TAIR BLAST Help

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General Information

BLAST stands for Basic Local Alignment Search Tool and was developed by Altschul et al. (1990) and significantly improved by Altschul et al. (1997). It is a very fast search algorithm that is used to separately search protein or DNA databases. BLAST is best used for sequence similarity searching, rather than for motif searching. For searches using a query sequence of fewer than twenty residues, PatMatch is the best choice. To search nonplant datasets, try NCBI BLAST.

A fairly complete on-line guide to BLAST searching can be found at the NCBI BLAST Help Manual. For a theoretical overview of BLAST, see the NCBI BLAST Course.

BLAST Methods

The NCBI BLAST family of programs includes:

blastp:compares an amino acid query sequence against a protein sequence database.blastncompares a nucleotide query sequence against a nucleotide sequence database.blastxcompares a nucleotide query sequence translated in all reading frames against a protein sequence database.tblastncompares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames.tblastxcompares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

TAIR Datasets

The following datasets are used in NCBI-BLAST, Patmatch and the bulk sequence download tools.

Entering query sequences

When pasting sequences into the text box, be aware that a single sequence is limited to 7000 characters in length; when you paste multiple sequences (up to five are allowed) you are limited to a total of 15,000 characters. These limitations may be changed in the future.  If you have a longer sequence, or many sequences, use the file upload feature.  This feature is not supported on some versions of Microsoft's Internet Explorer web browser.  If you do not see a "Browse..." button near the file upload text box (that displays your computer's filesystem directory when clicked), we suggest using Netscape or another browser supporting file uploading.

Multiple query sequences

To submit multiple query sequences, paste up to 5 sequences into the input box or,upload a file containing the concatenated sequences in FASTA format. For this option the files cannot be in raw format because they will be interpereted as a single query sequence. For NCBI-BLAST it may be possible to upload more than five sequences depending on the length of the query sequence and size of the target database.

Using the Browse option to upload a local file

NOTE:If you are uploading a file, make sure the file is in text format. If your file is a WORD document, open the file in Word and save again as text only format.

• Macintosh
  1. Click on Browse button
  2. Click on folders to open them, and on the file to upload it
• PC
  1. Click on the Browse button
  2. Change the file type from "HTML" to "all files"
  3. Click on folders to open them, and on the file to upload it
• UNIX
  1. Click on the Browse button
  2. Change *.html to * at the end of the string in the Filter box
  3. Click on a folder and then the Filter button to open the folder
  4. Click on a file and then the OK button to upload it

Word documents will not work unless saved as text first.

Input file size limits

For NCBI BLAST limits are imposed on the size of the input files based upon the type of query being performed and the size of the dataset being searched. For example, the limit for TBLASTX against a large data set such as GenBank AGI sequences is 1000 characters, whereas for a small dataset like TIGR CDS sequences the limit is 3000 characters. The following table lists the search type and limits for NCBI Blast.

Input Data Formats

• Raw text format

  An example sequence in raw format is:

  GGAAAAATCGAAGGATAATCTGTTTCTTCCAGCACAAGTTAACTTGCAAGAGAGAGCT
  CAAAGATGGAACCAACAGAAAAACCATCGACCAAACCATCTTCTCGGACTCTACCTAG
  AGACACTCGTGGCTCTCTCGAAGTATTCAACCCGTCAACTCAGCTGACCCGACCCGAT
  AACCCGGTGTTCCGTCCTGAACCACCAGCGTGGCAAAACTTGAGTGATCCACGTGGCA
  CCAGTCCTCAACCCCGACCACAACAAGAACCAGCTCCATCCAACCCTGTTCGGTCTGA
  TCAAGAAATCGCTGTCACGACCTCATGGATGGCTCTGAAAGACCCATCACCGGAGACA
  ATCTCCAAG
  

• FASTA format

  A sequence in FASTA format begins with a single-line description, followed by lines of sequence data. The description line is distinguished from the sequence data by a greater-than (">") symbol in the first column. It is recommended that all lines of text be shorter than 80 characters in length. An example sequence in FASTA format is:

