BLAST Command Line Applications User Manual
Contents
1. Page 10 4 5 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 as well as the cookbook 4 5 10 Improved software installation packages The BLAST applications are available 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 5 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 and windowmasker applications provide similar functionality for nucleotide sequences 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 5 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 extends2. 4 6 10 13 list Display BLAST databases available in the directory provided as an argument to this option 4 6 10 14 list_outfmt Allows for the specification of the output format for the list option a listing of the possible format types is available via the application s help option Unsupported output specifiers will be ignored This option s argument should be specified using double quotes if there are spaces in the output format specification 4 6 10 15 recursive Recursively traverse the directory provided to the list option to find and display available BLAST databases 4 6 10 16 show_blastdb_search_path Displays the default BLAST database search paths separated by colons 4 6 11 convert2blastmask This application extracts the lower case masks from its FASTA input and converts them to a file format suitable for specifying masking information to makeblastdb The following are its supported options 4 6 11 1 masking_algorithm The name of the masking algorithm used to create the masks e g dust seg windowmasker repeat 4 6 11 2 masking_options The options used to configure the masking algorithm 4 6 11 3 in Name of the input file by default is standard input 4 6 11 4 output Name of the output file by default is standard output 4 6 11 5 outfmt Output file format 4 6 11 6 parse_seqids Identical to makeblastdb 4 6 12 blastdbcheck This application performs tests on BLAST databases to check their integr
3. 0 47 0 Strand Plus Plus Query 1 ACGTCCGAGACGCGAGCAGCGAGCAGCAGAGCGACGAGCAGCGACGA 47 PEPE AA TEEPE PEEP EEE EEE EEE EE EEE EEE E EEE E EET E Sbjct 47 ACGTCCGGGACGCGAGCAGCGAGCAGCAGAGCGACGAGCAGCGACGA 93 Score 80 5 bits 43 Expect 3e 21 Identities 46 47 97 Gaps 1 47 2 Strand Plus Plus Query 1 ACGTCCGAGACGCGAGCAGCGAGCAGCAGAGCGACGAGCAGCGACGA 47 PEPE EE TEEPE PEEP E EEE EE EEE EEE EEE EE EEE EET Er 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 PEPPER OPE eA e E e PEL EEE PELE EEE ELE EEE E EE LETT 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 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
4. 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 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 27 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 r
5. 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 4 4 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 4 5 Improvements over C Toolkit BLAST command line applications 4 5 1 Query splitting This new feature in the BLAST applications provides substantial performance improvements particularly for blastx searches and it is automatically enabled by the software when deemed appropriate Below is a graph comparing the runtime of blastall and blastx when searching different size excerpts of NC_007113 varying from 10 kbases to about 10 Mbases against the human genome database experiments performed in July 2008 BLAST Command Line Applications User Manual dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d dieH 1SV1 d Page 8 BLASTX performance search human proteins 45000 40000 35000 3000
6. BLAST database format we need to use segmasker to generate the mask information file 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 We can generate the masking information with dustmasker using a single command line dustmasker in hs chr infmt blastdb parse_ segids outfmt maskinfo_asnl_bin out hs_chr_dust asnb Here we specify the input is a BLAST database named hs _ chr in hs_chr infimt 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 To generate the masking information using windowmasker from the BLAST database hs_chr we first need to generate a counts file BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 19 windowmasker in hs_chr infmt blastdb mk_counts parse seqids out hs_ chr mask counts Here we specify the input BLAST databas
7. 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 files experimental Current working directory The following is an example with comments describing the available parameters for configuration Start the BLAST Specifies section for BLAST configuration the path where BLAST databases are installed BLASTDB home guest blast db Specifies the data sources to use for automatic resolution for sequence identifiers DATA LOADERS blastdb Specifies BLASTDB_PROT_DATA_LOADER custom_protein_ database Specifies BLASTDB NUCL DATA LOADER home some_user my nucleotide db Windowmasker settings experimental WINDOW MAS ER WINDOW_MASKER_PATH home guest blast db windowmasker end of fil 4 7 1 Memory usage e the BLAST database to use resolve protein sequences the BLAST database to use resolve protein sequences 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 5 Cookbook 5 1 Query a BLAST d
