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BLAST Command Line Applications User Manual

1. 4 2 14 BLAST WindowMasker integration in BLAST search applications The BLAST search applications support integration with the windowmasker files via the window_masker_taxid and the WINDOW_MASKER PATH configuration parameter see Configuring BLAST or via the window_masker_db command line option In the first case the WINDOW _MASKER PATH configuration parameter should refer to a directory which contains subdirectories named after NCBI taxonomy IDs e g 9606 for human 10090 for mouse where the windowmasker unit counts data files should be placed with the following naming convention wmasker obinary for files generated with the obinary format and or wmasker oascii for files generated with the oascii format For an example on how to create these files please see the Cookbook Once these windowmasker files and the configuration file are in place this feature can be invoked by providing the taxonomy ID to the window_masker_taxid command line option Alternatively this feature can also be invoked by providing the path to the windowmasker unit counts data file via the window_masker_db Please see the Cookbook for a usage example of this feature 4 2 15 DELTA BLAST A tool for sensitive protein sequence search DELTA BLAST uses RPS BLAST to search for conserved domains matching to a query constructs a PSSM from the sequences associated with the matching domains and searches a sequence database Its sensitivity is comparable
2. mask_data hs_chr mfa asnb out hs_ chr mfa title Human chromosomes mfa Here we use the lowercase masked FASTA sequence file as input in hs_chr mfa its file type input_type fasta specify the database as nucleotide dbtype nucl enable parsing of sequence ids parse_seqids provide the masking data mask_data hs_chr_mfa asnb and name the resulting database as hs_chr_mfa out hs_chr_mfa Checking the database thus generated using blastdbcmd we have Database Human chromosomes mfa 24 sequences 3 095 677 412 total bases Date Aug 26 2009 11 41 AM Longest sequence 249 250 621 bases Available filtering algorithms applied to database sequences Algorithm ID Algorithm name Algorithm options 40 repeat repeatmasker lowercase Volumes export home tao hs_chr_mfa The algorithm name and algorithm options are the values we provided in step 5 2 1 4 5 2 3 Obtaining Sample data for this cookbook entry For input nucleotide sequences we use the BLAST database generated from a FASTA input file hs_chr fa containing complete human chromosomes from BUILD37 1 generated by inflating and combining the hs _ref_ fa gz files located at ftp ncbi nlm nih gov genomes H sapiens Assembled_chromosomes We use this command line to create the BLAST database from the input nucleotide sequences BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 17 makeblastdb i
3. blastdb_aliastool dblist nematode _ mrna nematode genomic dbtype nucl out nematode all title Nematode RefSeq mRNA Genomic 5 7 2 Create a subset of a BLAST database The nematode_mrna database contains RefSeq mRNAs for several species of round worms The best subset is from C elegance In most cases we want to search this subset instead of the complete collection Since the database entries are from NCBI nucleotide databases and the database is formatted with parse_seqids we can use the gilist c_elegance_mrna gi parameter value pair to limit the search to the subset of interest alternatively we can create a subset of the nematode _mrna database as follows blastdb_aliastool db nematode mrna gilist c_elegance mrna gi dbtype nucl out c_elegance _mrna title C elegans refseq mRNA entries Note one can also specify multiple databases using the db parameter of blastdb_aliastool 5 8 Reformat BLAST reports with blast_formatter It may be helpful to view the same BLAST results in different formats A user may first parse the tabular format looking for matches meeting a certain criteria then go back and examine the relevant alignments in the full BLAST report He may also first look at pair wise alignments then decide to use a query anchored view Viewing a BLAST report in different formats has been possible on the NCBI BLAST web site since 2000 but has not been possible with stand alone BLAST runs The
4. For both steps the input file can be a text file containing sequences in FASTA format or an existing BLAST database created using makeblastdb We will provide examples for both scenarios 5 2 1 Collect mask information files For nucleotide sequence data in FASTA files or BLAST database format we can generate the mask information files using windowmasker or dustmasker Windowmasker masks the over represented sequence data and it can also mask the low complexity sequence data using the built in dust algorithm through the dust option To mask low complexity sequences only we will need to use dustmasker For protein sequence data in FASTA files or BLAST database format we need to use segmasker to generate the mask information file BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 12 The following examples assume that BLAST databases listed in 5 2 3 are available in the current working directory Note that you should use the sequence id parsing consistently In all our examples we enable this function by including the parse_seqids in the command line arguments 5 2 1 1 Create masking information using dustmasker We can generate the masking information with dustmasker using a single command line dustmasker in hs_chr infmt blastdb parse_seqids outfmt maskinfo_asnl_bin out hs_chr_dust asnb Here we specify the input is a BLAST database named hs_chr in hs_
5. Strand Plus Plus Query 1 ACGTCCGAGACGCGAGCAGCGAGCAGCAGAGCGACGAGCAGCGACGA 47 IIIT TEEPE EEE EEE EEE EEE ETE EEE ETE T ETT E EEE E A Sbjct 1 ACGTCCG GACGCGAGCAGCGAGCAGCAGAGCGACGAGCAGCGACGA 46 Score 78 7 bits 42 Expect le 20 Identities 47 49 95 Gaps 2 49 4 Strand Plus Plus Query 1 ACGTCC GAGACGCGAGCAGCGAGCAGCAGAGCGACGAGCAGCGACGA 47 IITTI PEPE E EEE E PEPE EEE E EEE TEEPE EE ETE EEE EEE EEE Sbjct 94 ACGTCCGAGAGACGCGAGCAGCGAGCAGCAGAGCGACGAGCAGCGACGA 142 5 7 Use blastdb_aliastool to manage the BLAST databases Often we need to search multiple databases together or wish to search a specific subset of sequences within an existing database At the BLAST search level we can provide multiple BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 20 database names to the db parameter or to provide a GI file specifying the desired subset to the gilist parameter However for these types of searches a more convenient way to conduct them is by creating virtual BLAST databases for these Note When combining BLAST databases all the databases must be of the same molecule type The following examples assume that the two databases as well as the GI file are in the current working directory 5 7 1 Aggregate existing BLAST databases To combine the two nematode nucleotide databases named nematode_mrna and nematode_genomic we use the following command line
6. db Users joeuser My Documents Downloads mydb myotherdb info If the second database had contained a space it would have been necessary to surround it by quotes escaped by a backslash Under UNIX systems including LINUX and Mac OS X it is preferable to use a single quote in place of the escaped double quote blastdbcmd db path with spaces mydb info Multiple databases can also be listed within the single quotes similar to the procedure described for Microsoft Windows 5 16 Specifying a sequence as the multiple sequence alignment master in psiblast The in_msa psiblast option unlike blastpgp does not support the specification of a master sequence via the query option so if one wants to specify a sequence other than the first one in the multiple sequence alignment file to be the master sequence this has to be specified via the msa_master_idx option For instance in the example below the third sequence in the multiple sequence alignment would be used as the master sequence psiblast in_msa alignl db pataa msa_master idx 3 5 17 Ignoring the consensus sequence in the multiple sequence alignment in psiblast Often a consensus sequence is added to a multiple sequence alignment to be used as the master sequence in a PSI BLAST search The consensus sequence provides a good option to display the query subject alignment in the output and to define which MSA columns are to be converted to PSSM At the same time ad
7. for Seq entries in text ASN 1 format asn1_bin for Seq entries in binary ASN 1 format dbtype string prot Molecule type of input values can be nucl or prot title string none Title for BLAST database If not set the input file name will be used parse_seqids flag N A Parse bar delimited sequence identifiers e g gij129295 in FASTA input hash_index flag N A Create index of sequence hash values mask_data string none Comma separated list of input files containing masking data as produced by NCBI masking applications e g dustmasker segmasker windowmasker out string input file name Name of BLAST database to be created Input file name is used if none provided This field is required if input consists of multiple files max_file_size string 1GB Maximum file size to use for BLAST database taxid integer none Taxonomy ID to assign to all sequences taxid_map string none File mapping sequence IDs to taxonomy IDs logfile string none Program log file default is stderr BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E djeH LSV1d Page 36 Table C9 Makeprofiledb application options This application builds an RPS BLAST database An option of type flag takes no arguments but if present the argument is true COBALT a multiple sequence alignment program and DELTA BLAST both use RPS BLAST searches as part of their processing but use specialized versions of the database This application
8. title Human Chromosome Ref B37 1 Here we use the existing BLAST database as input file in hs_chr specify its type dbtype nucl enable parsing of sequence ids parse_seqids provide the masking data from step 5 2 1 2 mask_data hs_chr_mask asnb and name the output database with the same base name out hs_chr overwriting the existing one To use the original FASTA sequence file hs_chr fa as the input we need to use inhs_chr fa to instruct makeblastdb to use that FASTA file instead We can check the re created database to find out if the masking information was added properly using blastdbcmd with the following command line blastdbcmd db hs_chr info This command prints out a summary of the target database Database human chromosomes Ref B37 1 24 sequences 3 095 677 412 total bases Date Aug 13 2009 3 02 PM Longest sequence 249 250 621 bases Available filtering algorithms applied to database sequences Algorithm ID Algorithm name Algorithm options 30 windowmasker Volumes export home tao blast_test hs_chr Extra lines under the Available filtering algorithms describe the masking algorithms available The Algorithm ID field 30 in our case is what we need to use if we want to invoke database soft masking during an actual search through the db_soft_mask parameter Wecan apply additional masking data to an existing BLAST database with one type of masking informa
