Last Update: 12/24/2020

An Introduction to NCBI BLAST

Wilson Leung

Prerequisites
Detecting and Interpreting Genetic Homology: Lecture Notes on Alignment

Resources
The BLAST web server is available at https://blast.ncbi.nlm.nih.gov/Blast.cgi
Gene Record Finder is available at https://gander.wustl.edu/~wilson/dmelgenerecord/index.html

Files for this walkthrough

The package containing the files for this walkthrough are available through the “An Introduction
to NCBI BLAST” page on the GEP web site.

Introduction
The Basic Local Alignment Search Tool (BLAST) is a program that can detect sequence
similarity between a query sequence and sequences within a database. The ability to detect
sequence homology allows us to identify putative genes in a novel sequence. It also allows us to
determine if a gene or a protein is related to other known genes or proteins.

BLAST is popular because it can quickly identify regions of local similarity between two
sequences. More importantly, BLAST uses a robust statistical framework that can determine if
the alignment between two sequences is statistically significant. In this walkthrough, we will use
the National Center for Biotechnology Information (NCBI) BLAST service to help us annotate a
sequence from the Drosophila yakuba genome (unknown.fna in the walkthrough package).

The NCBI BLAST web interface

Before we begin the analysis, we should first familiarize ourselves with the NCBI BLAST web
interface. Open a new web browser window and navigate to the NCBI BLAST main page at
https://blast.ncbi.nlm.nih.gov/Blast.cgi. In this walkthrough, we will only use a few of the tools
available on the NCBI BLAST web site. To learn about the more advanced options available
(such as setting up My NCBI accounts), click on the “Help” link on the main navigation bar to
access the documentations for NCBI BLAST (Figure 1).

Figure 1. Click on the “Help” link to learn more about the NCBI BLAST web interface.

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All of the NCBI BLAST pages have the same header with four links:
Links Explanation
Home Link to the NCBI BLAST home page
Recent Results Link to results of the BLAST searches you have run previously
Saved Strategies NCBI BLAST search parameters you have previously saved to your My NCBI account
Help Documentation for NCBI BLAST

Besides the main toolbar, there are two other sections of the NCBI BLAST web interface that are
of interests: the “Web BLAST” section contains links to the common BLAST programs and the
“Specialized searches” section contains links to additional tools for performing sequence
searches (e.g., use CD-search to identify conserved domains within a query sequence). The type
of BLAST search you need to use will depend primarily on the type of query sequence and the
database you would like to search.

Four of the five common BLAST programs are available through the “Web BLAST” section of
the NCBI BLAST home page (Figure 2, top). The program tblastx, which translates the
nucleotide query and nucleotide database when it performs the sequence comparisons, is not
listed under the “Web BLAST” section. However, you can access this program by clicking on any
of the BLAST programs in the “Web BLAST” section and then click on the “tblastx” tab in the
NCBI BLAST search form (Figure 2, bottom).

Figure 2. The different BLAST programs available through the NCBI web server home page (top). The tblastx
program is available through the “tblastx” tab in the NCBI BLAST search form (bottom).

The basic BLAST programs are summarized below:

BLAST program Query Database
Nucleotide BLAST (blastn) Nucleotide Nucleotide
Protein BLAST (blastp) Protein Protein
blastx Translated Nucleotide Protein
tblastn Protein Translated Nucleotide
tblastx Translated Nucleotide Translated Nucleotide

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Instead of searching a query sequence against sequences in a database, you can also align two (or
more) sequences by selecting the “Align two or more sequences” checkbox at the bottom of the
“Enter Query Sequence” section (Figure 3). This feature is also known as BLAST 2 Sequences
(bl2seq).

Figure 3. Select the “Align two or more sequences” checkbox to compare a query sequence against a subject
sequence instead of a BLAST database.

Detecting sequence homology to mRNA using blastn

In this walkthrough, we will characterize an unknown genomic sequence (unknown.fna) and
determine if it has sequence similarity to any known genes. One strategy we can use is to search
for sequence similarity to mRNA sequences in the NCBI Reference Sequence (RefSeq) database.

