAGAAGCAAAAGAAAAATATTTAATTGGCTTTAATGAGCAGGAA
GCTGTTAGTGAGTTTGTAGAACAAGTAGAGGCAAATGACGAGGTCGCCATTCTCTCTGAGGAAGAGGAAG
TCGAAATTGAATTGCTTCATGAATTTGAAACGATTCCTGTTTTATCCGTTGAGTTAAGCCCAGAAGATGT
GGACGCGCTTGAACTCGATCCAGCGATTTCTTATATTGAAGAGGATGCAGAAGTAACGACAATGGCGCAA
TCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTG
TAAAAGTTGCTGTCCTCGATACAGGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTT
TGTACCAGGGGAACCATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCT
TTAAACAATTCGATTGGCGTTCTTGGCGTAGCGCCGAGCGCGGAACTATACGCTGTTAAAGTATTAGGGG
CGAGCGGTTCAGGTTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGT
TGCTAATTTGAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCGACTTCT
AGAGGGGTTCTTGTTGTAGCGGCATCTGGGAATTCAGGTGCAGGCTCAATCAGCTATCCGGCCCGTTATG
CGAACGCAATGGCAGTCGGAGCGACTGACCAAAACAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGG
GCTTGACATTGTCGCACCAGGTGTAAACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAAC
GGTACATCGATGGCTACTCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGT
CCAATGTACAAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAG
CGGACTTGTCAATGCAGAAGCGGCAACACGCTAATCAATAATAATAGGAGCTGTCCCAAAAGGTCATAGA
TAAATGACCTTTTGGGGTGGCTTTTTTACATTTGGATAAAAAAGCACAAAAAAATCGCCTCATCGTTTAA
AATGAAGGTACC

BLASTN search
Perform a BLAST search in the NR/NT database (BLASTN) using default settings. Remember
to set Expect threshold back to the default value, 10.

QUESTION 3.1:
(Once again remember to document your findings)

▪ Do we get any significant hits?

▪ What kind of genes (function) do we find? Similarities with bacillus and proteases

BLASTP search
Now let's try to do the same at the protein level.

▪ Find the best ORF using VirtualRibosome (hint: remember to search all
positive reading frames) and save of copy the sequence in FASTA
format.
https://services.healthtech.dtu.dk/service.php?VirtualRibosome-2.0

▪ BLAST the sequence (BLASTP) against the NR database.

QUESTION 3.2:
(Document!)

▪ Report your translated protein sequence in FASTA format.

PLETVLIKNEVEKIQST*R*YLFPNRQIFLTLIRLNYQLGQVGIKMRREPNEETVGENCRKHRTT
HFCCF*FIDRIGC*RSKRKIFNWL**AGSC**VCRTSRGK*RGRHSL*GRGSRN*IAS*I*NDSCFIR*VK
PRRCGRA*TRSSDFLY*RGCRSNDNGAISAMGN*PCASPSCP*PWIDRFWCKSCCPRYRYFHSSR
LKYSWWR*LCTRGTIHSRWEWAWHACGRDDCCFKQFDWRSWRSAERGTIRC*SIRGERFRFG
QLDCPRIGMGREQWHARC*FEFRKPFAKCHT*ASC**RDF*RGSCCSGIWEFRCRLNQLSGPLC
ERNGSRSD*PKQQPRQLFTVWRRA*HCRTRCKRAEHIPRFNVCQLKRYIDGYSSCCRCSSPC*T
KEPILVQCTNPQSSKEYGNELRKHELVWKRTCQCRSGNTLINNNRSCPKRS*INDLLGWLFYI
WIKKHKKIASSFKMKV

▪ Do we find any conserved protein domains? (Hint: Indicated at the very top of
the result page, and during the search or you can try with
https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)
 ▪ Identifying known protein domains can provide important clues to the function
of an unknown protein.
▪ Do we find any significant hits? (E-value?) E value equal to 0 up to 0.003
▪ Are all the best hits the same category of enzymes?
▪ From what you have seen, what is best for identifying intermediate quality hits -
DNA or Protein BLAST? The best way for identifying quality hits is protein
blast
BLAST example 2
In the previous section, we have been cheating a bit by using a sequence that was already in
the database - let's move on to the following sequence instead.