9. blastdb and the output of step 1 ustat hs_chr_mask counts We set the mask file format to binary asn 1 outfmt BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 13 maskinfo_asn1_bin enable the sequence ids parsing parse_seqids and save the masking data to hs_chr_mask asnb out hs_chr_mask asnb To use the FASTA file hs_chr fa we change the input file name and file type windowmasker in hs_chr fa infmt fasta ustat hs_chr counts outfmt maskinfo_asnl_bin parse_seqids out hs_chr_mask asnb 5 2 1 3 Create masking information using segmasker We can generate the masking information with segmasker using a single command line segmasker in refseq protein infmt blastdb parse segids outfmt maskinfo_asnl_ bin out refseq_seg asnb Here we specify the refseq_protein BLAST database in refseq_ protein infmt blastdb enable sequence ids parsing parse_seqids request the mask data in binary asn 1 format outfimt maskinfo_asn1_ bin and name the out file as refseq_seg asnb out refseq_seg asnb If the input format is the FASTA file we need to change the command line to specify the input format segmasker in refseq protein fa infmt fasta parse_seqids outfmt maskinfo_asnl_ bin out refseq_seg asnb 5 2 1 4 Extract masking information from FASTA sequences with lowercase masking We can also extract the masking information from a FASTA sequenc
10. can build databases for COBALT DELTA BLAST and a standard RPS BLAST search The dbtype option see entry in table determines which flavor of the database is built option type default value Description and notes in string stdin Input file that contains a list of scoremat files delimited by space tab or newline binary flag N A The scoremat files are binary ASN 1 title string none Title for RPS BLAST database If not set the input file name will be used threshold real 9 82 Threshold for RPSBLAST lookup table out string input file name Name of BLAST database to be created Input file name is used if none provided max_file_size string 1GB Maximum file size to use for BLAST database dbtype string rps Specifies use for RPSBLAST db One of rps cobalt or delta index flag N A Creates index files gapopen integer none Cost to open a gap Used only if scoremat files do not contain PSSM scores otherwise ignored gapextend integer none Cost to extend a gap by one residue Used only if scoremat files do not contain PSSM scores otherwise ignored scale real 100 PSSM scale factor matrix string BLOSUM62 Matrix to use in constructing PSSM One of BLOSUM45 BLOSUMS50 BLOSUM62 BLOSUM80 BLOSUM90 PAM250 PAM30 or PAM70 Used only if scoremat files do not contain PSSM scores otherwise ignored obsr_threshold real 6 Exclude domains with maximum number of independent observations bel
11. contain taxonomic information for each sequence include the identifier indices for lookups and can be up to four times smaller than the FASTA The original FASTA can be generated from the BLAST database using blastdbcmd 5 14 Limiting a Search with a List of Identifiers BLAST can now limit a database search by a list of text identifiers which should be specified one per line in a text file These identifiers referencing the sequences to include in BLAST search should not contain any whitespace and must be resolvable through the BLAST database ID lookup In some cases this means that the entire bar delimited format specified in http www ncbi nlm nih gov books NBK7183 rendertype table amp id ch_demo T5 must be used In other cases it is enough to simply specify an accession For the general example from section 5 13 a valid ID would be gnl MYDB 2 On the other hand if the identifier is gil 15674171 ref NP_268346 1 one of the following string is sufficient gi 15674171 ref NP_268346 1 15674171 refINP_ 268346 NP_268346 NP_268346 1 etc When the search is limited by a list of IDs the statistics of the BLAST database are re calculated to reflect the actual number of sequences and residuals base included in search BLAST has been able to limit a search by a list of GI s for a number of years It is important to note that the performance of a binary list of GI s will always be superior
12. dieH LSV1d Page 10 Int 6 Mm KFQYECGNYSGAAEBYLYFFRVLVpatdrnalsslwgklaseilmqnwdaamedltrlket 26SPS9 Hs lryagrqt ealkevagasknrsladfekaltdy S ess Hssecsssseceseses F57B9 Ce alkyngsdldamkaiaaaagkrslkdfqvafgsf YDLO97c_Sc akytketyqsrgidamkavaeaynnrslldfntalkqy YMJ5_ Ce ivaygqkspriiairsmadafrkrslkdfvkalaeh FUS6 ARATH asglahlelkkyklaarkfldvnpelgnsyneviapqdiatygglcalasfdrselkqkv cos41 8 Ci kfleaagrynelsyksaiheteqtkalekalncailapagqgqrsrmlatlfkdercqllp 644879 gailtklkcaaglaelaarkykqaakclllasfdhcdfpellspsnvaiygglcalatfd YPR108w_Sc llslisttaalqsissltislyasdyasyfpyllety eif 3pll0 Hs Seer Sse Seb sec Ss SSeS ast Sasa S SS nainn T23D8 A CQ qa e en ee eee YD95 Sp vlavlpqnesmssleacinslylcdysgffrtladve KIAA0107_ Hs ilevlhslpavrqylfslyecrysvffqslavv F49C12 8 Hs vqeqltgggingtlipvreylesyydchydrffiglaale s s4 ss 55 Int 6 Mm idnnsvssplqslqqrtwlihwslfvffnhpkgrdniidlflygqpqylnaiqtmcphilr 26SPS9 Hs a SO aaa YDLO97C C YMJ5 Ce FUS6 ARATH idninfrnflelvpdvrelindfyssryascleylasl COS41 8 Ci sfgilekmfldriiksdemeefar 644879 rqelqrnvissssfklflelepqvrdiifkfyeskyasclkmld
13. from the BLAST applications The third appendix is atable of BLAST options the type of input required and the default values for each application An introduction to BLAST is outside the scope of this manual more information on this subject can be found on http blast ncbi nlm nih gov Blast cgi CMD Web amp PAGE_TYPE BlastDocs Please feel free to contact us with any questions feedback or bug reports at blast help ncbi nlm nih gov 2 Installation Entries in the BLAST Help manual provide installation instructions for Windows and LINUX UNIX This section provides instructions for a few cases not covered by those entries The BLAST applications are distributed both as an executable and as source code For the executable formats we provide installers as well as tarballs the source code is only provided as a tarball These are freely available at ftp ftp ncbi nlm nih gov blast executables blast Please be sure to use the most recent available version this will be indicated in the file name for instance in the sections below version 2 2 18 is listed but this should be replaced accordingly 2 1 Windows Please see http www ncbi nlm nih gov books NBK52637 For users without administrator privileges follow the procedure described in http www ncbi nlm nih gov books NBK52640 For users with administrator privileges and machines MacOSX version 10 5 or higher Download the ncbi blast 2 2 18 dmg installer and double click
14. in every block Blocks are separated by one or more black lines Within a block there are no blank lines and each line consists of one sequence identifier followed by some whitespace followed by characters and gaps for that sequence in the multiple sequence alignment In each column all letters must be in upper case or all letters must be in lower case Example multiple sequence alignment file alignl 26SPS9 Hs IHAAEEKDWKTAYSYFYEAFEGYdsidspkaitslkymllckimlntpedvqalvsgkla F57B9 Ce LHAADEKDFKTAFSYFYEAFEGYdsvdekvsaltalkymllckvmldlpdevnsllsakl YDLO97c_Sc ILHCEDKDYKTAFSYFFESFESYhnitthnsyekacqvlkymllskimlnliddvkniln YMJ5_Ce LYSAEERDYKTSFSYFYEAFEGFasigdkinatsalkymilckimlneteqlagllaake FUS6 ARATH KNYIRTRDYCTTTKHIIHMCMNAilvsiemgqfthvtsyvnkaeqnpetlepmvnaklrc CoSs41 8 Ci SLDYKLKTYLTIARLYLEDEDPVgqaemyinrasllqnetadegqlqihykvcyarvldyrr 644879 KCYSRARDYCTSAKHVINMCLNVikvsvylqnwshvlsyvskaestpeiaeqrgerdsqt YPR108w_Sc IHCLAVRNFKEAAKLLVDSLATFtsieltsyesiatyasvtglftlertdlkskvidspe eif 3p110 Hs SKAMKMGDWKTCHSF I INEKMNGKVWasSss Sr sce sSe erase ssseseses esses T23D8 4 Ce SKAMLNGDWKKCQDYIVNDKMNQkvw YD95 Sp IYLMSIRNFSGAADLLLDCMSTFsstellpyydvvryavisgaisldrvdvktkivdspe KIAA0107_ Hs LYCVAIRDFKQAAELFLDTVSTFtsyelmdyktfvtytvyvsmialerpdlrekvikgae F49C12 8 Hs LYRMSVRDFAGAADLFLEAVPTFgsyelmtyenlilytvitttfaldrpdirtkvircne BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE
15. on it Double click the newly mounted ncbi blast 2 2 18 volume double click on ncbi blast 2 2 18 pkg and follow the instructions in the installer By default the BLAST applications are installed in usr local ncbi blast overwriting its previous contents an uninstaller is provided and it is recommended when upgrading a BLAST installation 2 3 RedHat Linux Download the appropriate rpm file for your platform and either install or upgrade the ncbi blast package as appropriate using the commands Install rpm ivh ncbi blast 2 2 18 1 x86 64 rpm Upgrade rpm Uvh ncbi blast 2 2 18 1 x86 64 rpm BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1G dieH LSV1d Page 3 Note one must have root privileges to run these commands If you do not have root privileges please use the procedure described in http www ncbi nlm nih gov books NBK52640 2 4 Other Unix platforms Please see http www ncbi nlm nih gov books NBK52640 2 5 Source tarball Use this approach to build the BLAST applications yourself Download the tarball expand it cd to the newly created directory and type the following commands Cd ett configure without debug with strip with mt with build root ReleaseMT cd ReleaseMT build make all_r The compiled executables will be found in c ReleaseMT bin In Windows extract the tarball and open the appropriate MSVC solution or project file e g ct compiler
16. parse_seqids flag when invoking makeblastdb to enable retrieval of sequences based upon sequence identifiers In this case each sequence must have a unique identifier and that identifier must have a specific format The identifier should begin right after the gt sign on the definition line contain no spaces and follow the formats described in http www ncbi nlm nih gov books NBK7183 rendertype table amp id ch_demo T5 User supplied sequences should make use of the local or general identifiers described in the above table A FASTA file with general IDs would look like cat mydb fsa gt gnl MYDB 1 this is sequence 1 GAATTCCCGCTACAGGGGGGGCCTGAGGCACTGCAGAAAGTGGGCCTGAGCCTCGAGGATGACGGTGCTGCAGGAAC CCG TCCAGGCTGCTATATGGCAAGCACTAAACCACTATGCTTACCGAGATGCGGTTTTCCTCGCAGAACGCCTTTATGCA GAA BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 24 GTACACTCAGAAGAAGCCTTGTTTTTACTGGCAACCTGTTATTACCGCTCAGGAAAGGCATATAAAGCATATAGACT CTT GAAAGGACACAGTTGTACTACACCGCAAT GCAAATACCTGCTTGCAAAATGTTGTGTTGATCTCAGCAAGCTTGCAG AAG GGGAACAAATCTTATCTGGTGGAGTGTTTAATAAGCAGAAAAGCCATGATGATATTGTTACTGAGTTTGGTGATTCA GCT TGCTTTACTCTTTCATTGTTGGGACATGTATATTGCAAGACAGATCGGCTTGCCAAAGGATCAGAATGTTACCAAAA GAG CCTTAGTTTAAATCCTTTCCTCTGGTCTCCCTTTGAATCATTATGTGAAATAGGTGAAAAGCCAGATCCTGACCAAA CAT TTAAATTCACATCTTTACAGAACTTTAGCAACTGTCTGCCCAACTCT TET CACAGACAGCCTGAGACAGTTCTTACGGAAACACCCCAGGACACAAT CAA gt gnl MYD
17. to PSI BLAST and does not require several iterations of searches against a large sequence database See section 5 18 for more information 4 3 Configuring BLAST The BLAST search applications can be configured by means of a configuration file named ncbire on Unix like platforms or ncbi ini on Windows This is a plain text file that contains sections and key value pairs to specify configuration parameters Lines starting with a semi colon are considered comments The application will search for the file in the following order and locations 1 Current working directory 2 User s HOME directory 3 Directory specified by the NCBI environment variable The search for this file will stop at the first location where it is found and the configurations settings from that file will be applied If the configuration file is not found default values will BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1G dieH LSV1d Page 8 apply The following are the possible configuration parameters that impact the BLAST applications Configuration Parameter Specifies Default value BLASTDB Path to BLAST databases Current working directory DATA_LOADERS Data loaders to use for automatic sequence identifier resolution This is a comma separated list of the following keywords blastdb genbank and none The none keyword disables this feature and takes precedence over any other keywords specified