When we set up a BLAST search, there are three basic decisions we must make: the BLAST
program we want to use, the query sequence we want to annotate, and the database we want to
search. In addition, we can change several optional parameters (such as the Expect threshold and
low complexity filters) in order to modify the behavior of BLAST.

In this case, we will set up our BLAST search using mostly default parameters. We will use the
blastn program to search our sequence (query) against the NCBI Reference Sequence (RefSeq)
RNA database (Figure 4).

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1. Navigate to the NCBI BLAST home page and click on the “Nucleotide BLAST”
image under the “Web BLAST” section
2. Under the “Enter Query Sequence” section, click on the “Browse” or the “Choose
File” button and select the file with the unknown sequence (unknown.fna)
3. Enter the Job Title “blastn search D. yakuba / RefSeq RNA”
4. In the “Choose Search Set” section, change the database to “Reference RNA
sequences (refseq_rna)”
5. Under “Program Selection”, select “Somewhat similar sequences (blastn)”
6. Check the box “Show results in a new window” next to the “BLAST” button
7. Click “BLAST”

Figure 4. Setting up our blastn search of the unknown sequence against the NCBI RefSeq RNA database.

Note: the blastn search may take a few minutes to complete when the NCBI web server is busy
(Figure 5).

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Figure 5. Waiting for the blastn search results

Once the search is complete, a new web page will appear with the BLAST report. For teaching
purposes, the BLAST output (blastnInitial.txt) is available in the package for this walkthrough.

The top left panel of the BLAST results page shows the parameters used in the BLAST search
(e.g., database name, query ID, query length). The controls in the top right panel can be used to
filter the BLAST hits by organism, percent identity, and Expect value (E-value). The details of
the BLAST results are organized into the four tabs below these two panels: “Descriptions”,
“Graphic Summary”, “Alignments”, and “Taxonomy”. We will go through each of these sections
in order to interpret our blastn output.

I. Descriptions
This tab shows the list of sequences in the database that have significant sequence homology
with our sequence (Figure 6). By default, the results are sorted by their E-value in ascending
order, where lower E-values denote more significant hits. You can click on the column headers
to sort the results by the other columns. You can also use the “Select columns” drop-down menu
on the main toolbar to show or hide each column.

Figure 6. List of blastn hits that produce significant alignments with our query sequence.

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Clicking on the accession number in the table will bring up a new page with the GenBank record
of the sequence. Clicking on the description of the hit will bring us to the corresponding
alignment in the BLAST output. Alternatively, you can click on the “Alignments” tab to jump to
the first alignment.

In addition to reviewing the records for individual sequences, you can also review multiple
sequence records by selecting the checkbox next to each match. The contents of the other tabs
will update automatically based on your selection. You can use the “Download” drop-down
menu on the main toolbar to download the selected hits in multiple formats (e.g., FASTA,
GenBank, Hit Table). For example, we can use the following steps to retrieve the GenBank
records for the first five blastn hits in the Descriptions table (Figure 7).

1. Uncheck the “select all” checkbox above the BLAST hit table
2. Select the checkboxes for the first five blastn hits
3. Click on the “Download” drop-down menu on the main toolbar, and then select
the “GenBank (complete sequence)” option

Figure 7. Click on the “GenBank (complete sequence)” link under the “Download” drop-down menu to retrieve the
GenBank records for the five selected mRNA sequences.

II. Graphic Summary

This tab provides a graphical overview of the alignments between the selected BLAST hits in the
Descriptions tab and the query sequence. The boxes correspond to regions in the query that have
sequence similarity to the sequences in the database. The color of the box corresponds to the
score, where hits with higher scores are more significant. When you move your mouse over a
BLAST hit, the title of the subject sequence will appear in a tooltip. Click on the color box and
then click on the “Alignment” link to jump to the alignments associated with that BLAST hit.

To examine the graphical overview for all the blastn hits, go back to the “Descriptions” tab and
then select the “select all” checkbox. Click on the “Graphic Summary” tab to view the updated
graphical overview (Figure 8).