8. 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_edge 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 4 5 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 se
9. g dieH 1SV1 d dieH 1SV1 d Page 21 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 information 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 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 type 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 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
10. hs_chr mfa dbtype nucl parse_seqids 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 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 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 23 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 makeblastdb in hs_
11. is the file name of the BLAST database usually provided as the db argument to other BLAST applications the second column represents the molecule BLAST Command Line Applications User Manual Page 30 type of the BLAST database This output is configurable via the list_outfmt command line option BLAST Command Line Applications User Manual
12. 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 list makeblastdb in hs_chr dbtype nucl parse seqids mask_ data hs_chr dust asnb hs_ chr mask asnb out hs_chr We can use the masking data file generated in step 5 2 1 3 to create a protein BLAST database makeblastdb in refseq protein dbtype prot parse_seqids mask data refseq _seg asnb out refseq protein title RefSeq Protein Database BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 22 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 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 We use the following command line which is very similar to that given in 5 2 2 1 makeblastdb in
13. output specifiers is available via the help command line option Unsupported output specifiers will be ignored This should be specified using double quotes if there are spaces in the output format specification e g outfmt 7 sseqid ssac qstart qend sstart send qseq evalue bitscore 4 2 27 parse_deflines Parse the query and subject deflines 4 2 28 query Name of the file containing the query sequence s or if these are provided on standard input 4 2 29 query_loc Location of the first query sequence to search in 1 based offsets Format start stop 4 2 30 remote Instructs the application to submit the search to NCBI for remote execution 4 2 31 searchsp Effective length of the search space 4 2 32 seg Arguments to SEG filtering algorithm use no to disable 4 2 33 show_gis Show NCBI GIs in deflines in the BLAST output 4 2 34 soft_masking Apply filtering locations as soft masks i e only when finding alignment seeds 4 2 35 subject Name of the file containing the subject sequence s to search 4 2 36 subject_loc Location of the first subject sequence to search in 1 based offsets Format start stop 4 2 37 strand Strand s of the query sequence to search 4 2 38 threshold Minimum word score such that the word is added to the BLAST lookup table 4 2 39 ungapped Perform ungapped alignments only 4 2 40 version Displays the application s version 4 2 41 window_size Size of the window fo
14. stdout by default 4 6 8 9 taxid Taxonomy ID to assign to all sequences 4 6 8 10 taxid_map Name of file which provides a mapping of sequence IDs to taxonomy IDs 4 6 9 blastdb_aliastool This application replaces part of the functionality offered by formatdb When formatting a large input FASTA sequence file into a BLAST database makeblastdb breaks up the resulting database into optimal sized volumes and links the volumes into a large virtual database through an automatically created BLAST database alias file We can use BLASTdatabase alias files under different scenarios to manage the collection of BLAST databases and facilitate BLAST searches For example we can create an alias file to combine an existing BLAST database with newly generated ones while leaving the original one undisturbed Also for an existing BLAST database we can create a BLAST database alias file based on a GI list so we can search a subset of it eliminating the need of creating a new database For examples of how to use this application please see the cookbook section This application supports three modes of operation 1 Gi file conversion Converts a text file containing GIs one per line to a more efficient binary format This can be provided as an argument to the gilist option of the BLAST search command line binaries or to the gilist option of BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d P