18. to a list of text IDs The binary list of GI s can be formatted to require minimal conversion at run time If all the sequences in the database have been assigned a GI a binary list of GI s should be used rather than a list of accessions BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E dieH LSV1d Page 26 5 15 Multiple databases vs spaces in filenames and paths BLAST has been able to search multiple databases since 1997 The databases can be listed after the db argument or in an alias file see section on blastdb_aliastool separated by spaces Many operating systems now allow spaces in filenames and directory paths so some care is required Basically one should always have two sets of quotes for any path containing a space Blastdbcmd is used as an example below but the same rules apply to makeblastdb as well as the search programs like blastn or blastp To access a BLAST database containing spaces under Microsoft Windows it is necessary to use two sets of double quotes escaping the innermost quotes with a backslash For example Users joeuser My Documents Downloads would be accessed by blastdbcmd db Users joeuser My Documents Downloads mydb info The first backslash escapes the beginning inner quote and the backslash following mydb escapes the ending inner quote A second database can be added to this command by including it within the outer pair of quotes blastdbcmd
19. 587 5671 2214 2130 5 6 2 Trace back operations BTOP The Blast trace back operations BTOP string describes the alignment produced by BLAST This string is similar to the CIGAR string produced in SAM format but there are important differences BTOP is a more flexible format that lists not only the aligned region but also matches and mismatches BTOP operations consist of 1 a number with a count of matching letters 2 two letters showing a mismatch e g AG means A was replaced by G or 3 a dash and a letter showing a gap The box below shows a blastn run first with BTOP output and then the same run with the BLAST report showing the alignments blastn query test_q fa subject test_s fa dust no outfmt 6 qseqid sseqid btop parse deflines BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 19 queryl q multi 7AG39 queryl q multi 7A 39 queryl q multi 6 G A41 blastn query test_q fa subject test_s fa dust no parse deflines BLASTN 2 2 24 Query queryl Length 47 Subject Length 142 Score 82 4 bits 44 Expect 9e 22 Identities 46 47 97 Gaps 0 47 0 Strand Plus Plus Query 1 ACGTCCGAGACGCGAGCAGCGAGCAGCAGAGCGACGAGCAGCGACGA 47 IIIT TEEPE EEE E EEE EEE PEPE EE EEE EE EEE EE EEE EEE Sbjct 47 ACGTCCGGGACGCGAGCAGCGAGCAGCAGAGCGACGAGCAGCGACGA 93 Score 80 5 bits 43 Expect 3e 21 Identities 46 47 97 Gaps 1747 2
20. 81 asn blast_formatter archive out 1786181l asn outfmt 7 qacc sacc evalue BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 21 qstart qend sstart send BLASTN 2 2 24 Query gi 1786181 gb AE000111 1 AE000111 Escherichia coli K 12 MG1655 section 1 of 400 Database ecoli Fields query acc subject acc evalue q start q end s start s end 85 hits found AE000 AE000111 0 0 1 10596 1 10596 AE000 AE000174 8e 30 5565 5671 6928 6821 AE000 AE000394 1e 27 5587 5671 135 219 AE000 AE000425 6e 26 5587 5671 8552 8468 AE000 AE000171 3e 24 5587 5671 2214 2130 AE000 AE000171 1e 23 5587 5670 10559 10642 AE000 AE000376 le 22 5587 5675 129 42 AE000 AE000268 le 22 5587 5671 6174 6090 AE000 AE000112 1e 22 10539 10596 1 58 AE000 AE000447 5e 22 5587 5670 681 598 AE000 AE000344 6e 21 5587 5671 4112 4196 AE000 AE000490 2e 20 5584 5671 4921 4835 AE000 AE000280 2e 20 5587 5670 12930 12847 5 9 Extract lowercase masked FASTA from a BLAST database with masking information Ifa BLAST database contains masking information this can be extracted using the blastdbcmd options db_mask and mask_sequence as follows blastdbcmd info db mask data db Database Mask data test 10 sequences 12 609 total residues Date Feb 17 2009 5 10 PM Longest sequence 1 694 residues Available filtering algorithms applied to database sequences Algorithm ID Algorithm
21. AATCCTTTCCTCTGGTCTCCCTTTGAATCATTATGTGAAATAGGTGAAAAGCCAGATCCTGACCAAA CAT TTAAATTCACATCTTTACAGAACTTTAGCAACTGTCTGCCCAACTCTTGCACAACACAAGTACCTAATCATAGTTTA TCTS BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 25 Makeblastdb can be invoked for this file as below makeblastdb in mydb fsa parse _segids dbtype nucl Building a new DB current time 01 28 2011 13 39 37 New DB name mydb fsa New DB title mydb fsa Sequence type Nucleotide Keep Linkouts T Keep MBits T Maximum file size 1073741824B Adding sequences from FASTA added 3 sequences in 0 00206995 seconds The FASTA file has three entries All entries are part of the MYDB database with the entries numbers 1 2 and 3 Makeblastdb will store this information properly and produce an index so that the sequences can be retrieved by these identifiers Makeblastdb stores the title portion of the definition line e g this is sequence 1 but will not parse it If the first token after the gt does not contain a bar it will be parsed as a local ID Use the full identifier string e g gnl MYDB 2 to retrieve sequences with a general ID The NCBI makes databases that are searchable on the NCBI web site such as nr refseq_rna and swissprot available on its FTP site It is better to download the preformatted databases rather than starting with FASTA The databases on the FTP site
22. AST search with a new style index requires that both the index and the corresponding BLAST database be present The index structure is described in PMID 18567917 Please cite this paper in any publication that uses makembindex option type default value Description and notes input string stdin Input file name or BLAST database name depending on the value of the iformat parameter For FASTA formatted input this parameter is optional and defaults to the program s standard input stream output string none The resulting index name The index itself can consist of multiple files called volumes called lt index_name gt 00 idx lt index_name gt 01 idx This option should not be used with new style indices iformat string fasta The input format selector Possible values are fasta and blastdb old_style_format boolean true If set to false the new style index is created New style indices require a BLAST database as input use iformat blastdb which can be downloaded from the NCBI FTP site or created with makeblastdb The option output is ignored for a new style index New style indices are always created at the same location as the corresponding BLAST database legacy boolean true This is a compatibility feature to support current production MegaBLAST If true then stride nmer and ws_hint are ignored The legacy format must be used for BLAST nmer integer 12 N mer size to use Ignored if legacy is s
23. B 2 this is sequence 2 GAATTCCCGCTACAGGGGGGGCCTGAGGCACTGCAGAAAGTGGGCCT CCG TCCAGGCTGCTATATGGCAAGCACTAAACCACTATGCTTACCGAGAT GAA TGCACAACACAAGTACCTAATCATAGTTTA TGAATTAAACAGATTGAATTTAGAATCTTC GAGCCTCGAGGATGACGGTGCTGCAGGAAC GCGGTTTTCCTCGCAGAACGCCTTTATGCA GTACACTCAGAAGAAGCCTTGTTTTTACTGGCAACCTGTTATTACCGCTCAGGAAAGGCATATAAAGCATATAGACT CTT GAAAGGACACAGTTGTACTACACCGCAATGCAAATACCTGCTTGCAAAATGTTGTGTTGATCTCAGCAAGCTTGCAG AAG GGGAACAAATCTTATCTGGTGGAGTGTTTAATAAGCAGAAAAGCCATGATGATATTGTTACTGAGTTTGGTGATTCA GCT TGCTTTACTCTTTCATTGTTGGGACATGTATATTGCAAGACAGATCGGCTTGCCAAAGGATCAGAATGTTACCAAAA GAG CCTTAGTTTAAATCCTTTCCTCTGGTCTCCCTTTGAATCATTATGTGAAATAGGTGAAAAGCCAGATCCTGACCAAA CAT TTAAATTCACATCTTTACAGAACTTTAGCAACTGTCTGCCCAACTCI TCT CACAGACAGCCTGAGACAGTTCTTACGGAAACACCCCAGGACACAAT CAA gt gnl MYDB 3 this is sequence 3 GAATTCCCGCTACAGGGGGGGCCTGAGGCACTGCAGAAAGTGGGCCT CCG TGCACAACACAAGTACCTAATCATAGTTTA TGAATTAAACAGATTGAATTTAGAATCTTC GAGCCTCGAGGATGACGGTGCTGCAGGAAC TCCAGGCTGCTATATGGCAAGCACTAAACCACTATGCTTACCGAGAT GAA GCGGTTTTCCTCGCAGAACGCCTTTATGCA GTACACTCAGAAGAAGCCTTGTTTTTACTGGCAACCTGTTATTACCGCTCAGGAAAGGCATATAAAGCATATAGACT CTT GAAAGGACACAGTTGTACTACACCGCAATGCAAATACCTGCTTGCAAAATGTTGTGTTGATCTCAGCAAGCTTGCAG AAG GGGAACAAATCTTATCTGGTGGAGTGTTTAATAAGCAGAAAAGCCATGATGATATTGTTACTGAGTTTGGTGATTCA GCT TGCTTTACTCTTTCATTGTTGGGACATGTATATTGCAAGACAGATCGGCTTGCCAAAGGATCAGAATGTTACCAAAA GAG CCTTAGTTTA
24. INMLADKLNMTPEEAERWIVNLI BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1G dieH LSV1d Page 11 26SPS9 Hs LDKKFHGILDQGEGVLIIFDEPP F57B9 Ce LDQKLSGSLDQGEGMLIVFEIAV YDLO97c_Sc LDKIFYGVLDQGNGWLYVYETPN YMJ5_Ce LDKKLMGSIDQHGDTVVVYPKAD FUS6 ARATH TDNQIQARIDSHNKILYARHADQ COS41 8 Ci CESRMKGHIDQIDGIVFFERRET 644879 LEGLISARVDSHSKILYARDVDO YPR108w_Sc PNKQLNCVIDRVNGIVETNRPDN eif 3p110 Hs INEELMASLDQPTOQTVVMHRTEP T23D8 4 Ce IQEELSATLDEPTDCLIMHRVEP YD95_ Sp PDNKLNCVIDRVNGVVFTNRPDE KIAA0107 Hs AAGRLHCKIDKVNEIVETNRPDS F49C12 8 Hs ATGQLQCRIDAVNGVIEVNHRDS Int 6 Mm RNARLDAKIDSKLGHVVMGNNAV 5 Cookbook 5 1 Query a BLAST database with a GI but exclude that GI from the results Extract a GI from the ecoli database blastdbcmd entry all db ecoli dbtype nucl outfmt g head 1 tee exclude_me 1786181 Run the restricted database search which shows there are no self hits blastn db ecoli negative gilist exclude_me show_gis num alignments 0 query exclude _ me grep cat exclude_me Query gi 1786181 gb AE000111 1 AE000111 5 2 Create a masked BLAST database Creating a masked BLAST database is a two step process a Generate the masking data using a sequence filtering utility like windowmasker or dustmasker b Generate the actual BLAST database using makeblastdb
25. Query accesion sseqid means Subject Seq id sallseqid means All subject Seq id s separated by a sgi means Subject GI sallgi means All subject GIs sacc means Subject accession sallacc means All subject accessions qstart means Start of alignment in query qend means End of alignment in query sstart means Start of alignment in subject send means End of alignment in subject qseq means Aligned part of query sequence sseq means Aligned part of subject sequence evalue means Expect value bitscore means Bit score score means Raw score length means Alignment length pident means Percentage of identical matches nident means Number of identical matches mismatch means Number of mismatches positive means Number of positive scoring matches gapopen means Number of gap openings gaps means Total number of gap ppos means Percentage of positive scoring matches frames means Query and subject frames separated by a qframe means Query frame sframe means Subject frame When not provided the default value is qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore which is equivalent to the keyword std Table C2 blastn application options The blastn application searches a nucleotide query against nucleotide subject sequences or a nucleotide database An option of type flag takes no arguments but if present the argument is true Four different tasks are supported 1 megablast for
