Basic Local Alignment Search Tool
   from Advanced Biocomputing, LLC

Index


Description

AB-BLAST 3.0 is a powerful software package for gene and protein identification, using sensitive, selective and rapid similarity searches of protein and nucleotide sequence databases. The feature list for AB-BLAST is long and continues to expand, while performance is improved. Much of this is outlined below. A complete suite of BLAST search programs (blastp, blastn, blastx, tblastn and tblastx) is provided in the package, along with several database management and support programs that include nrdb, patdb, xdformat, xdget, seg, dust and xnu.

AB-BLAST has been built to be the most trusted database search tool in your software toolbox, doing what you tell it, reporting precisely what it’s doing — even telling you what it could not do because of specific parameter restrictions you might wish to change — and able to handle the biggest jobs with aplomb. Users of other BLAST implementations have suffered every few years through a series of expensive and time-consuming rewrites, alpha releases, beta releases, output format changes, database format changes, specialized spin-off programs, and new program parameters and behaviors that may be important to NCBI operations but not to anyone else. Meanwhile AB-BLAST was built from scratch to offer performance, reliability and flexibility — plus backward compatibility with every AB-BLAST release for over 24 years.

AB-BLAST represents the most rigorous, sensitive implementation of the core BLAST algorithm available, yet it often runs faster than the rest. AB-BLAST has a simple, easy-to-use command line syntax; offers consistent behavior across all search modes; runs on general purpose computer hardware; can uniquely categorize and filter results based on biologically relevant criteria; and much more. All of these features help AB-BLAST users be more productive and save time and money.

AB-BLAST is not a re-hashed version of NCBI-BLAST. AB-BLAST shares virtually no code with NCBI-BLAST except for some portions that both packages copied from the public domain, ungapped BLAST version 1.4 released in 1994 (W. Gish, unpublished). A brief history of AB-BLAST development is available here. The AB-BLAST lineage includes the original gapped BLAST, first released in May 1996. Before that, its author created (and maintained) the "nonredundant" databases and BLAST Network Service but was asked by NCBI management not to publish this work.


Licensing

Please see https://blast.advbiocomp.com/licensing/ for complete licensing information.


Key Features

Some of the key features of AB-BLAST are described below.

To support XDF databases, the database formatting tool named xdformat is provided with AB-BLAST. Among other distinct capabilities and advantages to using XDF and xdformat are:

A reverse chronological list of changes to the AB-BLAST software is available in the file named HISTORY that comes bundled with the software. When possible, any bugs that have been found have typically been fixed within 24 hours of their being reported.

Please send us bug reports, questions, or suggestions.


Licensing

Full information about licensing of AB-BLAST is provided here.



Manifest

The AB-BLAST 3.0 package includes the following data analysis and utility programs:


AB-BLAST Command Line Options and Parameters

A complete list of command line options and parameters for modifying the behavior of the AB-BLAST search programs is available here.


Comparable AB/NCBI BLAST Parameters

A brief comparison of the some of the most important parameters for controlling sensitivity, selectivity and speed of AB-BLAST and NCBI BLAST is available here.


Environment Variables

AB-BLAST can utilize the settings of a few environment variables to adapt its behavior to different computing environments: BLASTDB, BLASTFILTER and BLASTMAT. To allow for triple AB/WU/NCBI BLAST installations, AB-BLAST also supports the environment variables ABBLASTDB, ABBLASTFILTER and ABBLASTMAT, as well as WUBLASTDB, WUBLASTFILTER and WUBLASTMAT. Settings of the AB versions of these variables take precedence over all others, and WU variable settings take precedence over the corresponding base name variables.

In AB-BLAST, the BLASTDB (or ABBLASTDB) environment variable can be a list of one or more directory names in which the programs are to look for database files. In UNIX parlance, such an environment variable might be called a path for the database files. Directory names should be delimited from one another by a colon (“:”) and listed in the order that they should be searched. If the BLASTDB environment variable is not set, the programs use a default path of .:/usr/ncbi/blast/db, such that the programs first look in the current working directory (“.”) for the requested database and then look in the /usr/ncbi/blast/db directory. For backward compatibility with programs that expect BLASTDB to be a single directory specification and not a path, if the user has set a value for BLASTDB but omitted the current working directory, AB-BLAST will still look for database files in the current working directory as a last resort. This usage is unchanged from NCBI/WU BLAST version 1.4 (1994), except multiple directories could be specified with the BLASTDB variable beginning with WU-BLAST 2.0 ca. 1997.