15. 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 For more details refer to the section titled Backwards compatibility script 3 2 For users of Web BLAST http blast ncbi nIm nih gov Users of Web BLAST can take advantage of the search strategies to quickly get started using the BLAST applications as these intend to allow seamless integration between the Web and command line BLAST tools For more details refer to the section on BLAST search strategies 3 3 For new users of BLAST 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 Nonetheless new users will benefit from the examples in the cookbook as well as reading the user manual 3 4 Downloading BLAST databases The BLAST databases are required to run BLAST locally and to support automatic resolution of sequence identifiers Documentation about these can be found ftp ftp ncbi nlm nih gov blast db README These databases may be retrieved automatically 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 vo
16. 0 25000 20000 e blastx m blastall 15000 10000 5000 0 2000 4000 6000 8000 10000 12000 Kbases of NC_007113 4 5 2 Tasks The concept of tasks has been added to support the notion of commonly performed tasks via the task command line option in blastn and blastp The following tasks are currently available Program 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 4 5 3 Megablast indexed searches Indexed searches for megablast are available and are faster than regular megablast The application to generate the database indices is called makembindex which is included in this distribution More information about it can be found at ftp ftp ncbi nlm nih gov pub agarwala indexed_megablast README usage 4 5 4 Partial sequence retrieval from BLAST databases Improvements to the BLAST database reading module allow it to fetch only the relevant portions of the subject sequence that are needed in the gap
17. AE000111 AE000174 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 5587 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 Note also that BTOP always shows the query on the plus strand whereas the CIGAR string always has the subject on the plus strand 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 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 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 26 Subject Length 142 Score 82 4 bits 44 Expect 9e 22 Identities 46 47 97 Gaps
18. ERGPGAPHAPAPGMRHDERGPPPAP PFR GPPPGLGRPT PDWERTRAGEYGPPS LHDGAEGRNAGGSASKSRRGPKAKD GSP WSAKGGVAAPGKNGKAST PFGTGVGAPVAAAGVGGGVGSKKGAAISLRPQ PLA ARAPSSRMVDEDYDEGAADALMGLAGAASAS SASVATAAPAPVSPVATSD PED FEHERGPPPPHOAGDLRYDSYSDGRDG ELEAAPAPPSPVPSSAGKKGRKTTSSRA EDOPDSRPGSPOSRRDAS PASSDGSNE RASSAEKRAESSLGKRPYAEEERAVDE SYKRAKSGSAAE IEADATSGGRLNGVSVSAKPEATAAEGTEQPKETRTETPPLAVAQATS PEAINGKAESESAVOPM 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 nabl000 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 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
19. KNGK ASTPFGTGVGAPVAAAGVGGGVGSKKGAAI SLRPOQEDQPDSRPGS POSRRDAS PASS DGSNEPLAARAPSSRMVD EDY DEGAADALMGLAGAASAS SASVATAAPAPVS PVATS DRAS SAEKRAESSLGKRPYAEEERAVDEPEDSYKRA KSGSAAE IEADATSGGRLNGVSVSAKPEATAAEGTEQPKETRTETPPLAVAQATS PEAINGKAESESAVOQPMDVD GREPSKAPSESATAMKDS PSTANPVVAAKASEPS PTAAP PATSMATSEAQPAKADSCEKNNNDEDEREEEEGQIH EDPIDAPAKRADEDGAK BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 25 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 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 AE000111 0 0 al 10596 if 10596
20. age 14 this program to create an alias file fora BLAST database see below 2 Alias file creation restricting with GI List Creates an alias for a BLAST database and a GI list which restricts this database This is useful if one often searches a subset of a database e g based on organism or a curated list The alias file makes the search appear as if one were searching a regular BLAST database rather than the subset of one 3 Alias file creation aggregating BLAST databases Creates an alias for multiple BLAST databases All databases must be of the same molecule type no validation is done The relevant options are dblist and num_volumes 4 6 9 1 gi_file_in Text file to convert should contain one GI per line 4 6 9 2 gi_file_out File name of converted GI file 4 6 9 3 title Title for BLAST database 4 6 9 4 gilist Name of the file containing the GIs to restrict the database provided in db 4 6 9 5 out Identical to makeblastdb 4 6 9 6 logfile Identical to makeblastdb Please note that when using GI lists the expect values in the BLAST results are based upon the sequences actually searched and not on the underlying database 4 6 10 blastdbcmd This application is the successor to fastacmd The following are its supported options 4 6 10 1 entry A comma delimited search string of sequence identifiers or the keyword all to select all sequences in the database 4 6 10 2 entry_batch Input file for batch pro