26. SRPGS POSRRDAS PASS DGSNE PLA ARAPSSRMVDEDY DEGAADALMGLAGAASAS SASVATAAPAPVS PVATS DRASSAEKRAESS LGKRP YAEEERAVDE PED SYKRAKSGSAAE IEADATSGGRLNGVSVSAKPEATAAEGTEQPKETRTETPPLAVAQATS PEAINGKAESESAVOQPM DVD GREPSKAPSESATAMKDS PSTANPVVAAKASEPS PTAAPPATSMATSEAQPAKADSCEKNNNDEDEREEEEGOIHED PID APAKRADEDGAK 5 10 Display the locations where BLAST will search for BLAST databases This is accomplished by using the show_blastdb_search_path option in blastdbcmd blastdbcmd show blastdb search path net nab1000 vol blast db blast1 net nab1000 vol blast db blast2 5 11 Display the available BLAST databases at a given directory This is accomplished by using the list option in blastdbcmd blastdbcmd list repeat recursive repeat repeat_3055 Nucleotide repeat repeat_31032 Nucleotide repeat repeat_35128 Nucleotide repeat repeat_3702 Nucleotide repeat repeat_40674 Nucleotide repeat repeat_4530 Nucleotide repeat repeat_4751 Nucleotide repeat repeat_6238 Nucleotide repeat repeat_6239 Nucleotide BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 23 repeat repeat_7165 Nucleotide repeat repeat_7227 Nucleotide repeat repeat_7719 Nucleotide repeat repeat_7955 Nucleotide repeat repeat_9606 Nucleotide repeat repeat_9989 Nucleotide The first column of the default output is the file name of the BLAST database usually provided as the db argument to other BLAST
27. Table C7 rpsblast application options The rpsblast application searches a protein query against the conserved domain database CDD which is a set of protein profiles Many of the common options such as matrix or word threshold are set when the CDD is built and cannot be changed by the rpsblast application A search ready CDD can be downloaded from ftp ftp ncbi nih gov pub mmdb cdd Option Type Default value Description and notes window_size integer 40 Multiple hits window size use 0 to specify 1 hit algorithm xdrop_ungap real 15 Heuristic value in bits for ungapped extensions xdrop_gap real 25 Heuristic value in bits for preliminary gapped extensions xdrop_gap final real 40 Heuristic value in bits for final gapped alignment seg string 12 2 2 2 5 Filter query sequence with SEG Format yes window locut hicut or no to disable soft_masking boolean false Apply filtering locations as soft masks i e only for finding initial matches Table C8 Makeblastdb application options This application builds a BLAST database An option of type flag takes no arguments but if present the argument is true option type default value Description and notes in string stdin Input file database name input_type string fasta Input file type it may be any of the following fasta for FASTA file s blastdb for BLAST database s asn1_txt
28. alue description and notes db string none BLAST database name query string stdin Query file name query_loc string none Location on the query sequence Format start stop out string stdout Output file name evalue real 10 0 Expect value E for saving hits subject string none File with subject sequence s to search subject_loc string none Location on the subject sequence Format start stop BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E dieH LSV1d Page 29 show_gis flag N A Show NCBI GIs in report num_descriptions integer 500 Show one line descriptions for this number of database sequences num_alignments integer 250 Show alignments for this number of database sequences max_target_seqs Integer 500 Number of aligned sequences to keep Use with report formats that do not have separate definition line and alignment sections such as tabular all outfmt gt 4 Not compatible with num_descriptions or num_alignments html flag N A Produce HTML output gilist string none Restrict search of database to GI s listed in this file Local searches only negative_gilist string none Restrict search of database to everything except the GI s listed in this file Local searches only entrez_query string none Restrict search with the given Entrez query Remote searches only culling limit integer none Delete a hit that is e
29. alue description and notes word_size integer 3 Word size for initial match matrix string BLOSUM62 Scoring matrix name threshold integer 13 Minimum word score to add the word to the BLAST lookup table seg string 122 225 Filter query sequence with SEG Format yes window locut hicut or no to disable soft_masking boolean false Apply filtering locations as soft masks i e only for finding initial matches lcase_masking flag N A Use lower case filtering in query and subject sequence s db_soft_mask integer none Filtering algorithm ID to apply to the BLAST database as soft mask i e only for finding initial matches db_hard_mask integer none Filtering algorithm ID to apply to the BLAST database as hard mask i e sequence is masked for all phases of search strand string both Query strand s to search against database subject sequences Choice of both minus or plus query_genetic_code integer 1 Genetic code to translate query see ftp ftp ncbi nih gov entrez misc data ge prt BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E djeH LSV1d Page 35 db_gen_code integer Genetic code to translate subject sequences see ftp ftp ncbi nih gov entrez misc data ge prt max_intron_length integer Length of the largest intron allowed in a translated nucleotide sequence when linking multiple distinct alignments a negative value disables linking
30. applications the second column represents the molecule type of the BLAST database This output is configurable via the list_outfmt command line option 5 12 Use Windowmasker to filter your BLAST search The blastn executable can filter a query sequence using the windowmasker data files This option can be used to mask interspersed repeats that may lead to spurious matches The windowmasker data files should be created as discussed in section 5 2 12 or downloaded from the NCBI FTP site Follow the instructions in Configuring BLAST to make sure BLAST will be able to find the windowmasker files in the examples below 1 Run BLAST search using Windowmasker for sequence filtering based upon taxid 9606 is the taxid for human blastn query input db database window_masker taxid 9606 out results txt 2 Run BLAST search using Windowmasker for sequence filtering based upon the windowmasker file name blastn query input db database window_masker db 9606 wmasker obinary 5 13 Building a BLAST database with local sequences The makeblastdb application produces BLAST databases from FASTA files In the simplest case the FASTA definition lines are not parsed by makeblastdb and may be completely unstructured The text in the definition line will be stored in the BLAST database and displayed in the BLAST report but it will not be possible to fetch individual sequences using blastdbcmd or to limit the search with the seqidlist option Use the
31. at can be used later to start a new search and greater flexibility in the formatting of tabular results The functionality of the BLAST applications is organized by search type As an example there is a blastp application that compares proteins queries to protein databases The blastx application translates a nucleotide query in six frames and searches it against a protein database This organization is different from that of the applications first released in 1997 e g blastall that supported all types of searches with one application but it resembles that of the NCBI BLAST web site An advantage of this design is that each application has only the options relevant to the searches it performs Additionally each application can compare a query to a set of FASTA sequences in a file bypassing the need to create a BLAST databases for small dieH LSV1d dieH LSV1E dieH LSV1G dieH LSV1d Page 2 and infrequently searched sets Finally a remote option permits each application to send off a search to the NCBI servers This manual has several sections It provides brief installation instructions a QuickStart a section describing BLAST features in more depth a Cook Book section on how to perform a number of tasks as well as three appendices The first appendix discusses tools to help with the transition from the older applications e g blastall to the BLAST applications The second appendix documents exit codes
32. blast_formatter allows this if the original search produced blast archive format using the outfmt 11 switch The query sequence the BLAST options the masking information the name of the database and the alignment are written out as ASN 1 a structured format similar to XML The max_target_seqs option should be used to control the number of matches recorded in the alignment The blast_formatter reads this information and formats a report The BLAST database used for the original search must be available or the sequences need to be fetched from the NCBI assuming the database contains sequences in the public dataset The box below illustrates the procedure A blastn run first produces the BLAST archive format and the blast_fomatter then reads the file and produces tabular output Blast_formatter will format stand alone searches performed with an earlier version of a database if both the search and formatting databases are prepared so that fetching by sequence ID is possible To enable fetching by sequence ID use the parse_seqids flag when running makeblastdb or if available download preformatted BLAST databases from ftp ftp ncbi nlm nih gov blast db using update_blastdb pl provided as part of the BLAST package Currently the blast archive format and blast_formatter do not work with database free searches i e subject rather than db was used for the original search echo 1786181 blastn db ecoli outfmt 11 out out 17861
33. blastdb genbank BLASTDB_PROT_DATA_LOADER Locally available BLAST database name to search when resolving protein sequences using BLAST databases Ignored if DATA_LOADERS does not include the blastdb keyword nr BLASTDB_NUCL_DATA_LOADER Locally available BLAST database name to search when resolving nucleotide sequences using BLAST databases Ignored if DATA_LOADERS does not include the blastdb keyword nt GENE_INFO PATH Path to gene information files NCBI only Current working directory WINDOW_MASKER_ PATH Path to windowmasker directory hierarchy Current working directory The following is an example with comments describing the available parameters for configuration Start the section for BLAST configuration BLAST Specifies the path where BLAST databases are installed BLASTDB home guest blast db Specifies t for sequence identifiers DATA LOADERS blastdb Specifies t BLASTDB_PROT_DATA_LOADER custom_protein_ database he data sources to use for automatic resolution he BLAST database to use resolve protein sequences Specifies the BLAST database to use resolve protein sequences BLASTDB NUCL DATA LOADER home some_user my nucleotide db Windowmasker settings WINDOW _MASKER WINDOW_MASKER_PATH home guest blast db windowmasker end of file 4 3 1 Controlling concatenation of queries BLAST works more efficiently by scanning the database once
34. ble via Windows and MacOSX installers as well as RPMs source and binary and unix tarballs For more details about these refer to the installation section 4 2 11 Sequence filtering applications The BLAST applications include a new set of sequence filtering applications namely segmasker dustmasker and windowmasker Segmasker is an application that identifies and masks low complexity regions of protein sequences The dustmasker application provides a similar functionality for nucleotide sequences Windowmasker uses a genome to identify sequences represented too often to be of interest to most users See ftp ftp ncbi nlm nih gov pub agarwala dustmasker README dustmasker and ftp ftp ncbi nlm nih gov pub agarwala windowmasker README windowmasker for more information 4 2 12 Best Hits filtering algorithm The Best Hit filtering algorithm is designed for use in applications that are searching for only the best matches for each query region reporting matches Its best_hit_overhang parameter H controls when an HSP is considered short enough to be filtered due to presence of another HSP For each HSP A that is filtered there exists another HSP B such that the query region of HSP A extends each end of the query region of HSP B by at most H times the length of the query region for B Additional requirements that must also be met in order to filter A on account of B are i evalue A gt evalue B ii score A length A lt 1 0 score_e