The BLASTFILTER (or ABBLASTFILTER) environment variable can be set to the directory containing the sequence filter programs, such as seg and xnu.

The BLASTMAT (or ABBLASTMAT) environment variable can be set to the parent directory for all scoring matrix files. The default directory location for scoring matrix files is beneath the matrix/ subdirectory of the AB-BLAST software installation. And beneath this directory exist 4 subdirectories to accommodate the 4 combinations of query-subject alphabets the search programs can use. Before looking anywhere else, though, the search programs first look for the requested scoring matrix in the current working directory.

For more information about environment variables, see the Installation instructions.


Filters and Masks

AB-BLAST provides a highly flexible means of applying both “hard” and “soft” masks to a query sequence, of supporting alternative, user-defined filter programs and non-standard parameters to the standard filters. The filter (for hard masking) and wordmask (for soft masking) command line options provide the basic interface. Multiple specifications of each type are acceptable on the BLAST command line and are executed in left-to-right order.

Individual filter and wordmask specifications may consist of pipelines of commands. For example, three filters are used in succession by this pipeline:

      filter="myfilter1 | myfilter2 | myfilter3 -x5 -"

The first two filters in this case expect to read their input from UN*X standard input (also known as stdin), whereas myfilter3 apparently needs to be told explicitly to read data from stdin, using the conventional “-” symbol for stdin. The standard output (stdout) from myfilter1 will be read via stdin by myfilter2, which in turn processes the query before handing its results to myfilter3; finally, myfilter3 reports its results to stdout, which the BLAST program itself reads to obtain the fully masked sequence. The final output from the filter pipeline is expected by the BLAST program to be in FASTA format.

Instead of running all 3 filters in the above example as part of one pipeline, they could instead be specified as three separate filter options like this:

    filter=myfilter1  filter=myfilter2  filter="myfilter3 -x5 -"

The same choice of running as a pipeline or running separately is available for wordmasks, too. Naturally, the two approaches can also be combined on the same command line. An advantage to using the pipeline approach is that all 3 filters in the example above may complete a little bit faster, because some I/O overhead is avoided. Furthermore, when used in a pipeline, there is no requirement that the output from myfilter1 and myfilter2 actually be in FASTA format. Those two programs could potentially pass information between themselves and to myfilter3 in a proprietary format. The only absolute format requirements are that the first filter in the pipeline must read FASTA data from stdin, and the last filter in the pipeline must output FASTA format. The final output from a filter or pipeline must also have the same length as the original input sequence.

It should be noted that with some filter programs, passing the query sequence sequentially through a pipeline of filters may yield a different result than processing the query independently with each filter and OR-ing the results. The script seg+xnu included in the filter/ directory provides an example with which to test this. Specifying filter=seg+xnu on the BLAST command line invokes a seg and xnu pipeline that is built-in to the search programs; whereas specifying filter="seg+xnu -" causes the seg+xnu script to be invoked on the query, which independently executes seg and xnu, then logically “ORs” the results with the bundled pmerge utility program. The built-in seg+xnu pipeline is historically the way these two filters have been invoked together, but the somewhat slower method employed by the seg+xnu script with pmerge may be more desirable.

The echofilter option can be used to display the filtered sequence near the beginning of search program output.


Precomputed Statistical Parameters

Nucleotide Scoring Systems

Precomputed values for λ, K and H are available for BLASTN searches with the following match,mismatch (M,N) scoring systems, using the sets of gap penalties {Q,R}:


Precomputed Nucleotide Scoring Systems
MN{Q,R}
+1−3 {3,3}  {3,2}  {3,1}  {7,2} 
+1−2 {2,2}  {2,1}  {1,1} 
+3−5 {10,5}  {6,3}  {5,5} 
+4−5 {10,5} 
+1−1 {3,1}  {2,1} 
+5−4 {20,10}  {10,10} 
+5−11 {22,22}  {22,11}  {12,2}  {11,11} 


Precomputed values are also available for a Purine-Pyrimidine scoring matrix named “pupy”:

PuPy Matrix
QR
2010
1010


Protein Scoring Systems

Precomputed values for λ, K and H are available for protein-level searches (BLASTP, BLASTX, TBLASTN and TBLASTX) with the following scoring matrix and gap penalty combinations (or gap penalty ranges for R) {Q, R}:


BLOSUM50
QR
161–4
151–4, 6, 8
141–5, 8
131–5, 8
122–5, 7
112–4, 6, 8
102–6, 8
93–5, 7
84–8
76, 7


BLOSUM55
QR
161–4
151–4, 6, 8
141–5, 7
132–5, 8
122–5, 8
112–6, 8
103–6, 9
93–5, 7
84–8
77


BLOSUM62
QR
121–3
111–3
101–4
91–5
82–7
72–6
63–5
55


BLOSUM80
QR
122–12
112–11
102–10
93–9
84–8
75–7


PAM40
QR
121, 2, 6
111, 2, 7
101–3, 7
91–3, 6
81–4
71–4
62–5
52–5
43, 4


PAM120
QR
121, 2, 4
111–3
101–3, 5
91–3, 5
81–4, 6
72–4, 6
62–5
53–5


PAM250
QR
161–4
151–5
141–6
131–6
122–7
112–7
103–8
93–7
85–7
77

Bugs

AB-BLAST is certainly not bug free, but historically bugs have been fixed typically within a day of their being reported. The currently known bugs are:

If you think you might be experiencing the effects of a bug, please contact us.

AB-BLAST exhibits a few different behaviors worth mentioning here, because they could trip up or confuse even the most knowledgeable of BLAST users. Any unexpected behavior might rightfully be construed as being a bug, so the following information is provided here in the Bugs section to help avoid the unexpected. If you should encounter problems or confusing areas other than those described below, or if you have questions or suggestions for improvement, please send them to us.


Supported Platforms

The computing platforms currently supported for AB-BLAST are listed below.

The list of supported platforms is subject to change without notice.
Through multithreading, multiple processors and processor cores are supported by AB-BLAST on all of the above platforms.

Installation

Prior to software installation, make sure you have obtained a license file named license.xml. This file will normally be provided as an attachment to a confidential e-mail from Advanced Biocomputing, LLC. A valid, active license is required to run all of the AB-BLAST search programs and many of the support programs (nrdb, patdb and all of the *2fasta programs). However, an active license will never be required to run the xdget and xdformat programs, to ensure you can always recover your data from AB-BLAST databases even after your license has expired; and to allow you to create searchable databases for AB-BLAST users who do have active licenses.

Proceed to download the UN*X tar archive appropriate for your computing platform from the Advanced Biocomputing, LLC website. A confidential URL to obtain the software will usually be included in the same e-mail that contained the license.xml file attachment. Scoring matrix files and documentation are included in each package. No databases are included, however.

The tar archives unpack into subdirectories named ab-blast-YYYYMMDD-platform-architecture. (This differs from WU-BLAST and earlier versions of AB-BLAST that would unpack in the current working directory.) The version of the AB-BLAST package is indicated by YYYYMMDD. The operating system is indicated by platform (e.g., linux or macos) and architecture describes the hardware architecture (e.g., x64).

Individual users of AB-BLAST must place their license.xml file in the directory ~/.config/ab-blast, where ~ (tilde) is a symbol for a user's home directory. If that directory does not exist, it must first be created. For site licensees only, the license.xml file can be conveniently placed in the same directory as the AB-BLAST software, to enable access for all users of the computer system.

Note that the programs blastp, blastn, blastx, tblastn and tblastx are actually “hard links” to the same executable program, blasta, that encodes the integrated capabilities of all 5 search methods. If desired, the links can be renamed, as long as the original names appear as substrings within the new names. Alphabetic case is unimportant. For instance, a link named ab-blastp will still invoke blasta in its blastp operational mode.

Similarly, the xdformat and xdget programs are hard links to the same program that operates differently depending on the name by which it is invoked.

A Note to Mac OS X Users
None of the programs bundled with AB-BLAST are intended to be double-clicked to execute. AB-BLAST software is intended to be invoked via a CLI (command line interface) instead. On a Mac, programs must be invoked either using a Terminal application (located in the /Applications/Utilities folder) or from within a script or other application.