The sequence is a DNA fragment from an unknown non-cultivatable microorganism. It was

cloned and sequenced directly from DNA extracted from a soil sample, and it goes by the
poetic name "CLONE12". It was amplified using degenerated PCR primers that target the
middle ("core cloning") of the sequence of a group of known enzymes. (I can guarantee this
particular sequence is not in the BLAST databases, since I have cloned and sequenced it
myself, and it has never been submitted to GenBank).
 LOCUS CLONE12.DNA 609 BP DS-DNA UPDATED 06/14/98 DEFINITION UWGCG
file capture
ACCESSION -
KEYWORDS -
SOURCE -
COMMENT Non-sequence data from original file:
BASE COUNT 174 A 116 C 162 G 157 T 0 OTHER
ORIGIN ?
clone12.dna Length: 609 Jun 13, 1998 - 03:39 PM Check: 6014 .. 1
AACGGGCACG GGACGCATGT AGCTGGAACA GTGGCAGCCG TAAATAATAA TGGTATCGGA 61
GTTGCCGGGG TTGCAGGAGG AAACGGCTCT ACCAATAGTG GAGCAAGGTT AATGTCCACA 121
CAAATTTTTA ATAGTGATGG GGATTATACA AATAGCGAAA CTCTTGTGTA CAGAGCCATT 181
GTTTATGGTG CAGATAACGG AGCTGTGATC TCGCAAAATA GCTGGGGTAG TCAGTCTCTG 241
ACTATTAAGG AGTTGCAGAA AGCTGCGATC GACTATTTCA TTGATTATGC AGGAATGGAC 301
GAAACAGGAG AAATACAGAC AGGCCCTATG AGGGGAGGTA TATTTATAGC TGCCGCCGGA 361
AACGATAACG TTTCCACTCC AAATATGCCT TCAGCTTATG AACGGGTTTT AGCTGTGGCC 421
TCAATGGGAC CAGATTTTAC TAAGGCAAGC TATAGCACTT TTGGAACATG GACTGATATT 481
ACTGCTCCTG GCGGAGATAT TGACAAATTT GATTTGTCAG AATACGGAGT TCTCAGCACT 541
TATGCCGATA ATTATTATGC TTATGGAGAG GGAACATCCA TGGCTTGTCC ACATGTCGCC 601
GGCGCCGCC
//

QUESTION 3.3 (Long question - read all):

Your task is now to find out what kind of enzyme this sequence is likely to
encode, using the methods you have learned.

INSTRUCTIONS: You are free to write the combined answer to this question in a free
style essay-like fashion - just be sure to include the subquestions in your answers. In
an exam situation, you will need to put all the clues together yourself, reason about the
tools/databases to use, and document your findings.

STEP 1 - cleaning up the sequence:

The sequence is (more or less) in GenBank format and the NCBI BLAST server
 expects the input to be in FASTA format, or to be "raw" unformatted sequence.

▪ There are two solutions to this:

▪ Copy the sequence into a text-editor and manually
create a FASTA file ("search and replace" and/or
"rectangular selection" is useful for the reformatting).
This is the most robust solution: it will always work.
(Look at the JEdit exercise for a reminder of how to
do this).
▪ Hope the creators of the web-server you're using
were kind enough to automatically remove non-DNA
letters (paste in ONLY the DNA lines) - this turns out
to be the case for both NCBI BLAST and
VirtualRibosome, but it cannot be universally relied
upon.
Subquestion: convert the sequence to FASTA format (manually, in JEdit
http://www.jedit.org/) and quote it in your report.

STEP 2 - thinking about the task:

Consider the following before you start on solving this task:

▪ Based on the information given: is the sequence protein

coding?
▪ If it is, can you trust it will contain both a START and STOP
codon?
▪ Do we know if the sequence is sense or anti-sense?

and think which consequences the answers to these questions should

have for your choice of methods and parameters.

Subquestion: Give a summary of your considerations.

STEP 3 - Performing the database search:

Significance: We will put the criteria for significance at 1e-10 (remember:

the higher the E-value, the worse the significance).

Subquestion:
Cover the following in your answer:

▪ What tool(s) and database(s) will be relevant to use? ▪

Protein blast and virtual ribosome

Document the results from the different BLAST searches -

what works and what does not work?
▪ You need to copy in small snippets of the BLAST results to
document what you observe.