35. cations of the search strategy file provided no other command line options will override the contents of the search strategy file 4 2 6 Negative GI lists Search applications support negative GI lists This feature provides a means to exclude GIs from a BLAST database search The expect values in the BLAST results are based upon the BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E dieH LSV1d Page 6 sequences actually searched and not on the underlying database For an example see the cookbook 4 2 7 Masking in BLAST databases It is now possible to create BLAST databases that contain filtered sequences also known as masking information or masks This filtering information can be used as soft masking for the subject sequences For instructions on creating masked BLAST databases please see the cookbook 4 2 8 Custom output formats for BLAST searches The BLAST search command line applications support custom output formats for the tabular and comma separated value output formats For more details see outfmt in Appendix C as well as the cookbook 4 2 9 Custom output formats to extract BLAST database data blastdbcmd supports custom output formats to extract data from BLAST databases via the outfmt command line option For more details see the blastdbcmd options in Appendix C as well as the cookbook 4 2 10 Improved software installation packages The BLAST applications are availa
36. chr infmt blastdb enable the sequence id parsing parse_seqids request the mask data in binary asn 1 format outfmt maskinfo_asn1_bin and name the output file as hs_chr_dust asnb out hs_chr_dust asnb If the input format is the original FASTA file hs_chr fa we need to change input to in and infmt options as follows dustmasker in hs_chr fa infmt fasta parse_seqids outfmt maskinfo_asnl_bin out hs_chr_dust asnb 5 2 1 2 Create masking information using windowmasker To generate the masking information using windowmasker from the BLAST database hs_chr we first need to generate a counts file windowmasker in hs_chr infmt blastdb mk_counts parse seqids out hs chr mask counts Here we specify the input BLAST database in hs_chr infmt blastdb request it to generate the counts mk_counts with sequence id parsing parse_seqids and save the output to a file named hs_chr_mask counts out hs_chr_mask counts To use the FASTA file hs_chr fa to generate the counts we need to change the input file name and format windowmasker in hs_chr fa infmt fasta mk_counts parse seqids out hs chr mask counts With the counts file we can then proceed to create the file containing the masking information as follows windowmasker in hs_chr infmt blastdb ustat hs_chr_mask count outfmt maskinfo_asnl_ bin parse seqids out hs_ chr _mask asnb Here we need to use the same input in hs_chr infmt
37. dge score B length B We consider 0 1 to 0 25 to be an acceptable range for the best_hit_overhang parameter and 0 05 to 0 25 to be an acceptable range for the best_hit_score_edge parameter Increasing the value of the overhang parameter eliminates a higher number of matches but increases the running time increasing the score_edge parameter removes smaller number of hits BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E dieH LSV1d Page 7 4 2 13 Automatic resolution of sequence identifiers The BLAST search applications support automatic resolution of query and subject sequence identifiers specified as GIs or accessions see the cookbook section for an example This feature enables the user to specify one or more sequence identifiers GIs and or accessions one per line in a file as the input to the query and subject command line options Upon encountering this type of input by default the BLAST search applications will try to resolve these sequence identifiers in locally available BLAST databases first then in the BLAST databases at NCBI and finally in Genbank the latter two data sources require a properly configured internet connection These data sources can be configured via the DATA_ LOADERS configuration option and the BLAST databases to search can be configured via the BLASTDB_PROT_ DATA LOADER and BLASTDB_NUCL_DATA LOADER configuration options see the section on Configuring BLAST
38. dieH LSV1E dieH LSV1gE dieH LSV1E dieH LSV1gG WV BLAST Help zZ O w BLAST Command Line Applications User Manual Christiam Camacho camacho ncbi nlm nih gov Thomas Madden madden ncbi nlm nih gov George Coulouris coulouri ncbi nlm nih gov Ning Ma maning ncbi nlm nih gov Tao Tao tao ncbi nlm nih gov Richa Agarwala HYPERLINK mailto richa ncbi nilm nih gov richa ncbi nlm nih gov Aleksandr Morgulis morgulis ncbi nlm nih gov NCBI NCBI NCBI NCBI NCBI NCBI NCBI 1 Introduction Sequence similarity searching is one of the more important bioinformatics activities and often provides the first evidence for the function of a newly sequenced gene or piece of sequence Basic Local Alignment Search Tool BLAST is probably the most popular similarity search tool The National Center For Biotechnology Information NCBI first introduced BLAST in 1989 The NCBI has continued to maintain and update BLAST since the first version In 2009 the NCBI introduced a new version of the stand alone BLAST applications BLAST The BLAST applications have a number of improvements that allow faster searches as well as more flexibility in output formats and in the search input These improvements include splitting of longer queries so as to reduce the memory usage and to take advantage of modern CPU architectures use of a database index to dramatically speed up the search the ability to save a search strategy th
39. ding the consensus sequence changes the statistical properties of the original alignment To avoid this the ignore_msa_master option can be used psiblast in_msa alignl db pataa ignore msa_master In this case the master sequence is displayed in the output but ignored when the PSSM scores are calculated BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1G dieH LSV1E dieH LSV1d Page 27 5 18 Performing a DELTA BLAST search DELTA BLAST searches a protein sequence database using a PSSM constructed from conserved domains matching a query It first searches the NCBI CDD database to construct the PSSM 5 18 1 Download the cdd_delta database Obtain this database from ftp ftp ncbi nlm nih gov blast db using the update_blastdb pl tool provided as part of the BLAST package Note that the cdd_delta database must be downloaded and installed to the standard BLAST database directory see Configuring BLAST or in the current working directory 5 18 2 Execute the deltablast search deltablast query query fsa db pataa Appendix A Conversion from C toolkit applications The functionality offered by the BLAST applications has been organized by program type The following graph depicts a correspondence between the NCBI C Toolkit BLAST command line applications and the BLAST applications The easiest way to get started using the BLAST command line applications is by means of the legacy_blast pl PERL script wh
40. e file with lowercase masking generated by various means using convert2blastmask utility An example command line follows convert2blastmask in hs_chr mfa parse seqids masking algorithm repeat masking options repeatmasker default outfmt maskinfo_asnl_bin out hs_chr_mfa asnb Here the input is hs_chr mfa in hs_chr mfa enable parsing of sequence ids specify the masking algorithm name masking algorithm repeat and its parameter masking_ options repeatmasker default and ask for asn 1 output outfmt maskinfo_asn1_bin to be saved in specified file out hs_chr_mfa asnb 5 2 2 Create BLAST database with the masking information Using the masking information data files generated in steps 5 2 1 1 5 2 1 2 5 2 1 3 and 5 2 1 4 we can create BLAST database with masking information incorporated Note we should use parse_seqids in a consistent manner either use it in both steps or not use it at all 5 2 2 1 Create BLAST database with masking information using an existing BLAST database or FASTA sequence file as input For example we can use the following command line to apply the masking information created in step 5 2 1 2 to the existing BLAST database generated in 5 2 3 BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 14 makeblastdb in hs_chr input_type blastdb dbtype nucl parse_ seqids mask_ data hs_chr_ mask asnb out hs_chr
41. em YPR1O8w Sc eif 3pll0 Hs sees Sseoshasses Sesser SsStanbresS Ssh nanne T23D8 4 Ce Sessa sssssss sess asses ease esses esses esses seeessss YD95 Sp KIAA0107_ Hs F49C12 8 Hs Int 6 Mm ylttavitnkdvrkrrqvlkdlvkviqgesytykdpitefveclyvnfdfdgaqkklrec 26SPS9 Hs RAELRDDPIISTHLAKLYDNLLEQNLIRVIEPFSRVQIEHISSLIKLSKADVERKLSOMI F57B9 Ce PQELOMDPVVRKHFHSLSERMLEKDLCRIIEPYSFVQIEHVAQQIGIDRSKVEKKLSOMI YDLO97c_Sc EKELMGDELTRSHFNALYDTLLESNLCKI IEPFECVEISHISKIIGLDTQQVEGKLSOQM YMJ5_Ce KIELVEDKVVAVHSQNLERNMLEKEISRVIEPYSEIELSYIARVIGMTVPPVERAIARMI FUS6 ARATH KSNLLLDIHLHDHVDTLYDQIRKKALIQYTLPFVSVDLSRMADAFKTSVSGLEKELEAL COS41 8 Ci QLMPHQKATTADGSNILHRAVTEHNLLSASKLYNNIRFTELGALLEI PHQMAEKVASOQM 644879 KDNLLLDMYLAPHVRTLYTOIRNRALIOYFS PYVSADMHRMAAAFNTTVAALEDELTOL YPR108w_Sc ANVLIPCKYLNRHADFFVREMRRKVYAQLLESYKTLSLKSMASAFGVSVAFLDNDLGKF eif 3p110 Hs DLFPEADKVRTMLVRKIQEESLRTYLFTYSSVYDSISMETLSDMFELDLPTVHSIISKMI T23D8 4 Ce NLFHNAETVKGMVVRRIQEESLRTYLLTYSTVYATVSLKKLADLFELSKKDVHSIISKM YD95 Sp VNHLKCDQFLVAHYRYYVREMRRRAYAQLLESYRALSIDSMAASFGVSVDYIDRDLASFI KIAA0107_ Hs EQEMKKDWLFAPHYRYYVREMRIHAYSQLLESYRSLTLGYMAEAFGVGVEFIDQELSRFI F49C12 8 Hs SERFKFDRYLSPHFNYYSRGMRHRAYEQFLTPYKTVRIDMMAKDFGVSRAFIDRELHRLI Int 6 Mm ESVLVNDFFLVACLEDFIENARLFIFETFCRIHQCIS
42. en a gap gapextend integer 1 Cost to extend a gap matrix string BLOSUM62 Scoring matrix name threshold integer 12 Minimum score to add a word to the BLAST lookup table seg string 12 2 2 2 5 Filter query sequence with SEG Format yes window locut hicut or no to disable soft_masking boolean false Apply filtering locations as soft masks i e only for finding initial matches lcase_masking flag N A Use lower case filtering in query and subject sequence s db_soft_mask integer none Filtering algorithm ID to apply to the BLAST database as soft mask i e only for finding initial matches db_hard_mask integer none Filtering algorithm ID to apply to the BLAST database as hard mask i e sequence is masked for all phases of search xdrop_gap_final real 25 Heuristic value in bits for final gapped alignment window_size integer 40 Multiple hits window size use 0 to specify 1 hit algorithm strand string both Query strand s to search against database subject Choice of both minus or plus query_genetic_code integer 1 Genetic code to translate query see ftp ftp ncbi nih gov entrez misc data ge prt max_intron_length integer 0 Length of the largest intron allowed in a translated nucleotide sequence when linking multiple distinct alignments a negative value disables linking comp_based_stats integer 2 Use composition based statistics for blastx D or d default equivalent to 2 0 or F or f no composition based statistic