If you already had AB-BLAST (or WU-BLAST) installed with BLAST-able databases, your installation or update of AB-BLAST is likely complete. If you did not have AB-BLAST or WU-BLAST already installed, read on...

Low-complexity sequence filters or masking programs — e.g., seg, xnu and dust — are included in AB-BLAST distributions. The bundled versions of these programs are precompiled and optimized. While these filter programs are not required for running the search programs, they can enormously reduce the amount of garbage output produced, memory used, and search time consumed.

NOTE: unlike NCBI-BLAST, the AB-BLAST search programs do not employ sequence complexity filtering by default. This behavior might change in the future, though. In case the search programs are updated to a version that does perform complexity filtering by default and you wish to guarantee an automated analysis pipeline will not perform this filtering, you can specify filter=none on the BLAST command line to maintain that behavior.

The databases themselves are not included with the AB-BLAST software. Once the source databases have been downloaded from any of many Internet sites, the database files are typically uncompressed and processed into FASTA format, if they are not in FASTA format already. Included in the tar archives are several utility programs for converting plain text database files into FASTA format:

The NCBI software Toolbox also contains some relevant parsers. One of these is asn2fsa, which converts both nucleotide and peptide sequences in GenBank ASN.1 format into FASTA format files. The asn2ff parser, which converts GenBank ASN.1 data into other flat file formats, may also come in handy, especially if you are inclined to parse GenBank into FASTA using your own routines or use the gb2fasta and gt2fasta programs mentioned above.

All of the above parsers can read from standard input (signified by a hyphen, “-”), so their input files can be maintained on disk in compressed format and streamed uncompressed directly into parsers with zcat, gunzip or other relevant decompression program. Because command line options themselves start with hyphens, if a hyphen is needed to specify standard input for the input file name, some of these programs require that a double-dash (--) be entered on the command line before the single-dash. This double-dash signifies the end of options and the start of the required filename arguments.

Once a source database is in FASTA format, the xdformat program should be used to convert it into “blastable” format. Concise usage instructions for xdformat (and xdget) can be obtained by invoking each program without any command line arguments. By default, xdformat produces 3 output files whose names are derived from the name of the FASTA input file. The 3 output files have distinct file name extensions and together comprise the blastable database. If sequence identifiers are optionally indexed during database creation, the blastable database will consist of a total of 4 output files. Databases formatted by xdformat contain full ambiguity code information within the blastable database files it produces.

By default, if any unrecognized amino acid or nucleotide codes are encountered or if the FASTA input file should otherwise appear corrupt, xdformat will emit an error message and halt. In such cases, if the blastable database was to be newly created, xdformat will remove the blastable database files it was creating before halting. If an existing blast database was being appended with new sequences when the error arose, the blastable database will be rolled back to its original state prior to the attempted update, with none of the new sequences appended.

While formatting a database, the xdformat program can optionally (-I option) index the sequence identifiers for later identifier-based retrieval with the xdget program. XDF databases that were formatted without an identifier index can have an index created post hoc by xdformat with its -X option. It may be of interest to note for the purposes of their maintenance that xdformat and xdget are actually one-and-the-same program file, merely invoked under the two different names to obtain the two different program behaviors. This helps ensure that the index created with xdformat will be compatible with xdget. See the file "FAQ-Indexing.html" for more details on identifier indexing.

For compatibility with legacy BLAST installations, the xdformat program can function in a setdb- and pressdb-compatibility mode, wherein its behavior is similar to that of setdb and pressdb. In its compatibility mode, a similar command line structure is used and the output files produced have the same names as those produced by setdb and pressdb. Compatibility mode is invoked when xdformat is renamed or has links pointing to it named setdb and pressdb. While the files produced in compatibility mode have the same file names as those produced by the original setdb and pressdb programs (setdb.real and pressdb.real), the content of these files is always XDF. Versions of the BLASTA search program dated on or after 1999-12-14 are able to work with the more-capable XDF databases.

Note that two XDF databases — one protein and one nucleotide — can be created with the exact same name and exist in the exact same directory, because the 3-letter file name extensions of XDF databases are completely distinct for protein and nucleotide sequence databases.

If xdformat and the legacy setdb and pressdb programs have all been used to create databases with the same name that reside in the same directory, the BLAST search programs will preferentially search the databases created with xdformat which will have the standard XDF database file name extensions. Note that two XDF databases of the same name — one protein and one nucleotide — can reside in the same directory, because the file name extensions of XDF databases are distinct for protein and nucleotide sequence databases.

