Practice retrieving data and running stand alone BLAST. 

 Step 1. Identify genes in the ABA biosynthesis pathway from the Arabidopsis Cyc database

http://www.arabidopsis.org/biocyc/index.jsp

Step 2. Identify subject databaseVitis vinifera (nucleotide)Solanum pennellii (EST)

Query: Select Pathway by name Enter: Abscisic AcidSubmit

Now what?

abscisic acid biosynthesis abscisic acid biosynthesisAT4G18350 AT4G18350AT3G24220 AT3G24220AT1G78390 AT1G78390AT1G78390 AT3G14440AT4G18350 AT1G30100AT3G24220 AT2G27150AT3G14440 AT1G52340AT1G30100AT2G27150AT1G52340

Filter for unique sequences (EXCEL: Data, Filter, Advanced Filter…)

Notepad ++ EDIT, LINE OPPERATIONS, JOIN LINES SEARCH, REPLACE, “space” with “spaceORsapce” Paste into ENTREZ Nucleotide search…

PERL

chomp;next if /^\s/; #(skip if there is a space in start of the line)next if /^Gene/; #(if line starts with “gene”, skip)my @temp = split /\t/; #(data set is tab delimited)$hash{$temp[0]} = 1; #(unique sequence i.d. #0 is first element of the array)

Then invoke BioPerl to query NCBI with the search string: TAIR:AT### AND “complete cds”

Where AT### are the unique accession numbers from AraCyc and “complete cds” eliminates genomic sequence (e.g. complete Ath chrom 4)

See complete script on class site….

Do we want this much sequence?

Use the push pin to highlight all boxes for mRNA (22 sequences) so we don’t get chromosome 4 genomic sequences

Try: Use Unix to verify that the file contains all the sequences…

Q: What command would you use?

A: $ grep –c “>” filename

(lycopersicum [ORGN] AND EST) AND "Solanum pennellii"[porgn:__txid28526]

Try: Use Unix to verify that the file contains all the sequences…

Vitis [ORGN] AND ESTNucleotide

Note syntax of ENTREZ search invoked by organism tree link

For class, I recommend downloading the smaller Nucleotide data set…

Try: Use Unix to verify that the file contains all the sequences…

Now what?

Which file needs to be formatted for BLAST (formatdb)?

Which file will be the query file?

What is the syntax for the BLAST (including PATH)?

Formatdb

$ /path/formatdb -i /path/filename –p F

Run nucleotide BLAST (blastn)

$ /path/blastall -p blastn -d /path/filename -i /path/filename –o filename –e 0.01