Identification of Organism place in a place in a
conical tube sealable bag
1. Sample Collection with absolute with desiccants
2. DNA Extraction ethanol
- DNA Fragments/Primers Store at room
a. Forward sequence temperature,
b. Reverse Sequence refrigerator
3. DNA Amplification
- Multiplying
Blast Query Database Alignmen General Use
- Amplicons (End products)
Type Sequence t
4. DNA Sequencing
a. Good Quality
b. Bad Quality
5. DNA Analysis
BLASTn nucleotid nucleotid nucleotide Sequence
Directly Proportional e e identificatio
n
Inversely Proportional ;
Percent Query Cover Expected Value Useful for
Identification all taxa
Percent of Percent of If query cover categories
nucleotides sequence is high, E-value BLASTx nucleotid protein protein Identificatio
or amino align from must be low or e n of
acids that sequences in 0.0. potential
are identical GenBank proteins
between the Similarities encoded by
query
aligned Accepted sequences
query and value is ;
the database 75%- 100% Detection of
sequences novel virus
Must be BLASTp protein protein protein Sequence
98%-100% identificatio
Example. If the Example. If A lower E n
two sequences you have a value
are align, and query suggests that
out of 100 sequence of the match is tBLASTx nucleotid nucleotid protein Identificatio
positions, 80 200 base more e e n of
are identical so pairs and it significant or nucleotide
the percent aligns with meaningful, sequences
identity would 160 base indicating a similar to the
be 80%. It query based
pairs of a lower
on their
gives an idea subject probability coding
on how closely sequence, the of the match potential
related or query cover occurring by tBLASTn protein nucleotid protein Identificatio
similar the would be random e n of database
sequences are. 80%. chance. sequences
encoding
proteins
similar to the
query.
For Animal Sample For Plant Sample
After collection, After collection,
Max score Total Score
Highest score The sum of
calculated from alignment scores
matches and for all of the
mismatches sequence
found in local segments or
alignments local alignments
The higher the When the max
max score, the score and total
better the score are the
alignment same, there is
one global
alignment
between the
query and its
match in the
database.
This means that
the sequences
can be aligned
without long
insertions or
deletions
Possible Q’s
Importance of Bioinformatics
Explain the concepts of Percent Identity,
Query Cover, and E value
How does bootstrap value help analyze
phylogenetic trees?
What is Bioinformatics
(manipulator era ksksks)
Open the sequences in chromas.
Click export. Then FASTA format Click BLAST
Click Nucleotide BLAST
Open NCBI
Choose first the Forward Sequence. Check if the
option Standard databases and Highly similar
sequences are checked.
Then click BLAST and wait.
Click nucleotide
Click nucleotide
Select the organism with Percent Identity of 98%-
100%, Query Cover of 75%-100%, E-value of 0.0.
Click BLAST
Then Click.
Click Nucleotide BLAST
Do the same for reverse sequence.
Open NCBI
Choose first the Reverse Sequence. Check if the
option Standard databases and Highly similar
sequences are checked.
Then click BLAST and wait.
Click nucleotide
Select the organism with Percent Identity of 98%-
100%, Query Cover of 75%-100%, E-value of 0.0.
Click BLAST
Click Nucleotide BLAST
Click Align two or more sequences.
For both sequences and Gene Editing
Open NCBI
In the reverse sequence click the reverse. Ensure that
the reverse complement is checked indicating that the
sequence is already is reversed.
Add the FASTA format of the Forward and
Reverse Sequences. Check if the option Standard
databases and Highly similar sequences are checked.
Then click BLAST and wait. Then start editing based on the alignment from NCBI.
Copy a alignment from the NCBI, then click Find in
chromas for both forward and reverse sequence to find
the sequence to edit.
Change the nucleotide (use small letter) base from the
nucleotide with higher quality of the two.
Click the alignment.
Then edit mo na lahat ksksks.
MEGA software
Open.
Click Align then select Edit/ Build Alignment
Now we have the alignment. Go back to chromas.
Click Create new Alignment, then OK. Click Web, then choose Query Genbank
Click DNA.
Input e.g Chanos Chanos
Click Edit. Then select “Insert Sequence from a
File”
Select hsp20. FASTA sequence file
Choose the closest bp with hsp20. And for this
example ika-26th
Then Ctrl+A
Click Alignment, then Align by ClustalW
Click “Send to” then select “Coding Sequence”
Click ok
Then OK.
Then Click Data, then select Export File, then Then close. Then open again the MEGA software.
choose MEGA format
Click Phylogeny. Then choose Maximum
Likelihood Tree
Then OK
Select the file you save a while ago.
Then click Yes.
Click the Test of Phylogeny. Then select Bootstrap
Method. Then dapat 500 according kay Ma’am. Then
Ok
Then wait
Results
