TBLASTn and Protein Sequence Analysis - Revision history

Macampbell at 14:53, 13 February 2013

2013-02-13T14:53:43Z

Dycoughtreydavenport: /* When Nucleotides Don't Work */

2011-02-24T15:38:31Z

‎When Nucleotides Don't Work

Dycoughtreydavenport: /* When Nucleotides Don't Work */

2011-02-24T14:52:17Z

‎When Nucleotides Don't Work

Dycoughtreydavenport: /* Interpreting Your Results */

2011-02-24T14:44:07Z

‎Interpreting Your Results

Dycoughtreydavenport: /* Interpreting Your Results */

2011-02-24T14:42:20Z

‎Interpreting Your Results

Dycoughtreydavenport: /* Using tBLASTn */

2011-02-24T04:30:09Z

‎Using tBLASTn

Dycoughtreydavenport: /* Interpreting Your Results */

2011-02-24T04:14:25Z

‎Interpreting Your Results

Dycoughtreydavenport: /* Protein BLASTing */

2011-02-24T03:40:14Z

‎Protein BLASTing

Dycoughtreydavenport: /* When Nucleotides Don't Work */

2011-02-24T03:30:30Z

‎When Nucleotides Don't Work

Dycoughtreydavenport: /* Using tBLASTn */

2011-02-24T03:30:05Z

‎Using tBLASTn

← Older revision		Revision as of 14:53, 13 February 2013
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−	tBLASTn works in much the same way that BLASTn does, but adds in an extra step. Before BLASTing, the tBLASTn program must convert the ~~amino acid sequence provided~~ into a ~~nucleotide sequence. Because~~ amino ~~acids can be signified by multiple codons, it is impossible to reverse translate into a single nucleotide sequence~~. ~~In order to capture all possible sequences~~ tBLASTn will ~~search~~ with ~~every possible configuration of nucleotides that could potentially form~~ the ~~protein being queried~~. ~~This~~ allows any genes with silent mutations to be ~~counted~~ as ~~homologous sequences~~.	+	tBLASTn works in much the same way that BLASTn does, but adds in an extra step. Before BLASTing, the tBLASTn program must convert the database of nucleotide sequences into a amino acid sequences. tBLASTn will query with your protein sequence against the translated nucleotide sequences. tBLASTn allows any genes with silent mutations to be detected as an ortholog.

@@ Line 11: / Line 11: @@
 :'''''First''''' -- Check to make sure that your database is made correctly.
-::*You should have three additional files made from the original fasta file:
+::*You should have three additional files made from the original fasta file: -.nsq, -.nin, and -.nhr
 :'''''Second''''' -- Check that your commands are entered correctly.

@@ Line 13: / Line 13: @@
 ::*You should have three additional files made from the original fasta file:
-:'''''Second''''' -- Check that your commands are entered correct.
+:'''''Second''''' -- Check that your commands are entered correctly.
 ::*Ensure that you are in the correct directory (i.e. Desktop) and BLASTing with the correct file name
 ::*Sequences being blasted should be in TextEdit and saved without any extension (such as .txt)

← Older revision		Revision as of 14:44, 24 February 2011
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	[[File:Screenshot.jpg]]		[[File:Screenshot.jpg]]
		+
		+	As can be seen, the Contig and nucleotide range are provided for easy location and the e-value shows how strong the hit is.

← Older revision		Revision as of 14:42, 24 February 2011
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	=== Interpreting Your Results ===		=== Interpreting Your Results ===
	Results are provided in exactly the same way that an ordinary BLAST would output. The location and e-value of each hit is provided. This makes it easy to establish the validity of the find and actually locate the gene sequence within the genome.		Results are provided in exactly the same way that an ordinary BLAST would output. The location and e-value of each hit is provided. This makes it easy to establish the validity of the find and actually locate the gene sequence within the genome.
		+
		+
		+	The example below provides the result of when the Myb Consensus Sequence shown above was BLASTed against a new genome.
		+
		+	[[File:Screenshot.jpg]]