21. 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 1786181 asn blast_formatter archive out 1786181l asn outfmt 7 qacc sacc evalue 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 AE000111 AE000111 0 0 I 10596 al 10596 AE000111 AE000174 8e 30 5565 5671 6928 6821 AE000111 AE000394 le 27 5587 5671 135 219 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 28 AE000 AE000425 6e 26 5587 567 8552 8468 AE000 AE000171 3e 24 5587 567 2214 2130 AE000 AE000171 le 23 5587 5670 0559 10642 AEO00 AE000376 le 22 5587 5675 29 42 AE000 AE000268 le 22 5587 567 6174 6090 AE000 AE000112 le 22 10539 10596 58 AE000 AE000447 5e 22 5587 5670 681 598 AE000 AE000344 6e 21 5587 567 4112 4196 AEO00 AE000490 2e 20 5584 567 4921 4835 AEO00 AE000280 2e 20 5587 5670 2930 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 da
22. arch can be configured via the BLASTDB_ PROT DATA LOADER and BLASTDB_NUCL_DATA LOADER configuration options see the section on Configuring BLAST BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 11 4 6 Options by program type 4 6 1 blastp 4 6 2 blastn 4 6 3 blastx 4 6 1 1 task Specify the task to execute For more details refer to the section on tasks 4 6 1 2 comp_based_stats Select the appropriate composition based statistics mode applicable only to blastp and tblastn Available choices and references are available by invoking the application with help option 4 6 1 3 use_sw_tback Instead of using the X dropoff gapped alignment algorithm use Smith Waterman to compute locally optimal alignments 4 6 2 1 task Specify the task to execute For more details refer to the section on tasks 4 6 2 2 penalty Penalty for a nucleotide mismatch 4 6 2 3 reward Reward for a nucleotide match 4 6 2 4 use_index Use a megablast database index 4 6 2 5 index_name Name of the megablast database index 4 6 2 6 perc_identity Minimum percent identity of matches to report 4 6 2 7 dust Arguments to DUST filtering algorithm use no to disable 4 6 2 8 filtering _db Name of BLAST database containing filtering elements i e repeats 4 6 2 9 window_masker_taxid Enable WindowMasker filtering using a Taxonomic ID experimental 4 6 2 10 window_masker_db Enable Wi
23. ask 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 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 24 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 blastdbemd db nr entry all outfmt g 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 5 5 Custom data extraction and formatting from a BLAST database The following examples show how to extract selected info
24. atabase 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 tee exclude_me 1786181 Run the restricted database search head 1 which shows there are no self hits blastn db ecoli negative gilist exclude_me show_gis num alignments 0 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 18 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 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
25. c on Unix like platforms or ncbi ini on Windows This is a plain text file which contains sections and key value pairs to specify configuration parameters Lines starting with a semi colon are considered comments This file will be searched 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 apply The following are the possible configuration parameters that impact the BLAST applications BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 17 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 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 BLASTDB_NUCL_DATA_LOADER Locally available BLAST
26. cessing entries must be provided one per line If input is provided on standard input a should be used to indicate this 4 6 10 3 pig PIG Protein Identity Group to retrieve 4 6 10 4 info Print BLAST database information overrides all other options 4 6 10 5 range Selects the range of a sequence to extract in 1 based offsets Format start stop 4 6 10 6 strand Strand of nucleotide sequence to extract BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 15 4 6 10 7 outfmt Output format string For a list of available format specifiers invoke the application with its help option Note that for all format specifiers except of each line of output will correspond to a single sequence This should be specified using double quotes if there are spaces in the output format specification e g outfmt g t 4 6 10 8 target_only The definition line of the sequence should contain target GI only 4 6 10 9 get_dups Retrieve duplicate accessions 4 6 10 10 line_length Line length for output applicable only with FASTA output format 4 6 10 11 ctrl_a Use Ctrl A as the non redundant defline separator applicable only with FASTA output format 4 6 10 12 mask_sequence_with Allows the specification of a filtering algorithm ID from the BLAST database to apply to the sequence data Applicable only with FASTA and sequence data output formats f and s respectively