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Figure 8. The “Graphic Summary” tab shows the graphical overview for the selected BLAST hits in the
“Descriptions” tab. Select the “select all” checkbox in the “Descriptions” tab and then navigate back to the “Graphic
Summary” tab to view the graphical overview for all the BLAST hits.

III. Alignments
This tab contains the alignments between the selected BLAST hits in the Descriptions tab and the
query sequence. The sequence alignments show us how well our query sequence match the
subject sequence in the database. Because we will rely on sequence alignments heavily in our
annotation efforts, we will examine this Alignment tab more closely.

Alignments to different subject sequences in the database are separated by a blue toolbar that
contains options to manipulate the alignment results and to retrieve additional information for
that specific BLAST hit (Figure 9). For example, we can use the “Download” drop-down menu
on this toolbar to obtain the FASTA sequence or the GenBank record for a specific hit. We can
use the navigation links at the right side of the toolbar to quickly navigate to the next or the
previous BLAST hit.

Figure 9. Alignments to different subject sequences in the database are separated by a blue toolbar with options to
manipulate and download the alignment results.

In addition, we can click on the “Graphics” link to examine the location of each alignment block
relative to the subject sequence (Figure 10).

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Figure 10. The “Graphics” link allows us to see a graphical view of the alignment blocks relative to the subject
sequence (e.g., the D. melanogaster legless mRNA).

As its name suggests, BLAST is designed to identify local regions of sequence similarity. This
means that BLAST might report multiple distinct regions of sequence similarity when we align a
query against a subject sequence in a database. For example, if we were to align a processed
mRNA sequence to a genomic sequence, we would expect to see multiple alignment blocks
(many of which correspond to transcribed exons) in our BLAST output. Each alignment block
demarcates a local region of similarity between the query and the subject sequences. Regions of
the genomic sequence without significant alignments that fall between these alignment blocks
would likely correspond to intronic sequences.

The “Number of Matches” field beneath the name of the sequence shows the number of
alignment blocks identified by blastn. For example, the seventh blastn hit contains 6 different
alignment blocks to the subject sequence — the legless mRNA from D. melanogaster (Figure
11). Each alignment block represents a region of the D. melanogaster legless gene that shows
sequence homology with our genomic sequence from D. yakuba.

Figure 11. blastn detected 6 distinct alignment blocks between the D. melanogaster legless mRNA and the
D. yakuba genomic sequence.

You can use the “Sort by” drop-down box (red arrow in Figure 11) on the toolbar above each
BLAST hit to sort the alignment blocks based on different criteria (e.g., by E-value, query start
position, subject start position). Each alignment block begins with a line that has the following
format: “Range #:start to end” (where # is the alignment block number). You can use the “Next
Match” and “Previous Match” links to navigate to the different alignment blocks within the same
BLAST hit.

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Depending on the database you use, there might be additional links to other parts of NCBI listed
under the “Related Information” panel next to the sequence alignments. For example, there are
links to Entrez Gene, GEO Profiles, PubChem BioAssay, and the Genome Data Viewer for the
seventh hit “Drosophila melanogaster legless (lgs), mRNA” (NM_143665.4; Figure 12). Entrez
Gene provides us with an overview of the gene and links to literature references. GEO Profiles
allow us to access expression data associated with the gene. PubChem BioAssay contains
bioactivity and toxicity data derived from small-molecule and RNAi screens. The Genome Data
Viewer allow us to view the BLAST alignments in a genome browser with other evidence tracks
(e.g., gene annotations, RNA-Seq data, repeats).

Figure 12. You can learn more about the blastn match using the links under the “Related Information” section.

What about the alignments themselves? Each alignment block begins with a summary, including
the Expect value (i.e. the statistical significance of the alignment), sequence identity (number of
identical bases between the query and the subject sequence), the number of gaps in the
alignment, and the orientation of the query relative to the subject sequence. The alignment
consists of three lines: the query sequence, the matching sequence, and the subject sequence
(Figure 13).

Figure 13. The key characteristics of a typical BLAST alignment.

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The - character in either the query or the subject sequence denotes a gap in the alignment
(Figure 14).

Figure 14. Gaps in the alignment are represented by the ‘-’ character.