▪ In conclusion: What kind of enzyme is CLONE12? Gather as

much evidence as possible.
Serina peptidasa de la familia S8 [Bacteroides sp.].
Genes (total) :: 3.485
CDS (total) :: 3.456
Genes (codificación) :: 3.052
CDS (con proteína) :: 3.052
 Genes (ARN) :: 29
ARNt :: 27
ncRNA :: 2
Pseudo Genes (total) :: 404
CDSs (sin proteína) :: 404
Pseudogenes (residuos ambiguos) :: 0 de 404
Pseudo Genes (frameshifted) :: 310 de 404
Pseudo Genes (incompleto) :: 42 de 404
Pseudo Genes (parada interna) :: 142 de 404
Pseudo Genes (problemas múltiples) :: 81 de 404
##Genoma-Anotación-Datos-FIN##

Part 4: BLAST'ing Genomes

So far we have been using BLAST to search in big broad databases that cover a huge set
of sequences from a large range of organisms. In this final part of the exercise, we will be
doing some more focused searches in smaller databases by targeting specific genomes.

Typically, this will be useful if you have a gene of known function from one organism (say a
cell-cycle controlling gene from Yeast, Saccharomyces cerevisiae) and want to find the human
homolog/ortholog to this gene (genes that control cell division are often involved in cancer).

When you have been performing the BLAST searches, you have probably already noticed
that it is possible to search specifically in the Human and Mouse genomes (these
databases only contain sequences from Human/Mouse). It's also possible to restrict the
output from searches in the large databases (e.g. NR) to specific organisms.

A growing number of organisms have been fully sequenced, and the research teams
responsible for a large-scale genome project typically put up their own Web resources for
accessing the data. For example the Yeast genome is principally hosted in the Saccharomyces
Genome Database (SGD - www.yeastgenome.org) - it should be noted that SGD also offers
BLAST as a means to search the database.

Genome-specific analysis of histones

SGD

Let's do a small study of the relationship between the histones found in Yeast and in
humans (evolutionary distance: ~1-1.5 billion years).

Look up the HTA2 gene in SGD (http://www.yeastgenome.org - use the search box at the top
of the page). Notice that a brief description of the function of the gene and its protein product is
displayed (a huge amount of additional information can be found further down the page - much
of it Yeast specific).
QUESTION 4.1:
What information is given about the relationship between this gene and the gene
"HTA1"?
Browse the page and locate the link to the protein sequence. Save the sequence as a
file, we'll need it in a moment.
NCBI
Now return to the NCBI blastp page. S
 et Database to "Reference proteins (refseq_protein)", and
enter Saccharomyces cerevisiae in the Organism field (and accept the
suggestion with taxid:4932).

QUESTION 4.2:
(Remember to document your answers)

▪ How many high-confidence hits do we get?

▪ Do the hits make sense, from what you have read about HTA2 at the SGD
webpage?

Tip: click on the Gene links under Related Information (to the right of the alignments) to
see the gene names for the protein hits.

The next step is to search the translated version of the human genome.

Do as before, still with Database set to "Reference proteins

(refseq_protein)", just enter Human in the Organism field.

QUESTION 4.3:

-10
▪ How many high-confidence hits (with E-value better than 10 ) are found?
(Approximately)
▪ What are all the high-confidence hits called?

Concluding remarks
Today we have been using BLAST to find a number of homologous genes (and protein
products). If we want to go even deeper into the analysis of the homologs, the next logical step
would be to build a dataset of the full-length versions of the sequences we have found (not just
the part found by the local alignment in BLAST).
A further analysis could consist of a series of pairwise alignments (for finding out what is
similar/different between pairs of sequences) or a multiple alignment which could form the
basis of establishing the evolutionary relationship between the entire set of sequences.

BLAST can also be used as a way to build a dataset of sequences based on a known
"seed" sequence. As we saw in the GenBank exercise, free-text searching in the GenBank
can be difficult, and if we for instance wanted to build a dataset of variants of the insulin
gene, an easy way to go around this would be to BLAST the normal version of the insulin
against the sequence database of choice, and pick the best matching hits from here.