27. 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_megablast_m
28. dbtype Molecule type stored or to store in a BLAST database BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 5 4 2 6 db_soft_mask Filtering algorithm ID to apply to the database as soft masking for subject sequences The algorithm IDs for a given BLAST database can be obtained by invoking blastdbcmd with its info flag only shown if such filtering in the BLAST database is available For more details see the section Masking in BLAST databases 4 2 7 culling limit Ensures that more than the specified number of HSPs are not aligned to the same part of the query This option was designed for searches with a lot of repetitive matches but if possible it is probably more efficient to mask the query to remove the repetitive sequences 4 2 8 entrez_query Restrict the search of the BLAST database to the results of the Entrez query provided 4 2 9 evalue Expectation value threshold for saving hits 4 2 10 export_search_strategy Name of the file where to save the search strategy see section titled BLAST search strategies 4 2 11 gapextend Cost to extend a gap 4 2 12 gapopen Cost to open a gap 4 2 13 gilist File containing a list of GIs to restrict the BLAST database to search The expect values in the BLAST results are based upon the sequences actually searched and not on the underlying database 4 2 14 h Displays the application s brief documentation 4 2 15 help Disp
29. dieH LSV1 d dieH 1SV1 d dieH 1SV1 d dieH 1SV1 d WV zZ w L BLAST Help azie BLAST Command Line Applications User Manual Christiam Camacho NCBI camacho ncbi nlm nih gov Thomas Madden NCBI madden ncbi nlm nih gov George Coulouris NCBI coulouri ncbi nlm nih gov Vahram Avagyan NCBI avagyanv ncbi nlm nih gov Ning Ma NCBI maning ncbi nlm nih gov Tao Tao NCBI tao ncbi nlm nih gov Richa Agarwala NCBI richa ncbi nlm nih gov 1 Introduction This manual documents the BLAST Basic Local Alignment Search Tool command line applications developed at the National Center for Biotechnology Information NCBI These applications have been revamped to provide an improved user interface new features and performance improvements compared to its counterparts in the NCBI C Toolkit Hereafter we shall distinguish the C Toolkit BLAST command line applications from these command line applications by referring to the latter as the BLAST applications which have been developed using the NCBI C Toolkit http www ncbi nlm nih gov books bv fegi rid toolkit TOC amp depth 2 Please feel free to contact us with any questions feedback or bug reports at blast help ncbi nlm nih gov 2 Installation The BLAST applications are distributed in executable and source code format For the executable formats we provide installers as well as tarballs the source code is only provided as a tarball These are free
30. e 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 blastdb and the output of step 1 ustat hs_chr_mask counts We set the mask file format to binary asn 1 outfmt 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 We can generate the masking information with segmasker using a single command line segmasker in refseq protein infmt blastdb parse_seqids outfmt maskinfo_asnl_ bin out refseq_seg asnb Here we specify the refseq_protein BLAST database in refseq_protein infmt blastdb enable sequence
31. e to use as source the data type is automatically detected Note that multiple input files BLAST databases can be provided each must be separated by white space in a string quoted with single quotation marks Multiple input files BLAST databases which contain white space in them should be quoted with double quotation marks inside the white space separated single quoted string e g in C My Documents seqs fsa E Users Joe Smith myfasta fsa 4 6 8 2 title Title for the BLAST database to create 4 6 8 3 parse_seqids Parse the Seq id s in the FASTA input provided Please note that this option should be provided consistently among the various applications involved in creating BLAST databases For instance the filtering applications as well as convert2blastmask should use this option if makeblastdb uses it also 4 6 8 4 hash_index Enables the creation of sequence hash values These hash values can then be used to quickly determine if a given sequence data exists in this BLAST database 4 6 8 5 mask_data Comma separated list of input files containing masking data to apply to the sequences being added to the BLAST database being created For more information see Masking in BLAST databases and the examples 4 6 8 6 out Name of the BLAST database to create 4 6 8 7 max_file_sz Maximum file size for any of the BLAST database files created 4 6 8 8 logfile Name of the file to which the program log should be redirected