By default, NCBI BLAST automatically masks low complexity sequences in the query sequence.
Depending on your BLAST search settings, these masked bases may appear as either grey
lowercase letters (Figure 15) or as X’s. The matching sequence consists of a combination of | and
empty spaces, where | denotes a matching base between the query and subject sequences and the
empty space denotes a mismatched base.

Figure 15. Bases masked by the low complexity filter appear as lowercase grey letters by default.

IV. Taxonomy
This tab shows the taxonomy of the selected BLAST hits in the Descriptions tab. The Taxonomy
tab organizes the selected BLAST hits in three different report formats: Lineage, Organism, and
Taxonomy (Figure 16).

Figure 16. Use the buttons next to the “Reports” label on the main toolbar of the Taxonomy tab to view the Lineage,
Organism, and Taxonomy reports for the selected BLAST hits.

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The Lineage report provides an overview of the number of selected BLAST hits that are at each
taxonomic level. The level of indentation in the “Organism” column corresponds to the
taxonomic level. The value in the “Score” column corresponds to the maximum score for the
BLAST hits of a terminal node. The value in the “Number of Hits” column shows the number of
selected hits that are at the corresponding taxonomic level (Figure 17).

Figure 17. The Lineage report under the Taxonomy tab shows that 51 of the 100 selected blastn hits are in the
melanogaster subgroup.

The Organism report groups the selected BLAST hits by organism. The BLAST hits for the
different species are separated by a blue header. Within each species, the BLAST hits are sorted
by E-value in ascending order (Figure 18).

Figure 18. The Organism report under the Taxonomy tab allows one to quickly identify the best match to the query
sequence in each species.

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The Taxonomy report has a similar layout compared to the Lineage report. However, the
Taxonomy report provides additional controls (the +/- icons under the “Taxonomy” column) to
expand or collapse the non-leaf nodes (Figure 19). It also includes the number of organisms with
BLAST hits at each taxonomic level.

Figure 19. Click on the “-” icon next to the taxonomic level under the “Taxonomy” column to collapse a non-leaf
node (red arrow). Click on the “+” icon next to the taxonomic level to expand the non-leaf node (purple arrow).

Interpreting the blastn search result

Now that we have a better understanding of how the BLAST report is organized, we are ready to
interpret the blastn results. The “Descriptions” and the “Graphic Summary” tabs (Figure 6 and
Figure 8) show that the top 27 hits are much more significant (with E-values of 0.0) than the rest
of the blastn hits. Most of these top hits contain regions of sequence similarity that span the
entire length of the query sequence (Figure 8). Looking at the descriptions and the corresponding
GenBank records, it appears that these blastn hits correspond to the gene legless (also known as
BCL9) in different Drosophila species.

Among these significant matches, only the D. melanogaster hit has an accession number that
begins with the prefix “NM_” (Figure 20). The accession numbers for the matches to the other
Drosophila species all have the prefix “XM_”. The main difference between these two prefixes
is the type of information available to support the RefSeq mRNAs. The “NM_” prefix indicates
that the RefSeq mRNA record is supported by experimental evidence, whereas the “XM_” prefix
indicates that the record is based solely on computational predictions. Because we would prefer
to base our inferences on a gene model that is supported by experimental evidence, we will use
the D. melanogaster model in this analysis.

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Figure 20. The best manually curated RefSeq match to the query sequence is the D. melanogaster legless (lgs)
mRNA (with the accession number NM_143665.4).

From the blastn hit list, click on the description that corresponds to the D. melanogaster hit to
jump to the alignment section. Our analysis above has shown that there are six alignment blocks
(Figure 11). We also notice that the D. melanogaster mRNA has a total length of 5357 bases, so
the first question we would like to address is whether the entire mRNA aligns to our sequence.

To address this question, we will examine the subject coordinates of the alignment blocks from
the D. melanogaster legless mRNA. We find that these blocks span from 3209-1200, 5357-3394,
699-2, 987-699, 1204-990, and 3395-3198. Re-ordering the coordinates of the alignment blocks
with respect to our subject sequence produces the following list of alignments (coordinates of the
query sequence are in parenthesis): 2-699 (9853-9167), 699-987 (9107-8819), 990-1204 (8314-
8100), 1200-3209 (5374-3359), 3198-3395 (2809-2606), and 3394-5357 (2552-586).

