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--- blast_protocol [2021/09/02 16:19] – 38.20.199.40
+++ blast_protocol [2022/09/06 14:49] (current) – 134.190.232.106
@@ Line 7: / Line 7: @@
   - __tblastx__: search translated nucleotide database with translated nucleotide sequence query
-//Note: blastp and blastx can usually provide better hit alignments than blastn, especially for distantly related species.This is because amino acids sequences are more conserved than nucleotides (Koonin and Galperin, 2002).//
+{{:blast.png?400|}}
+//**blastp** can usually provide better hit alignments than blastn, especially for distantly related species.This is partially because amino acids sequences are more conserved than nucleotides (Koonin and Galperin, 2002).//
+//**blastx** translates the query sequence in all six reading frames and provides combined significance statistics for hits to different frames, it is particularly useful __when the reading frame of the query sequence is unknown or it contains errors that may lead to frame shifts or other coding errors__. Thus blastx is often the first analysis performed with a newly determined nucleotide sequence.//
+//**tblastn** is useful for __finding homologous protein coding regions in unannotated nucleotide sequences such as expressed sequence tags (ESTs)__ and draft genome records, ESTs are short, single-read cDNA sequences. They comprise the largest pool of sequence data for many organisms and contain portions of transcripts from many uncharacterized genes. __Since ESTs have no annotated coding sequences, there are no corresponding protein translations in the BLAST protein databases.__ Hence a tblastn search is the only way to search for these potential coding regions at the protein level.//
+Courtesy of the web source: https://guides.lib.berkeley.edu/ncbi/blast
 **General bugs**
@@ Line 34: / Line 41: @@
 Using BLASTP search option to blast the amino acid sequences against uniport_db database.
 <code>
-> ./blastp -query XXX.fasta -db uniprot_db -out BLASTP_XXX_uniprot.xml -evalue 1e-5 -outfmt 5
+./blastp -query XXX.fasta -db uniprot_db -out BLASTP_XXX_uniprot.xml -evalue 1e-5 -outfmt 5
 </code>
@@ Line 77: / Line 84: @@
 salltitles    All subject titles, separated by '&lt;&gt;'
-    $ python blastxml_to_tabular.py -o output.tabular -c std input.xml
+    > python blastxml_to_tabular.py -o output.tabular -c std input.xml
-    $ python blastxml_to_tabular.py -o output.tabular -c ext input.xml
+    > python blastxml_to_tabular.py -o output.tabular -c ext input.xml
-    $ python blastxml_to_tabular.py -o output.tabular -c qseqid,qlen,salltitles,sseqid,slen,bitscore,qframe,pident,evalue,qstart,qend,sstart,send,length input.xml
+    > python blastxml_to_tabular.py -o output.tabular -c qseqid,qlen,salltitles,sseqid,slen,bitscore,qframe,pident,evalue,qstart,qend,sstart,send,length input.xml
 </code>
-**V5 database**
+#This is another way to parse BLAST outputs via using -outfmt '6 qseqid sseqid ...'
+<code>
+#!/bin/bash
+#$ -S /bin/bash
+. /etc/profile
+#$ -pe threaded 2
+#$ -cwd
+source activate blast
+export BLASTDB= /misc/scratch3/rogerlab_databases/other_dbs/nr_010621
+DB=nr
+query=ATCG00670.1.fasta
+blastp -db $DB -query $query -out /scratch2/xizhang/BLASTP_nr.tsv -num_threads 2 -outfmt '6 qseqid sseqid evalue pident qcovs length slen qlen qstart qend sstart send stitle'
+source deactivate
+</code>
+Sep 6th,2022 Since Diamond is faster on BLASTP and BLASTx, this is another way using Diamond
+<code>
+#!/bin/bash
+#$ -S /bin/bash
+. /etc/profile
+#$ -pe threaded 40
+#$ -cwd
+source activate /scratch2/software/anaconda/envs/diamond-2.0.7
+#DB=nr
+while read line
+do
+diamond blastp -p 40 -k 5 -e 1e-10 -f 6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore stitle salltitles --header -d /misc/scratch3/rogerlab_databases/other_dbs/nr_02032022/diamond_nr.dmnd -q $line -o BLASTP_nr.$line.tsv --sensitive
+done <$1
+conda deactivate
+</code>
+**V5 NCBI database**
+The latest blast+ package can be found via https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/
+{{:blast_screenshot.png?nolink&600|}}
+The V5 NCBI database can be found via https://ftp.ncbi.nlm.nih.gov/blast/db/v5. The good thing about V5 than V4 database is not just the former is faster, but also the option to screen out your interested taxonomy.
+In order to limit your BLAST+ search by taxonomy, you’ll need to obtain the taxid(s) for your organism(s). Two options can be used here: "taxids" or "taxidlist"
+This is to acquire the taxid list for your interested organism e.g.,bacteria
+<code>
+./get_species_taxids.sh -n bacteria
+</code>
+get_species_taxids.sh script is from the blast+ package under the bin directory.
+Taxid for bacteria is 2. Then acquire a list of taxonomy ids from bacteria species.
+<code>
+./get_species_taxids.sh -t 2 > 2.txids
+</code>
+Using 2.txids to limit the NCBI v5 database search scope is far more efficient.
+<code>
+./blastp –db nr –query QUERY –taxidlist 2.txids –outfmt 5 –out OUTPUT.tab
+./blastp –db nr –query QUERY –taxids 1117,1118,1119,1121 –outfmt 5 –out OUTPUT.tab
+</code>
+If use "taxids" option, use comma to separate different organisms.e.g., different cyanobacteria organisms: 1117,1118,1119,1121
+Note: Please refer to the guide for the most updated information. https://ftp.ncbi.nlm.nih.gov/blast/db/v5/v5/blastdbv5.pdf
+{{:22-you-got-this-meme-5.jpg?nolink&200|}}
+<Last updated by Xi Zhang on Sep 3rd,2021>