32. ect your search strategy file then click on View to load it into the submission page 4 5 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 specifications of the search strategy file provided no other command line options will override the contents of the search strategy file 4 5 6 Negative GI lists Negative GI lists are available on search applications and they provide a means to exclude GIs from a BLAST database search The expect values in the BLAST results are based upon the sequences actually searched and not on the underlying database For an example see the cookbook 4 5 7 Masking in BLAST databases It is now possible to create BLAST databases which 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 5 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 the common options as well as the cookbook BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d
33. elevant 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 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 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
34. ids parsing parse_seqids request the mask data in binary asn 1 format outfmt 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 We can also extract the masking information from a FASTA sequence 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 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 20 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 manne
35. ity The following are its supported options BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 16 4 6 12 1 dir Name of the directory where to look for BLAST databases 4 6 12 2 recursive Flag to specify whether to recursively search for BLAST databases in the directory specified above 4 6 12 3 full Check every database sequence 4 6 12 4 stride Check every Nth database sequence 4 6 12 5 samples Check a randomly selected set of N sequences 4 6 12 6 ends Check the beginning and ending N sequences in the database 4 6 12 7 isam Set to true to perform ISAM file checking on each of the selected sequences 4 6 13 blast_formatter This application formats both local and remote BLAST results An RID is required to format remote BLAST results The RID may be obtained either from a search submitted to the NCBI BLAST web page or by using the remote switch with one of the applications mentioned above The blast formatter accepts the BLAST archive format for stand alone formatting The BLAST archive format can be produced by using outfmt 11 argument with the stand alone applications The following are its supported options 4 6 13 1 rid BLAST RID of the report to be formatted 4 6 13 2 archive File produced by BLAST application using outfmt 11 4 7 Configuring BLAST The BLAST search applications can be configured by means of a configuration file named ncbir
36. l to blastx 4 6 5 4 comp_based_stats Identical to blastp 4 6 5 5 use_sw_tback Identical to blastp 4 6 5 6 in_pssm Checkpoint file to initiate PSI TBLASTN currently unimplemented 4 6 6 1 comp_based_stats Identical to blastp with the exception that only composition based statistics mode 1 is valid when a PSSM is the input either when restarting from a checkpoint file or when performing multiple PSI BLAST iterations 4 6 6 2 gap_trigger Number of bits to trigger gapping 4 6 6 3 use_sw_tback Identical to blastp 4 6 6 4 num_iterations Number of iterations to perform 4 6 6 5 out_pssm Name of the file to store checkpoint file containing a PSSM 4 6 6 6 out_ascii_pssm Name of the file to stire ASCII version of PSSM 4 6 6 7 in_msa Name of the file containing multiple sequence alignment to restart PSI BLAST 4 6 6 8 in_pssm Checkpoint file to re start PSI BLAST 4 6 6 9 pseudocount Pseudo count value used when constructing the PSSM 4 6 6 10 inclusion_ethresh E value inclusion threshold for pairwise alignments to be considered to build the PSSM 4 6 6 11 phi_pattern Name of the file containing a PHI BLAST pattern to search 4 6 7 rpstblastn 4 6 7 1 query_gencode Identical to blastx 4 6 8 makeblastdb This application serves as a replacement for formatdb BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 13 4 6 8 1 in Input file or BLAST database nam
37. lays the application s detailed documentation 4 2 16 html Enables the generation of HTML output suitable for viewing in a web browser 4 2 17 import_search_strategy Name of the file where to read the search strategy to execute see section titled BLAST search strategies 4 2 18 lcase_masking Interpret lowercase letters in query sequence s as masked 4 2 19 matrix Name of the scoring matrix to use 4 2 720 max_target_seqs Maximum number of aligned sequences to keep from the BLAST database 4 2 21 negative_gilist File containing a list of GIs to exclude from the BLAST database 4 2 22 num_alignments Number of alignments to show in the BLAST output 4 2 23 num_descriptions Number of one line descriptions to show in the BLAST output 4 2 24 num_threads Number of threads to use during the search 4 2 25 out Name of the file to write the application s output Defaults to stdout 4 2 26 outfmt Allows for the specification of the search application s output format A listing of the possible format types is available via the search application s help option If a custom output format is desired this can be specified by providing a quoted string composed of the desired output format tabular tabular with comments or comma separated value a space BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 6 anda space delimited list of output specifiers The list of supported