Despite some minor overlaps and missing bases, we can account for most of the mRNA
sequence in this collection of alignments. Note that all the alignment blocks are collinear with
respect to our query sequence (i.e. all the alignment blocks are in the reverse orientation relative
to the subject mRNA) and show a high degree of sequence similarity (with sequence identity that
ranges from 75–92% at the nucleotide level).

Detecting Coding Regions Using blastx

Because the RefSeq mRNA sequence consists of both translated and untranslated regions (i.e. 5’
and 3’ UTRs), the next step in our analysis is to identify the coding region in our sequence. We
will set up a blastx search in order to compare a nucleotide genomic sequence against a protein
database. Because every mRNA in the RefSeq RNA database has a corresponding sequence in
the RefSeq Protein database, we will search our D. yakuba sequence against the RefSeq Protein
(refseq_protein) database. We now have all the information we need to setup the blastx search.

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1. Navigate to the NCBI BLAST home page and click on the “blastx” image
2. Under the “Enter Query Sequence” section, click on the “Browse” or the “Choose
File” button and select our sequence (unknown.fna).
3. Enter the Job Title “blastx search D. yakuba / RefSeq Protein”
4. In the “Choose Search Set” section, change the database to “Reference proteins
(refseq_protein)”.
5. Check the box “Show results in a new window” next to the “BLAST” button
6. Click “BLAST” (Figure 21)

Figure 21. Configure our blastx search of the unknown sequence against the NCBI RefSeq Protein database.

For teaching purposes, the blastx search result (blastxRefSeqProtein.txt) is available in

the package for this walkthrough. (Note that this blastx search can take several minutes to
complete.)

The blastx report is similar to the blastn report. It consists of the “Descriptions”,
“Graphic Summary”, “Alignments”, and “Taxonomy” tabs. The “Graphic Summary” tab
shows the highly significant hits to the legless protein in D. melanogaster and the
homologous protein in the other Drosophila species. It also shows a few significant hits
to transposases in the region between 6000-8000 bp of our sequence (Figure 22).

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Figure 22. Multiple blastx hits in the region between 6000-8000 bp in our sequence.

These hits suggest our sequence contains a type of repetitious element called a transposable
element. In future walkthroughs, we will learn how we can reduce the number of spurious hits in
our BLAST reports by masking these elements prior to performing the BLAST search. For now,
we will ignore these additional matches and focus on the best manually curated RefSeq hit — the
D. melanogaster legless protein (NP_651922.1; Figure 23).

Figure 23. The blastx result shows that our sequence is very similar to the D. melanogaster legless protein.

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We can analyze the blastx alignments in the same way that we have previously analyzed the
blastn report. However, because blastx translates the input sequence in all 6 reading frames
before comparing our sequence with the protein database, there is an additional “Frame” field in
each alignment block. The frame begins with either + or -, which corresponds to the relative
orientation of our sequence compared to the protein. The number following the relative
orientation in the frame field ranges from 1 to 3, which reflects the reading frame that produces
the translated peptide sequence. Collectively, the relative orientation and the number can be used
to represent all 6 reading frames. A frame shift between two alignment blocks in the blastx
match often indicates that the two alignment blocks correspond to different coding exons. The
“Positives” field corresponds to the number of amino acids that are either identical or have
similar chemical properties between the translated query and the subject sequences (Figure 24).

Figure 24. The key characteristics of a blastx alignment.

Similar to the blastn alignment, each alignment block in our blastx report also consists of three
lines: the query sequence, the matching sequence, and the subject sequence. Note that the query
sequence has been translated into the corresponding amino acid sequence in the reading frame
specified by the “Frame” field. However, the coordinates of the query sequence are still relative
to the original nucleotide sequence. Like our blastn alignment, the grey lowercase residues in the
query sequence correspond to low complexity sequences that were masked by BLAST.