43. ence data without defline a means accession g means gi 0 means ordinal id OID t means sequence title l means sequence length T means taxid L means common taxonomic name S means scientific name P means PIG mX means sequence masking data where X is an optional comma separated list of integers to specify the algorithm ID s to display or all masks if absent or invalid specification Masking data will be displayed as a series of N M values separated by or the word none if none are available For every format except f each line of output will correspond to a sequence target_only flag N A Definition line should contain target GI only get_dups flag N A Retrieve duplicate accessions line_length integer 80 Line length for output ctrl_a flag N A Use Ctrl A as the non redundant definition line separator Table C11 Makembindex application options The indexed databases created by makembindex are used by production MegaBLAST software and by a new srsearch utility designed to quickly search for nearly exact matches up to one mismatch of short queries against a genomic database When a FASTA formatted file is used as the input then masking by lower case letters is incorporated in the index Makembindex can currently build two types of indices called old style and new style indexing The NCBI offers full support for the new style and has deprecated the old style A MegaBL
44. for multiple queries This feature is knows as concatenation and speeds up MegaBLAST searches the most as they spend little time on tasks that consume CPU and most of the time streaming through the database BLASTN and discontiguous MegaBLAST searches also run faster with concatenation though the effect is less pronounced Unfortunately for some searches the concatenation values are not optimal too many queries are searched at once and the process can consume too much memory This is most often a sign that the query or database has not been masked for interspersed repeats and that should be done Windowmasker and repeatmasker are two options for masking The concatenation values have been adjusted lower for the 2 2 27 release For that release the following values are used for the blastn application megaBLAST task 5 BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 9 million bases discontiguous megaBLAST task 500 000 bases BLASTN task 100 000 bases It is possible to control these values by setting the BATCH_SIZE environment variable Setting the value too low will degrade performance dramatically so this environment variable should be used with caution NCBI staff are working on improvements to prevent memory exhaustion in future releases of the software Query concatenation also means that BLAST will produce no output until the first set of concatenated queries have been processed S
45. formation from a BLAST database and how to format it Extract the accession sequence length and masked locations for GI 71022837 blastdbemd entry 71022837 db Test mask data db outfmt a 1 m XP_761648 1 1292 119 139 140 144 147 152 154 160 161 216 5 6 Display BLAST search results with custom output format The outfmt option permits formatting arbitrary fields from the BLAST tabular format Use the help option on the command line application e g blastn to see the supported fields The max_target_seqs option should be used with any tabular output to control the number of matches reported 5 6 1 Example of custom output format The following example shows how to display the results of a BLAST search using a custom output format The tabular output format with comments is used but only the query accession subject accession evalue query start query stop subject start and subject stop are requested For brevity only the first 10 lines of output are shown echo 1786181 blastn db ecoli outfmt 7 qacc sacc evalue qstart gend sstart send BLASTN 2 2 18 Query gi 1786181 gb AE000111 1 AE000111 Database ecoli Fields query acc subject acc evalue q start q end s start s end 85 hits found AE000111 AEO00111 0 0 1 10596 1 10596 AE000111 AEO00174 8e 30 5565 5671 6928 6821 AE000111 AE000394 le 27 5587 5671 135 219 AE000111 AE000425 6e 26 5587 5671 8552 8468 AE000111 AE000171 3e 24 5
46. ich is bundled along with the BLAST applications To utilize this script simply prefix it to the invocation of the C toolkit BLAST command line application and append the path option pointing to the installation directory of the BLAST applications For example instead of using blastall i query d nr o blast out use legacy blast pl blastall i query d nr o blast out path opt blast bin The purpose of the legacy_blast pl PERL script is to help users make the transition from the C Toolkit BLAST command line applications to the BLAST applications This script produces its own documentation by invoking it without any arguments The legacy_blast pl script supports two modes of operation one in which the C Toolkit BLAST command line invocation is converted and executed on behalf of the user and another which solely displays the BLAST application equivalent to what was provided without executing the command BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1G djeH LSV1d Page 28 The first mode of operation is achieved by specifying the C Toolkit BLAST command line application invocation and optionally providing the path argument after the command line to convert if the installation path for the BLAST applications differs from the default available by invoking the script without arguments See example in the first section of the Quick start The second mode of operation is achieved by specify
47. ing the C Toolkit BLAST command line application invocation and appending the print_only command line option as follows legacy blast pl megablast i query fsa d nt o mb out print_only opt ncbi blast bin blastn query query fsa db nt out mb out Appendix B Exit codes All BLAST applications have consistent exit codes to signify the exit status of the application The possible exit codes along with their meaning are detailed in the table below Exit Code Meaning 0 Success 1 Error in query sequence s or BLAST options 2 Error in BLAST database 3 Error in BLAST engine 4 Out of memory 255 Unknown error In the case of BLAST database applications the possible exit codes are 0 indicating success and 1 indicating failure Appendix C Options for the command line applications This appendix consists of several tables that list option names types default values and a short description of the option These tables were first published as an appendix to an article in BMC Bioinformatics BLAST architecture and applications They have been updated for this manual Table C1 Options common to all BLAST search applications An option of type flag takes no argument but if present is true Some options are valid only for a local search remote option not used others are valid only for a remote search remote option used option type default v
48. ist makeblastdb in hs_chr input_type blastdb dbtype nucl parse_seqids mask_data hs_chr_dust asnb hs_chr_mask asnb out hs_chr 5 2 2 2 Create a protein BLAST database with masking information We can use the masking data file generated in step 5 2 1 3 to create a protein BLAST database makeblastdb in refseq_protein input_type blastdb dbtype prot parse_seqids mask data refseq seg asnb out refseq protein title RefSeq Protein Database Using blastdbcmd we can check the database thus generated blastdbcmd db refseq_ protein info This produces the following summary which includes the masking information Database RefSeq Protein Database 7 044 477 sequences 2 469 203 411 total residues Date Sep 1 2009 10 50 AM Longest sequence 36 805 residues Available filtering algorithms applied to database sequences BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 16 Algorithm ID Algorithm name Algorithm options 21 seg window 12 locut 2 2 hicut 2 5 Volumes export home tao blast_test refseq protein2 00 export home tao blast_test refseq protein2 01 export home tao blast_test refseq protein2 02 5 2 2 3 Create a nucleotide BLAST database using the masking information extracted from lower case masked FASTA file We use the following command line which is very similar to that given in 5 2 2 1 makeblastdb in hs_chr mfa dbtype nucl parse_seqids
49. k to the public NCBI server if desired The BLAST package offers three categories of applications 1 search tools 2 BLAST database tools and 3 sequence filtering tools The blastn blastp blastx tblastx tblastn psiblast rpsblast and rpstblastn are considered search applications as they execute a BLAST search whereas makeblastdb blastdb_aliastool makeprofiledb and blastdbcmd are considered BLAST database applications as they either create or examine BLAST databases There is also a new set of sequence filtering applications described in the section Sequence filtering applications and an application to build database indices that greatly speed up megablast in some cases see section titled Megablast indexed searches 4 2 BLAST features 4 2 1 Tasks The blastn and blastp applications have a task option This option sets the parameters e g word size or gap values to typical values for a specific type of search For example the megablast task is optimized for intraspecies comparison as it uses a large word size whereas blastn is better suited for interspecies comparisons with a shorter word size These tasks resemble the Program Selection section of the BLAST web pages and do not preclude the user from setting other options to override those specified by the task See Appendix C for documentation on parameter values for different tasks The following tasks are currently available P
50. n hs_chr fa dbtype nucl parse_seqids out hs_chr title Human chromosomes Ref B37 1 For input nucleotide sequences with lowercase masking we use the FASTA file hs_chr mfa containing the complete human chromosomes from BUILD37 1 generated by inflating and combining the hs_ref_ mfa gz files located in the same ftp directory For input protein sequences we use the preformatted refseq_protein database from the NCBI blast db ftp directory ftp ncbi nlm nih gov blast db refseq protein 00 tar gz ftp ncbi nlm nih gov blast db refseq protein 0l tar gz ftp ncbi nlm nih gov blast db refseq protein 02 tar gz 5 3 Search the database with database soft masking information To enable the database masking during a BLAST search we need to get the Algorithm ID using the info parameter of blastdbcmd For the database generated in step 5 2 2 2 we can use the following command line to activate one of the database soft masking created by windowmasker blastn query HTT gene task megablast db hs_chr db_soft_mask 30 outfimt 7 out HTT megablast_mask out num_threads 4 Here we use the blastn program to search a nucleotide query HTT_gene query HTT_gene with megablast algorithm task megablast against the database created in step 5 2 2 1 db hs_chr We invoke the soft database masking db_soft_mask 30 set the result format to tabular output outfmt 7 and save the result to a file named HTT_megablast_mask tab out HTT_megablas