38. lume 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 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 BLAST Command Line Applications User Manual Page 4 4 User manual 4 1 Functionality offered by BLAST applications dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d dieH 1SV1 d The functionality offered by the BLAST applications has been organized by program type as to more closely resemble Web BLAST The following graph depicts a correspondence between the NCBI C Toolkit BLAST command line applications and the BLAST applications makeblastdb blastdb_aliastool 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 instead of blastall 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 network to the public NCBI server if desired The blastn blas
39. ly 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 dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 2 2 1 Windows Download the executable installer ncbi blast 2 2 18 exe and double click on it After accepting the license agreement select the install location and click Install and then Close 2 2 MacOSX For users without administrator privileges Download the ncbi blast 2 2 18 universal macosx tar gz tarball and follow the procedure described in Other Unix platforms 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 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 blas
40. ndowMasker filtering using this repeats database experimental 4 6 2 1 no_greedy Use non greedy dynamic programming extension 4 6 2 12 min_raw_gapped_score Minimum raw gapped score to keep an alignment in the preliminary gapped and traceback stages 4 6 2 13 template_type Discontiguous megablast template type 4 6 2 14 template_length Discontiguous megablast template length 4 6 2 15 off_diagonal_range Maximum number of diagonals separating two hits used to initiate an extension Increasing values of this parameter lead to a longer run time but more sensitive results If this parameter is set a value of five is suggested Only discontiguous megablast uses two hits by default 4 6 3 1 query_gencode Genetic code to use to translate the query sequence s 4 6 3 2 frame_shift_penalty Frame shift penalty for use with out of frame gapped alignments BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d 4 6 4 tblastx 4 6 5 tblasin 4 6 6 psiblast Page 12 4 6 3 3 max_intron_length Length of the largest intron allowed in a translated nucleotide sequence when linking multiple distinct alignments a negative value disables linking 4 6 4 1 db_gencode Genetic code to use to translate database subjects 4 6 4 2 max_intron_length Identical to blastx 4 6 5 1 db_gencode dentical to tblastx 4 6 5 2 frame_shift_penalty Identical to blastx 4 6 5 3 max_intron_length Identica
41. ped alignment stage providing a substantial improvement in runtime The following example compares 103 mouse EST sequences against the human genome shows example run in July 2008 after the database had already been loaded into memory time megablast d 9606 genomic i est 500 in r 2 q 3 F mL m 9 o old out W 11 t 18 G 5 E 2 341 455u 65 242s 6 47 99 99 6 0 0k 0 0io Opf 0w time blastn task dc megablast db 9606 genomic query BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 9 est 500 in outfmt 7 out new out 218 540u 11 632s 3 50 53 99 8 0 0k 0 0io Opf 0Ow Similar gains in performance should be expected in BLAST databases which contain very large sequences and many very short queries 4 5 5 BLAST search strategies BLAST search strategies are files which 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 5 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 5 5 2 Exporting search strategies with BLAST applications Add the export_search_strategy along with a file name to the command line options 4 5 5 3 Importing search strategies on Web BLAST Go to the Saved Strategies tab click on Browse to sel
42. r either use it in both steps or not use it at all 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 makeblastdb in hs_chr dbtype nucl parse_seqids mask_ data hs_ chr mask asnb out hs_ chr 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 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1