@@ Line 50: / Line 50: @@
 :*Ensure that the TextEdit file contains an amino acid sequence.  Remove any "Met" designations and replace with "M".  Also be sure that there are no stop codons in the sequence.
 :*Replace the '''blastn''' in the command with '''tblastn'''
-::Normal BLAST ''/usr/local/ncbi/blast/bin/'''blastn''' -query scf1453.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"''
+::Normal BLAST ''/usr/local/ncbi/blast/bin/'''blastn''' -query FILE_NAME -db DATABASE_NAME -outfmt "7 qacc sacc evalue qstart qend sstart send"''
-::Protein BLAST ''/usr/local/ncbi/blast/bin/'''tblastn''' -query scf1453.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"''
+::Protein BLAST ''/usr/local/ncbi/blast/bin/'''tblastn''' -query FILE_NAME -db DATABASE_NAME -outfmt "7 qacc sacc evalue qstart qend sstart send"''
 === Interpreting Your Results ===
 Results are provided in exactly the same way that an ordinary BLAST would output.  The location and e-value of each hit is provided.  This makes it easy to establish the validity of the find and actually locate the gene sequence within the genome.

← Older revision		Revision as of 04:14, 24 February 2011
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	=== Interpreting Your Results ===		=== Interpreting Your Results ===
		+	Results are provided in exactly the same way that an ordinary BLAST would output. The location and e-value of each hit is provided. This makes it easy to establish the validity of the find and actually locate the gene sequence within the genome.

← Older revision		Revision as of 03:40, 24 February 2011
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	::Normal BLAST ''/usr/local/ncbi/blast/bin/'''blastn''' -query scf1453.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"''		::Normal BLAST ''/usr/local/ncbi/blast/bin/'''blastn''' -query scf1453.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"''
	::Protein BLAST ''/usr/local/ncbi/blast/bin/'''tblastn''' -query scf1453.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"''		::Protein BLAST ''/usr/local/ncbi/blast/bin/'''tblastn''' -query scf1453.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"''
		+
		+	=== Interpreting Your Results ===

@@ Line 10: / Line 10: @@
 But assuming that you're working with a gene that should be highly conserved and you have allowed for the exon/intron issue, it may be time to go through some extra steps to make sure everything is working as it should:
-::'''''First''''' -- Check to make sure that your database is made correctly.
+:'''''First''''' -- Check to make sure that your database is made correctly.
-:::*You should have three additional files made from the original fasta file:
+::*You should have three additional files made from the original fasta file:
-::'''''Second''''' -- Check that your commands are entered correct.
+:'''''Second''''' -- Check that your commands are entered correct.
-:::*Ensure that you are in the correct directory (i.e. Desktop) and BLASTing with the correct file name
+::*Ensure that you are in the correct directory (i.e. Desktop) and BLASTing with the correct file name
-:::*Sequences being blasted should be in TextEdit and saved without any extension (such as .txt)
+::*Sequences being blasted should be in TextEdit and saved without any extension (such as .txt)
-::'''''Third''''' -- Ensure that the databases/commands are working.
+:'''''Third''''' -- Ensure that the databases/commands are working.
-:::*Take a known sequence from the database being blasted
+::*Take a known sequence from the database being blasted
-::::*Typically copy a small portion of the genome into a separate text file
+:::*Typically copy a small portion of the genome into a separate text file
-:::*Run a BLAST with that sequence against the database it was originally in
+::*Run a BLAST with that sequence against the database it was originally in
-::::*If the sequence doesn't show a hit in the scaffold that you took it from, something is wrong in your programming
+:::*If the sequence doesn't show a hit in the scaffold that you took it from, something is wrong in your programming

@@ Line 48: / Line 48: @@
 In order to use the tBLASTn program, only a few basic changes are necessary to the standard BLAST program:
-::*Ensure that the TextEdit file contains an amino acid sequence.  Remove any "Met" designations and replace with "M".  Also be sure that there are no stop codons in the sequence.
+:*Ensure that the TextEdit file contains an amino acid sequence.  Remove any "Met" designations and replace with "M".  Also be sure that there are no stop codons in the sequence.
-::*Replace the '''blastn''' in the command with '''tblastn'''
+:*Replace the '''blastn''' in the command with '''tblastn'''
 ::Normal BLAST ''/usr/local/ncbi/blast/bin/'''blastn''' -query scf1453.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"''
-:::Normal BLAST ''/usr/local/ncbi/blast/bin/'''blastn''' -query scf1453.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"''
+::Protein BLAST ''/usr/local/ncbi/blast/bin/'''tblastn''' -query scf1453.fasta -db bb_latest_assembly.fasta -outfmt "7 qacc sacc evalue qstart qend sstart send"''