51. nally seg all string no Filter query sequence with SEG Format yes window locut hicut or no to disable soft_masking blastp boolean false Apply filtering locations as soft masks i e only for finding initial matches Icase_masking all flag N A Use lower case filtering in query and subject sequence s db_soft_mask all integer none Filtering algorithm ID to apply to the BLAST database as soft mask i e only for finding initial matches db_hard_mask all integer none Filtering algorithm ID to apply to the BLAST database as hard mask i e sequence is masked for all phases of search BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E djeH LSV1d Page 33 xdrop_gap_final all real 25 Heuristic value in bits for final gapped alignment window_size blastp integer 40 Multiple hits window size use 0 to specify 1 hit algorithm window_size blastp short integer 15 Multiple hits window size use 0 to specify 1 hit algorithm use_sw_tback all flag N A Compute locally optimal Smith Waterman alignments Table C4 blastx application options The blastx application translates a nucleotide query and searches it against protein subject sequences or a protein database option type default value description and notes word_size integer 3 Word size for initial match gapopen integer 11 Cost to op
52. name Algorithm options 20 seg default options used 40 repeat species Desmodus_rotundus Volumes mask data db blastdbcmd db mask data db mask_sequence_with 20 entry 71022837 gt gi 71022837 ref XP_761648 1 hypothetical protein UM05501 1 Ustilago maydis 521 MPPSARHSAHPSHHPHAGGRDLHHAAGGPPPOGGPGMPPGPGNGPMHHPHSS YAQSMPPPPGLPPHAMNGINGPPPS THG GPPPRMVMADGPGGAGGPPPPPPPHI PRSSSAQSRIMEAaggpagpppagppastspavOk1lslANEaawvsIGsaa etm BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 22 EdydralsayeaalrhnpysvpalsaiagvhrtldnfekavdyfqrvlnivpengdTWGSMGHCYLMMDDLORAYTA YQO ALYHLPNPKEPKLWYGIGILYDRYGSLEHAEEAFASVVRMDPNYEKANEIYFRLGIIYKOQONKFPASLECFRYILDN PPR PLTEIDIWFOIGHVYEQOKEFNAAKEAYERVLAENPNHAKVLOOLGWLYHLSNAGFNNQERAIQFLTKSLESDPNDA Qs X Cc Q 4 LLGRAYMAGONYNKAYEAYQOQAVYRDGKNPTFWCSIGVLYYQINOYRDALDAYSRAIRLNPYISEVWFDLGSLYEA NN SDAIHAYERAADLDPDNPOQIQORLOLLRNAEAKGGELPEAPVPQDVHPTAYANNNGMAPGPPTOQIGGGPGPSYPP LV GPQLAGNGGGRGDLSDRDLPGPGHLGSSHS PPPFRGPPGTDDRGARGP PHGALAPMVGGPGGPEPLGRGGFSHSRGP SPG PPRMDPYGRRLGSPPRRS PPPPLRS DVHDGHGAP PHVHGOQGHGOGHGQGHGOQGHGOQGHGQSHGHSHGGEFRGPPPLA AAG PGGPPPPLDHYGRPMGGPMSEREREMEWEREREREREREQAARGY PASGRITPKNEPGYARSQHGGSNAPS PAFGRP PVY GRDEGRDY YNNSHPGSGPGGPRGGYERGPGAPHAPAPGMRHDERGPP PAPFEHERGPPPPHQAGDLRYDSYSDGRDG PFR GPPPGLGRPT PDWERTRAGEYGPPSLHDGAEGRNAGGSASKSRRGPKAKDELEAAPAPPS PVPSSAGKKGKTTSSRA GSP WSAKGGVAAPGKNGKAST PFGTGVGAPVAAAGVGGGVGSKKGAAISLRPQEDQPD
53. nveloped by at least this many higher scoring hits best_hit_overhang real none Best Hit algorithm overhang value recommended value 0 1 best_hit_score_edge real none Best Hit algorithm score edge value recommended value 0 1 dbsize integer none Effective size of the database searchsp integer none Effective length of the search space import_search_strategy string none Search strategy file to read export_search strategy string none Record search strategy to this file parse_deflines flag N A Parse query and subject bar delimited sequence identifiers e g gi 129295 num_threads integer 1 Number of threads CPUs to use in blast search remote flag N A Execute search on NCBI servers BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E djeH LSV1d Page 30 oufmt string 0 alignment view options 0 pairwise 1 query anchored showing identities 2 query anchored no identities 3 flat query anchored show identities 4 flat query anchored no identities 5 XML Blast output 6 tabular 7 tabular with comment lines 8 Text ASN 1 9 Binary ASN 1 10 Comma separated values 11 BLAST archive format ASN 1 Options 6 7 and 10 can be additionally configured to produce a custom format specified by space delimited format specifiers The supported format specifiers are qseqid means Query Seq id qgi means Query GI qacc means
54. ome users find this disconcerting but it is not a problem The new lower concatenation values mean that BLAST will start to produce output sooner 4 3 2 Memory usage The BLAST search programs can exhaust all memory on a machine if the input is too large or if there are too many hits to the BLAST database If this is the case please see your operating system documentation to limit the memory used by a program e g ulimit on Unix like platforms Setting the BATCH_SIZE environment variable as described above may help 4 4 Input formats to BLAST 4 4 1 Multiple sequence alignment The in_msa psiblast option provides a way to jump start psiblast from a master slave multiple sequence alignment computed outside psiblast The multiple sequence alignment must contain the query sequence as one of its sequences but it need not be the first sequence The multiple sequence alignment must be specified in a format that is derived from Clustal but without some headers and trailers see example below The rules are also described by the following words Suppose the multiple sequence alignment has N sequences It may be presented in one or more blocks where each block presents a range of columns from the multiple sequence alignment E g the first block might have columns 1 60 the second block might have columns 61 95 the third block might have columns 96 128 Each block should have N rows one row per sequence The sequences should be in the same order
55. on index_name megablast string none MegaBLAST database index name xdrop_ungap all real 20 Heuristic value in bits for ungapped extensions xdrop_gap all real 30 Heuristic value in bits for preliminary gapped extensions xdrop_gap_final all real 100 Heuristic value in bits for final gapped alignment no_greedy megablast flag N A Use non greedy dynamic programming extension BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E djeH LSV1d Page 32 min_raw_gapped_score all integer none Minimum raw gapped score to keep an alignment in the preliminary gapped and trace back stages Normally set based upon expect value ungapped all flag N A Perform ungapped alignment window_size dc megablast integer 40 Multiple hits window size use 0 to specify 1 hit algorithm Table C3 blastp application options The blastp application searches a protein sequence against protein subject sequences or a protein database An option of type flag takes no arguments but if present the argument is true Two different tasks are supported 1 blastp for standard protein protein comparisons 2 blastp short optimized for query sequences shorter than 30 residues This table reflects the 2 2 27 BLAST release option task type default value description and notes w
56. ord_size blastp integer 3 Word size of initial match word_size blastp short integer 2 Word size of initial match gapopen blastp integer 11 Cost to open a gap gapextend blastp integer 1 Cost to extend a gap gapopen blastp short integer 9 Cost to open a gap gapextend blastp short integer 1 Cost to extend a gap matrix blastp string BLOSUM62 Scoring matrix name matrix blastp short string PAM30 Scoring matrix name threshold blastp integer 11 Minimum score to add a word to the BLAST lookup table threshold blastp short integer 16 Minimum score to add a word to the BLAST lookup table comp_based_stats blastp string 2 Use composition based statistics D or d default equivalent to 2 0 or F or f no composition based statistics 1 Composition based statistics as in NAR 29 2994 3005 2001 2 or T or t Composition based score adjustment as in Bioinformatics 21 902 911 2005 conditioned on sequence properties 3 Composition based score adjustment as in Bioinformatics 21 902 911 2005 unconditionally comp_based_stats blastp short string 0 Use composition based statistics D or d default equivalent to 2 0 or F or f no composition based statistics 1 Composition based statistics as in NAR 29 2994 3005 2001 2 or T or t Composition based score adjustment as in Bioinformatics 21 902 911 2005 conditioned on sequence properties 3 Composition based score adjustment as in Bioinformatics 21 902 911 2005 unconditio
57. ow this value for use in DELTA BLAST searches exclude_invalid real true Exclude domains that do not pass validation test for use in DELTA BLAST searches logfile string none Program log file default is stderr Table C10 Blastdbcmd application options This application reads a BLAST database and produces reports option type default value description and notes db string nr BLAST database name dbtype string guess Molecule type stored in BLAST database one of nucl prot or guess entry string none Comma delimited search string s of sequence identifiers e g 555 AC147927 gnl dbname tag or all to select all sequences in the database entry_batch string none Input file for batch processing Format one entry per line pig integer none PIG protein identity group to retrieve info flag N A Print BLAST database information range string none Range of sequence to extract Format start stop strand string plus Strand of nucleotide sequence to extract Choice of plus or minus mask_sequence_with string none Produce lower case masked FASTA using the algorithm IDs specified BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E djeH LSV1d Page 37 out string stdout Output file name outfmt string f Output format where the available format specifiers are f means sequence in FASTA format s means sequ
58. pecified ws_hint integer 28 This is an optimization hint for makembindex that indicates an expected minimum match size in searches that use the index Ifn is the value of nmer parameter and s is the value of stride parameter then the value of ws_hint must be at least n s 1 stride integer 5 makembindex will index every stride th N mer of the database volsize integer 1536 Target index volume size in megabytes BLAST Command Line Applications User Manual
59. pply to the BLAST database as hard mask i e sequence is masked for all phases of search xdrop_gap_final real 25 Heuristic value in bits for final gapped alignment window_size integer 40 Multiple hits window size use 0 to specify 1 hit algorithm db_gen_code integer 1 Genetic code to translate subject sequences see ftp ftp ncbi nih gov entrez misc data ge prt max_intron_length integer 0 Length of the largest intron allowed in a translated nucleotide sequence when linking multiple distinct alignments a negative value disables linking comp_based_stats string 2 Use composition based statistics for tblastn D or d default equivalent to 2 0 or F or f no composition based statistics 1 Composition based statistics as in NAR 29 2994 3005 2001 2 or T or t Composition based score adjustment as in Bioinformatics 21 902 911 2005 conditioned on sequence properties 3 Composition based score adjustment as in Bioinformatics 21 902 911 2005 unconditionally Default 2 Table C6 tblastx application options The tblastx application searches a translated nucleotide query against translated nucleotide subject sequences or a translated nucleotide database An option of type flag takes no arguments but if present the argument is true This table reflects the 2 2 27 BLAST release Only ungapped searches are supported for tblastx option type default v
60. r a nucleotide mismatch reward blastn short integer 1 Reward for a nucleotide match penalty blastn short integer 3 Penalty for a nucleotide mismatch strand all string both Query strand s to search against database subject Choice of both minus or plus dust all string 20641 Filter query sequence with dust filtering_db all string none Mask query using the sequences in this database window_masker_taxid all integer none Enable WindowMasker filtering using a Taxonomic ID window_masker_db all string none Enable WindowMasker filtering using this file soft_masking all boolean true Apply filtering locations as soft masks i e only for finding initial matches Icase_masking all flag N A Use lower case filtering in query and subject sequence s db_soft_mask all integer none Filtering algorithm ID to apply to the BLAST database as soft mask i e only for finding initial matches db_hard_mask all integer none Filtering algorithm ID to apply to the BLAST database as hard mask i e sequence is masked for all phases of search perc_identity all integer 0 Percent identity cutoff template_type dc megablast string coding Discontiguous MegaBLAST template type Allowed values are coding optimal and coding _and_optimal template_length dc megablast integer 18 Discontiguous MegaBLAST template length use_index megablast boolean false Use MegaBLAST database index Indices may be created with the makembindex applicati