43. r multiple hits algorithm use 0 to specify 1 hit algorithm 4 2 42 word_size Word size for word finder algorithm 4 2 43 xdrop_gap X dropoff value in bits for preliminary gapped extensions 4 2 44 xdrop_gap_final X dropoff value in bits for final gapped alignment 4 2 45 xdrop_ungap X dropoff value in bits for ungapped extensions 4 3 Backwards compatibility script 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 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d Page 7 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 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 specifying the C Toolkit
44. rmation 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 Sm XP_761648 1 1292 119 139 140 144 147 152 154 160 161 216 Extract the masked FASTA for GI 71022837 blastdbcemd entry 71022837 db Test mask data db mask_ sequence with 20 40 target_only gt gi 71022837 ref XP_761648 1 hypothetical protein UM05501 1 MPPSARHSAHPSHHPHAGGRDLHHAAGGPPPOGGPGMP PGPGNGPMHHPHSS YAQSMPPPPGLPPHAMNGINGPP PSTHGGPPPRMVMADGPGGAGGPPPPPPPHI PRSSSAQSRIMEAaggpagpppagppastspavqklslaNEaaw vsiGsaaetmedydralsayeaalrhnpysvpalsaiagvhrtldnfekavdyfqrvlnivpengdtWGSMGHCY LMMDDLORAY TAY QQALYHLPNPKEPKLWYGIGILYDRYGSLEHAEEAFASVVRMDPNYEKANEIYFRLGIIYKQ ONKFPASLECFRYILDNPPRPLTEI DIWFOQIGHVYEQOQKEFNAAKEAYERVLAENPNHAKVLOOQLGWLYHLSNAG FNNOERAIQFLTKSLESDPNDAQSWY LLGRAYMAGONY NKAYEAYQOQAVYRDGKNPTFWCSIGVLYYQINOYRDA LDAYSRAIRLNPYISEVWFDLGSLYEACNNOQISDAIHAYERAADLDPDNPQIQOQRLOLLRNAEAKGGELPEAPVP QDVHPTAYANNNGMAPGPPTOQIGGGPGPSY PPPLVGPOQLAGNGGGRGDLSDRDLPGPGHLGSSHSPPPFRGPPGT DDRGARGP PHGALAPMVGGPGGPEPLGRGGFSHSRGPSPGPPRMDPYGRRLGSPPRRS PPPPLRSDVHDGHGAPP HVHGQGHGQGHGOGHGOGHGOGHGOQS HGHSHGGEFRGPPPLAAAGPGGPPPPLDHYGRPMGGPMSEREREMEWER EREREREREQAARGY PASGRITPKNEPGYARSQHGGSNAPS PAFGRPPVYGRDEGRDY YNNSHPGSGPGGPRGGY ERGPGAPHAPAPGMRHDERGPPPAPFEHERGPPPPHOQAGDLRYDSYSDGRDGPFRGPPPGLGRPTPDWERTRAGE YGPPSLHDGAEGRNAGGSASKSRRGPKAKDELEAAPAPPS PVPSSAGKKGRKTTSSRAGS PWSAKGGVAAPG
45. t 2 2 18 1 x86 64 rpm Note one must have root privileges to run these commands If you do not have root privileges please use the procedure described in Other Unix platforms 2 4 Other Unix platforms Download the tarball and expand it in the location of your choice 2 5 Source tarball Use this approach if you would like to build the BLAST applications yourself Download the tarball expand it and in the expanded directory type the following commands od oft configure without debug 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 compilers 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 BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 d dieH 1SV1 d dieH 1SV1 d Page 3 3 Quick start 3 1 For users of NCBI C Toolkit BLAST The easiest way to get started using these command line applications is by means of the legacy_blast pl PERL script which is bundled along with the BLAST applications To utilize this script simply prefix it to the invocation of
46. ta 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 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 MPPSARHSAHPSHHPHAGGRDLHHAAGGPPPOGGPGMPPGPGNGPMHHPHSSYAQSMPPPPGLPPHAMNGINGPPPS THG GPPPRMVMADGPGGAGGPPPPPPPHIPRSSSAQSRIMEAaggpagpppagppastspavQklslANEaawvsIGsaa etm EdydralsayeaalrhnpysvpalsaiagvhrtldnfekavdyfqrvlnivpengdTWGSMGHCYLMMDDLQORAYTA YQQ ALYHLPNPKEPKLWYGIGILYDRYGSLEHAEEAFASVVRMDPNYEKANEIYFRLGIIYKQQNKFPASLECFRYILDN PPR PLTEIDIWFQIGHVYEQQKEFNAAKEAYERVLAENPNHAKVLOQLGWLYHLSNAGFNNQERAIQFLTKSLESDPNDA Qs Y Cc Q P LLGRAYMAGONYNKAYEAYQOAVYRDGKNPTFWCSIGVLYYQINOYRDALDAYSRAIRLNPYISEVWFDLGSLYEA NN SDATHAYERAADLDPDNPOITQORLOLLRNAEAKGGELPEAPVPQDVHPTAYANNNGMAPGPPTOIGGGPGPSYPP LV GPQLAGNGGGRGDLSDRDLPGPGHLGSSHS PPPFRGPPGTDDRGARGP PHGALAPMVGGPGGPEPLGRGGFSHSRGP BLAST Command Line Applications User Manual dieH 1SV1 d dieH 1SV1 g dieH 1SV1 d dieH 1SV1 d SPG Page 29 PPRMDPYGRRLGSPPRRS PPPPLRS DVHDGHGAP PHVHGOGHGOGHGQGHGOGHGOGHGOQSHGHSHGGEFRGPPPLA AAG PGGPPPPLDHYGRPMGGPMSEREREMEWEREREREREREQAARGY PASGRITPKNEPGYARSQHGGSNAPS PAFGRP PVY GRDEGRDY YNNSHPGSGPGGPRGGY
47. tp blastx tblastx tblastn psiblast rpsblast and rpstblastn are considered search applications as they execute a BLAST search whereas makeblastdb blastdb_aliastool 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 Please note that the NCBI C Toolkit applications seedtop and blastclust are not available in this release 4 2 Common options The following is a listing of options that are common to the majority of BLAST applications followed by a brief description of what they do 4 2 1 best_hit_overhang Overhang value for Best Hit algorithm For more details see the section Best Hits filtering algorithm 4 2 2 best_hit_score_edge Score edge value for Best Hit algorithm For more details see the section Best Hits filtering algorithm 4 2 3 db File name of BLAST database to search the query against Unless an absolute path is used the database will be searched relative to the current working directory first then relative to the value specified by the BLASTDB environment variable then relative to the BLASTDB configuration value specified in the configuration file 4 2 4 dbsize Effective length of the database 4 2 5
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