There are some minor differences in the matching sequence of the blastn and blastx outputs.
Residues in the matching sequence represent amino acids that are identical between the query
and subject sequences. The “+” character denotes amino acids that are different between the
query and subject but these different amino acids have similar chemical properties. A space
indicates that the two aligned amino acid in the query and subject are different and they have
different chemical properties.

When investigating the blastx alignment with the D. melanogaster legless protein, the first
question is whether there are matches to the entire legless protein. We see from the “Length”
field underneath the sequence name that the D. melanogaster legless protein has 1469 residues.
Sorting the alignment blocks by the subject start position, we see matches to the protein sequence
at 1-158 (9344-8814), 148-229 (8332-8099), 228-897 (5374-3359), 888-959 (2827-2606), and
959-1469 (2553-1018). In addition, the coordinates relative to our query sequence (in

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parenthesis) are consistent with the results from our previous blastn search. Based on both the
blastn and blastx results, we can determine the approximate coordinates of the UTRs and the
coding regions in our D. yakuba sequence. Hence it appears that our D. yakuba sequence
contains an ortholog of the D. melanogaster legless gene.

While the alignment generally looks good, there are a few problems with some of the blastx
alignment blocks. Looking at the alignment block that corresponds to the first 158 amino acids of
the protein sequence (9344-8814 in our query sequence), we noticed a large gap beginning at
residue 61 (9167 in our query sequence) (Figure 25). Furthermore, the translation of the query in
this region contains a stop codon (the * character). One possible explanation for the stop codon is
that blastx might have combined two separate exons into the same alignment. If that were the
case, the intron between the two exons would also be translated by blastx.

Figure 25. The blastx alignment between the unknown sequence (query) and the D. melanogaster legless protein
(subject) shows a large gap and an in-frame stop codon (*).

Another problem with the alignments is the substantial amount of overlap between two adjacent
alignment blocks; this occurs with blocks 1-158 and 148-229, and with blocks 228-897 and 888-
959. However, examination of the beginning of the alignment block that spans from 148-229
shows that the first 10 residues in the alignment block have much weaker sequence similarity
than the rest of the residues in the alignment (Figure 26). We also see a similar pattern in the
alignment block beginning at 888 compared to the block that ends at residue 897. Hence our
observations suggest that blastx might have over-extended the alignments in both cases.

Figure 26. The beginning of the alignment shows a much lower degree of sequence homology.

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Define the Intron-Exon Boundaries with Gene Record Finder and bl2seq
Based on our previous blastn and blastx analyses, our current hypothesis is that we have
identified the putative ortholog of the legless gene in our D. yakuba sequence. However, in order
to construct a complete gene model, we must resolve the discrepancies in the alignments of our
blastn and blastx output. Because the coding region is under strong selective pressure and is
likely to be more conserved than other regions of the genome, our first step is to identify the
coding regions of our putative gene.

To begin the more detailed analysis, we will perform a series of BLAST searches using the amino
acid sequence of each exon in the D. melanogaster version of the legless gene. It will be helpful
to our annotation efforts if we can obtain the amino acid sequence that corresponds to each exon
individually. Fortunately, we can easily obtain the individual exon sequences using the Gene
Record Finder.

1. Navigate to the F Element project page on the GEP web site

2. Click on the “Gene Record Finder” link under “Resources & Tools”
3. Type “lgs” (the official FlyBase symbol for the legless gene) in the textbox and
click on the “Find Record” button (Figure 27)

Figure 27. Access the Gene Record Finder from the F Element project page on the GEP web site

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In the “mRNA Details” section of the gene report, we notice that there is only one
isoform of the legless gene in D. melanogaster (lgs-RA, the A isoform of lgs). We can
access all the transcribed exons through the “Transcript Details” tab and all the translated
exons through the “Polypeptide Details” tab (Figure 28).

Figure 28. Coding exons for the selected isoform of lgs is listed under the “Polypeptide Details” section

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To retrieve the amino acid sequence for each coding exon (CDS), click on the row that
corresponds to the coding exon in the CDS table (Figure 29).