61. rogram Task Name Description blastp blastp Traditional BLASTP to compare a protein query to a protein database blastp short BLASTP optimized for queries shorter than 30 residues blastn blastn Traditional BLASTN requiring an exact match of 11 blastn short BLASTN program optimized for sequences shorter than 50 bases megablast Traditional megablast used to find very similar e g intraspecies or closely related species sequences dc megablast Discontiguous megablast used to find more distant e g interspecies sequences BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E dieH LSV1d Page 5 4 2 2 Megablast indexed searches Indexing provides an alternative way to search for initial matches in nucleotide nucleotide searches blastn and megablast by pre indexing the N mer locations in a special data structure called a database index Using an index can improve search times significantly under certain conditions It is most beneficial when the queries are much shorter than the database and works best for queries under 1 Mbases long The advantage comes from the fact that the whole database does not have to be scanned during the search Indices can capture masking information thereby enabling search against databases masked for repeats low complexity etc There are however limitations to using indexed search in blast Index files are about four times larger than the blast da
62. s 1 Composition based statistics as in NAR 29 2994 3005 2001 2 or T or t Composition based score adjustment as in Bioinformatics 21 902 911 2005 conditioned on sequence properties 3 Composition based score adjustment as in Bioinformatics 21 902 911 2005 unconditionally Default 2 Table C5 tblastn application options The tblastn application searches a protein query against nucleotide subject sequences or a nucleotide database translated at search time option type default value description and notes word_size integer 3 Word size for initial match BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E djeH LSV1d Page 34 gapopen integer 11 Cost to open a gap gapextend integer 1 Cost to extend a gap matrix string BLOSUM62 Scoring matrix name threshold integer 13 Minimum score to add a word to the BLAST lookup table seg string 12 2 22 5 Filter query sequence with SEG Format yes window locut hicut or no to disable soft_masking boolean false Apply filtering locations as soft masks i e only for finding initial matches Icase_masking flag N A Use lower case filtering in query and subject sequence s db_soft_mask integer none Filtering algorithm ID to apply to the BLAST database as soft mask i e only for finding initial matches db_hard_mask integer none Filtering algorithm ID to a
63. s msvc800_prj static build build the CONFIGURE project click on Reload when prompted by the development environment and then build the BUILD ALL project The compiled executables will be found in the directory corresponding to the build configuration selected e g c compilers msvc800_prj static bin debugdll Information on using and compiling the NCBI C toolkit is available at http www ncbi nlm nih gov books NBK7160 Please send questions about compiling the NCBI C toolkit to toolbox ncbi nlm nih gov 3 Quick start A BLAST search against a database requires at least a query and db option The command blastn db nt query nt fsa out results out will run a search of nt fsa a nucleotide sequence in FASTA format against the nt database printing results to the file results out If out results out had been left off the results would have been printed to stdout i e the screen The blastn application searches a nucleotide query against a nucleotide database The BLAST applications print documentation when invoked with the h or help option The h option provides abbreviated help and the help flag provides more extensive documentation The BLAST databases are required to run BLAST locally and to support automatic resolution of sequence identifiers Documentation about these identifiers can be found at http www ncbi nlm nih gov books NBK7183 table ch_demo T5 The databases may be retrieved a
64. t_mask tab We also activated the multi thread feature of blastn to speed up the search by using 4 CPUs num_threads 4 This is a genomic fragment containing the HTT gene from human including 5 kb up and down stream of the transcribed region It is represented by NG_009378 The number to use under in your run will depend on the number of CPUs your system has In a test run under a 64 bits Linux machine the above search takes 9 828 seconds real time while the same run without database soft masking invoked takes 31 minutes 44 651 seconds 5 4 Extract all human sequences from the nr database Although one cannot select GIs by taxonomy from a database a combination of unix command line tools will accomplish this blastdbcmd db nr entry all outfmt Sg ST awk if 2 9606 print 1 blastdbcmd db nr entry_ batch out human_sequences txt The first blastdbcmd invocation produces 2 entries per sequence GI and taxonomy ID the awk command selects from the output of that command those sequences which have a taxonomy ID of 9606 human and prints its GIs and finally the second blastdbcmd invocation uses those GIs to print the sequence data for the human sequences in the nr database BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E dieH LSV1d Page 18 5 5 Custom data extraction and formatting from a BLAST database The following examples show how to extract selected in
65. tabases If an index does not fit into computer operating memory then the advantage of using it is eliminated e Word size must be set to 16 or more in order to use an indexed search e Discontiguous search is not supported Reference Morgulis A Coulouris G Raytselis Y Madden TL Agarwala R Schaffer AA Database Indexing for Production MegaBLAST Searches Bioinformatics 2008 24 16 1757 64 PMID 18567917 4 2 3 BLAST search strategies BLAST search strategies are files that encode the inputs necessary to perform a BLAST search The purpose of these files is to be able to seamlessly reproduce a BLAST search in various environments Web BLAST command line applications etc 4 2 5 1 Exporting search strategies on the Web BLAST Click on download next to the RID saved strategy in the Recent Results or Saved Strategies tabs 4 2 5 2 Exporting search strategies with BLAST applications Add the export_search_ strategy along with a file name to the command line options 4 2 5 3 Importing search strategies on Web BLAST Go to the Saved Strategies tab click on Browse to select your search strategy file then click on View to load it into the submission page 4 2 5 4 Importing search strategies with BLAST applications Add the import_search_strategy along with a file name containing the search strategy file Note that if provided the query db use_index and index_name command line options will override the specifi
66. tion already added For example we can apply the dust masking generated in step 5 2 1 1 to the database generated in step 5 2 2 1 we can use this command line makeblastdb in hs_chr input_type blastdb dbtype nucl parse_seqids mask_ data hs_ chr dust asnb out hs_ chr title Human Chromosome Ref B37 1 Here we use the existing database as input file in hs_chr specify its input and molecule type input_type blastdb dbtype nucl enable parsing of sequence ids parse_seqids provide the masking data from step 5 2 1 1 mask_data hs_chr_dust asnb naming the database with the same based name out hs_chr overwriting the existing one Checking the re generated database with blastdbcmd BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1gE dieH LSV1d Page 15 blastdbcmd db hs_chr info we can see that both sets of masking information are available Database Human Chromosome Ref B37 1 24 sequences 3 095 677 412 total bases Date Aug 25 2009 4 43 PM Longest sequence 249 250 621 bases Available filtering algorithms applied to database sequences Algorithm ID Algorithm name Algorithm options 11 dust window 64 level 20 linker 1 30 windowmasker Volumes net gizmo4 export home tao blast_test hs_chr A more straightforward approach to apply multiple sets of masking information in a single makeblastdb run by providing multiple set of masking data files in a comma delimited l
67. utomatically with the update_blastdb pl PERL script which is included as part of this distribution This script will download multiple tar files for each BLAST database volume if necessary without having to designate each volume For example update_blastdb pl htgs will download all the relevant HTGs tar files htgs 00 tar gz htgs N tar gz BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1G dieH LSV1E djeH LSV1d Page 4 The script can also compare your local copy of the database tar file s and only download tar files if the date stamp has changed reflecting a newer version of the database This will allow the script run on a schedule and only download tar files when needed Documentation for the update_blastdb pl script can be obtained by running the script without any arguments perl is required RPS BLAST ready databases are available at ftp ftp ncbi nih gov pub mmdb cdd 4 User manual 4 1 Functionality offered by BLAST applications The functionality offered by the BLAST applications has been organized by program type as to more closely resemble Web BLAST As an example to run a search of a nucleotide query translated on the fly by BLAST against a protein database one would use the blastx application The blastx application will also work in Blast2Sequences mode i e accept FASTA sequences instead of a BLAST database as targets and can also send BLAST searches over the networ
68. very similar sequences e g sequencing errors 2 dc megablast typically used for inter species comparisons 3 blastn the traditional program used for inter species comparisons 4 blastn short optimized for sequences less than 30 nucleotides option task s type default value description and notes word_size megablast integer 28 Length of initial exact match word_size dc megablast integer 11 Number of matching nucleotides in initial match dc megablast allows non consecutive letters to match word size blastn integer 11 Length of initial exact match BLAST Command Line Applications User Manual dieH LSV1d dieH LSV1E dieH LSV1E dieH LSV1 E Page 31 word_size blastn short integer 7 Length of initial exact match gapopen megablast integer 0 Cost to open a gap gapextend megablast integer none Cost to extend a gap This default is a function of reward penalty value gapopen blastn blastn short dc megablast integer 5 Cost to open a gap gapextend blastn blastn short dc megablast integer 2 Cost to extend a gap reward megablast integer 1 Reward for a nucleotide match penalty megablast integer 2 Penalty for a nucleotide mismatch reward blastn dc megablast integer 2 Reward for a nucleotide match penalty blastn dc megablast integer 3 Penalty fo

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