  >gi|1122533|gb|AAB05099.1| BELL1
  MARDQFYGHNNHHHQEQQHQMINQIQGFDETNQNPTDHHHYNHQIFGSNSNMGMMIDFSKQQQIRMTSGS
  DHHHHHHQTSGGTDQNQLLEDSSSAMRLCNVNNDFPSEVNDERPPQRPSQGLSLSLSSSNPTSISLQSFE
  LRPQQQQQGYSGNKSTQHQNLQHTQMMMMMMNSHHQNNNNNNHQHHNHHQFQIGSSKYLSPAQELLSEFC
  SLGVKESDEEVMMMKHKKKQKGKQQEEWDTSHHSNNDQHDQSATTSSKKHVPPLHSLEFMELQKRKAKLL
  SMLEELKRRYGHYREQMRVAAAAFEAAVGLGGAEIYTALASRAMSRHFRCLKDGLVGQIQATSQALGERE
  EDNRAVSIAARGETPRLRLLDQALRQQKSYRQMTLVDAHPWRPQRGLPERAVTTLRAWLFEHFLHPYPSD
  VDKHILARQTGLSRSQVSNWFINARVRLWKPMIEEMYCEETRSEQMEITNPMMIDTKPDPDQLIRVEPES
  LSSIVTNPTSKSGHNSTHGTMSLGSTFDFSLYGNQAVTYAGEGGPRGDVSLTLGLQRNDGNGGVSLALSP
  VTAQGGQLFYGRDHIEEGPVQYSASMLDDDQVQNLPYRNLMGAQLLHDIV
  
  

• GCG format

  An example sequence in GCG format is:

  !!NA_SEQUENCE 1.0
  nga361 
  
  
  nga361.seq  Length: 204  February 22, 1999 12:09  Type: N  Check: 234  ..
  
         1  TTATATGATA TATATAGTTA TGTATGTTNC AAGAATNCGA TATGGNACGC 
  
        51  ATGATTGAAG AATAATGATT GAGGAATTTT NCTGTAACAA AAAAATTNGA 
  
       101  NATAAACAAN TNTGTGGCTA AGAACTTAAC AAGGNCACAT GTTGATATGT 
  
       151  GAANTAGGAA TCTCATNATA AGGANCACAC GGTTGACAGC AAACGGGCNT 
  
       201  NTAC
  
  

• RSF format

  A Rich Sequence Format (RSF) file contains one or more sequences that may or may not be related. In addition to the sequence data, each sequence can be richly annotated with descriptive sequence information such as creator/author of the sequence, sequence weight, creation date, one-line description of the sequence, offset, or the number of leading gaps in a sequence that is part of an alignment or fragment assembly project, and known sequence features. An example sequence in RSF format is:

  !!RICH_SEQUENCE 1.0
  ..
  {
  name  Hs70_Plafa
  descrip    PileUp of: @Hsp70.List
  type    PROTEIN
  longname  Gendocdisk:[Gcgdoc.Program_Manual]Hsp70.Msf{Hs70_Plafa}
  checksum    1012
  creation-date 10/15/96  8:40:33
  strand  1
  sequence  
    ~~~~~~~~~~~~~~~MASAKGSKPNLPESNIAIGIDLGTTYSCVGVWRNENVDIIANDQG
    NRTTPSYVAFT.DTERLIGDAAKNQVARNPENTVFDAKRLIGRKFTESSVQSDMKHWPFT
    VKSGVDEKPMIEVTYQGEKKLFHPEEISSMVLQKMKENAEAFLGKSIKNAVITVPAYFND
    SQRQATKDAGTIAGLNVMRIINEPTAAAIAYGLHKKG..KGEKNILIFDLGGGTFDVSLL
    TIED...G.IFEVKATAGDTHLGGEDFDNRLVNFCVEDFKRKNRGKDLSKNSRALRRLRT
    QCERAKRTLSSSTQATIEIDSLFEGID....YSVTVSRARFEELCIDYFRDTLIPVEKVL
    KDAMMDKKSVHEVVLVGGSTRIPKIQTLIKEFFNGKEACRSINPDEAVAYGAAVQAAILS
    G.DQSNAVQDLLLLDVCSLSLGLETAGGVMTKLIERNTTIPAKKSQIFTTYADNQPGVLI
    QVYEGERALTKDNNLLGKFHLDGIPPAPRKVPQIEVTFDIDANGILNVTAVEKSTGKQNH
    ITITNDKGRLSQDEIDRMVNDAEKYKAEDEENRKRIEARNSLENYCYGVKSSLEDQKIKE
    KLQPAEIETCMKTITTILEWLEKN.QLAGKDEYEAKQKEAESVCAPIMSKIYQDAAGAAG
    .GMPGGMP..GGMPGGMPSGMPGGMNFPGGMPGAGMPGNAPAGSGPTVEEVD~~~~~~
  }
  
  

Filtering

Filtering masks off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (Computers and Chemistry, 1993) or, for BLASTN, by the DUST program of Tatusov and Lipman (in preparation). Filtering can eliminate statistically significant but biologically uninteresting reports from the BLAST output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.

Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs. It is not unusual for nothing at all to be masked by SEG, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect.

Results Options

Output titleType in the title you would like to appear at the top of your BLAST output. E-mail address:Entering your email address is suggested even if requesting a browser reply, and may be mandatory depending on server configuration options.  Depending on server loading, or the expected execution time of your request, the server may convert your request to an email reply and auto-select the E-mail URL option. This avoids the frustration of wondering when your job will finish, and allows you to request more jobs quicker. Return Results:

To your web browserYour job will run immediately and return results directly to your Web browser in HTML format.  Accession numbers that appear in the query and target loci names and descriptions will be hyperlinked to allow easy access to additional sequence information.  NOTE: Attempting to view large result files may cause your browser to "blank out".  If you have trouble viewing results, particularly if you've asked for many scores and alignments, or submitted many queries in one job, your browser's "memory" and "disk" cache settings may need to be increased.  See your browser's help and preference menus for details.  To prevent loss of a large HTML result file, you might wish to request emailing a URL to it rather than a browser reply.  This way you'll be able to experiment with your browser's cache settings and retrieve the output as many times as you wish without waiting for your request to be re-run. By E-mail messageThe results are sent within the body of a normal email message, to the email address you enter.  Any comments you type will appear as the Subject of the email message. Generally, you should use this option only if your mail system can handle large messages, and you've asked for textual output.  If requesting HTML format by email, either of the following choices may be better if your mail reader software is not HTML-aware. Result Formats:HTML hypertext(file type "htm")HTML format is used by web browsers.  Accession numbers that appear in the query and target loci names and descriptions will be hyperlinked to allow easy access to additional sequence information.  Embedded Java "Applets" may be used to render graphical information (e.g., the ClustalW dendrogram), which will not appear unless your browser is set to permit these applets to run.  Normal text(file type "txt")The results are returned as conventional human-readable text.

Protein:
RANK, STATUS, SCORE, E-VALUE, PROGRAM, Gap Penalties (Existence), Gap Penalty (Extension), EMPTY, EMTPY, MATRIX, TEMPFILENAME, QUERY LENGTH, empty, QUERY NAME, DATASET, Target length, empty, DESCRIPTION, empty, empty, empty, empty, empty, empty, empty, empty, empty, Identities, Positives, Gaps, Percentage ratio of identical matches to the length of the alignment, Percentage ratio of identical matches to the length of the query, unknown, unknown, Percentage ratio of identical matches to the length of the target, unknown, unknown, Query Start, Query End, Target Start, Target End, empty, QUERY NT, COMPARISON, TARGET NT

Advanced parameters

Max ScoresRestricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. See also Expectation. Max AlignmentsRestricts database sequences to the number specified for which high-scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see Expectation), only the matches ascribed the greatest statistical significance are reported. ExpectationThe statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECTATION threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. Query and Database Genetic CodeGenetic code to be used in BLASTX translation of the query. Gapped AlignmentsWhether to allow gapped alignments; either ON or OFF. Gap Opening PenaltyDefault Setting (option name: gapopen)Cost to open a gap; a 0 in the field means to use the default. Supported values for BLASTP, BLASTX, TBLASTN, and TBLASTX are limited. Gap Extension PenaltyDefault Setting (option name: gapextend)Cost to extend a gap, a 0 in this field means to use the default. Supported values for BLASTP, BLASTX, TBLASTN, and TBLASTX are limited. Nucleic MismatchPenalty for a mismatch in the BLAST™ portion of run. Nucleic MatchReward for a match in the BLAST™ portion of run. MatrixThe amino acid substitution matrix to be used for protein comparisons. Both BLOSUM and PAM matrices are available at several different levels of evolutionary distance. Extension ThresholdDefault Setting (option name: threshold)The threshold above which BLAST™ will extend a hit found. The hit is based on finding a word of a certain size (see Word Size) Word SizeDefault Setting (option name: word_size)The size of the initial word that must be matched between the database and the query sequence.