Figure 29. Click on a row in the CDS table to retrieve the amino acid sequence for the corresponding coding exon.

The first problem in our blastx results is the stop codon in the alignment block that spans from 1-
158 of the translated protein sequence. To determine the locations of the coding exons, we will
perform BLAST searches to compare the individual exons with our sequence. Because we are
comparing a protein sequence against a nucleotide sequence, we will use the tblastn program for
our search. In order to prevent BLAST from masking low complexity regions in our protein, we
will turn off the low complexity filter. In addition, because we are only comparing two
sequences, we will also turn off compositional adjustments under scoring parameters.

1. Select the first CDS (1_9490_0) from the Gene Record Finder CDS table and
copy the sequence to the clipboard
2. Open a new web browser tab and navigate to the NCBI BLAST home page; click
on the “tblastn” image under the “Web BLAST” section
3. Select the checkbox “Align two or more sequences” under the “Enter Query
Sequence” section
4. Paste the CDS sequence for 1_9490_0 into the “Enter Query Sequence” field
5. For the “Subject Sequence”, click on the “Browse” or the “Choose File” button
and select our unknown sequence (unknown.fna)
6. Click on the “Algorithm parameters” link to expand this section. Verify that the
“Word size” parameter is set to 3.
7. Change the “Compositional adjustments” field to “No adjustment” under the
“Scoring Parameters” section
8. Uncheck the box “Low complexity regions” under “Filters and Masking”
9. Click “BLAST” (Figure 30).

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Figure 30. Use the “Align two or more sequences” feature with tblastn to align the first coding exon of lgs against
our sequence with the low complexity filter turned off and no compositional adjustment.

For teaching purposes, the BLAST output (bl2lgsExon1_tbn.txt) is available in the

package for this walkthrough.

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From the “Alignments” tab of the tblastn output, we see that the first coding exon has a
length of 60 amino acids and corresponds to 9344-9168 of the query sequence when it is
translated in frame -2 (Figure 31).

Figure 31. bl2seq results showing the tblastn alignment of the first coding exon with our unknown sequence

We can use the same strategy to map the rest of the coding exons:
Exon # Protein Alignment Our Sequence Alignment Frame
(Number of complete codons) (Start-End) (Start-End)
1 (60) 1-60 9344-9168 -2
2 (96) 1-95 9104-8820 -2
3 (69) 1-69 8311-8105 -3
4 (667) 1-667 5371-3365 -3
5 (63) 1-63 2800-2606 -3
6 (511) 1-511 2550-1015 -1

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The results of our exon-by-exon bl2seq analyses suggest we can account for all of the
coding exons of the D. melanogaster legless gene in our sequence. Furthermore, we were
able to resolve the problem with the first exon in our initial blastx search: the alignment
block that spans from 9344-8814 actually consists of two separate exons, one that spans
approximately from 9344-9168 and the other from 9104-8820.

For your own annotation projects, it will be advantageous to save the bl2seq outputs as you
construct your gene model so that you can revisit the results later (e.g., via the “Download All”
drop-down menu in the RID field of the BLAST results page). Note that we have yet to generate a
complete gene model for this putative gene in our D. yakuba sequence. In future walkthroughs,
we will learn how we can use the UCSC Genome Browser to identify intron splice sites and to
define the exact exon boundaries.

We could use the same strategy to map the locations of the putative UTRs using bl2seq with the
blastn program. We can then compare the exon-by-exon search results with our initial blastn
search against the RefSeq RNA database. To retrieve the exon sequences for each transcribed
exon, select the “Transcript Details” tab in the Gene Record Finder and then click on a row in
the exon table (Figure 32). Mapping of the untranslated exons is left as an exercise for the reader.

Figure 32. The “Transcript Details” tab of the Gene Record Finder shows the list of transcribed exons for the
D. melanogaster gene legless

Conclusion
In this walkthrough, we have used multiple BLAST programs to identify and characterize a
putative gene in a genomic sequence from D. yakuba. You are now ready to tackle some of the
more challenging BLAST exercises on the GEP web site:

• Detecting and Interpreting Genetic Homology

• Using mRNA and EST Evidence in Annotation

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