Using the -t option to xdformat, a descriptive name or title can be assigned to a database that will appear in BLAST search output. The title of an existing database can be changed after its creation, by appending an empty FASTA database and specifying the -t option with the desired new title. For example,


     xdformat -n -a mydb -t "Fancy New Title" /dev/null

The blastable database files can be placed anywhere, but for convenience the BLASTDB environment variable should include their directory location. If the BLASTDB environment variable is not set, the programs look for databases by default in /usr/ncbi/blast/db and in the current working directory. If the old pressdb program (instead of xdformat) is used to create the blastable database, the associated nucleotide sequence FASTA file must be located in the same directory as the three output files from pressdb, if the BLAST search programs are to find the FASTA file. It may sometimes be useful to maintain the FASTA files in a separate directory — even on another disk partition — and provide UNIX soft links in the BLASTDB directory that point to the real location of the FASTA files. In addition, on systems where NCBI BLAST will not be in use, blastable databases can be maintained in multiple directories listed in the BLASTDB environment variable, with each directory name delimited from the next by a colon (:), just as directory names are often delimited in the PATH environment variable.

On multi-processor computer systems, the search programs will employ as many CPUs as are installed; when more than about 4 CPUs are used, this default behavior cause efficiency of hardware utilization to be quite low, compared to running individual single-threaded BLAST jobs on each CPU. Memory use also increases linearly with the number of CPUs or threads employed. One way to govern the number of processors employed is to wrap the search programs in a shell script that sets a lower number of CPUs via the cpus=# command line option. Another, simpler approach to changing the default number of CPUs for all users follows below, for implementation by BLAST system managers possessing “root” or “SuperUser” privileges.

Distributions of AB-BLAST include a sample file named sysblast.sample, that illustrates the system-wide configuration parameters that can be established to govern the execution of BLAST jobs and, thereby, provide a more productive, trouble-free level of service. When the sysblast file is installed under the name /etc/sysblast, all BLAST jobs executed on a given computer system can be made subject to the parameters:

The sysblast file is only effective when installed in the /etc directory. The /etc directory generally resides locally to any given computer system, so parameter settings can be tailored to each computer, even if the BLAST software is maintained on a shared disk partition. The /etc directory should only be writable by “root”. Unlike the shell script wrapper approach described above, the limits set in /etc/sysblast typically can not be circumvented by normal (non-root) users of a computer system. See the comments included in the sample sysblast file for further details.


Differences between AB-BLAST and WU-BLAST

Apart from bug fixes, the most outward differences in usage and appearance of AB-BLAST and WU-BLAST include:


Citing BLAST

Citations or acknowledgments of AB-BLAST usage are greatly appreciated, as are any personal accounts of how the software is being used that you might wish to share. When URLs are acceptable, please cite with:

   Gish, W. (1996-2019) http://blast.advbiocomp.com

When URLs are not acceptable, please use:

   Gish, W. (unpublished).

In scientific communications, it is important to report both the program name and the specific version used. In the case of AB-BLAST 3.0, the version is a combination of the version number, release date, target platform, and build date. The release date is the first (left-most) date displayed on the first line of output and corresponds to the freeze date of the source code. The build date is the second date reported and corresponds to the date and time the executables were built for the indicated target platform. Both dates are reported in program output in ISO 8601 format.

For example, consider this introductory line of output from AB-BLAST 3.0:

  BLASTN 3.0 [2018-12-16] [macos-x64 2018-12-19T22:14:33]

Here the program name is BLASTN, the software version is “3.0”, the release date is December 16, 2018, and the build date and time of the 64-bit macOS X64 binary is December 19, 2018, at 10:14PM.


Historical Notes


References

Altschul, SF, and W Gish (1996). Local alignment statistics. ed. R. Doolittle. Methods Enzymol. 266:460–80.

Altschul, SF, and DJ Lipman (1990). Protein database searches for multiple alignments. Proc. Natl. Acad. Sci. USA 87:5509–13.

Altschul, SF, Gish, W, Miller, W, Myers, EW, and DJ Lipman (1990). Basic local alignment search tool. J. Mol. Biol. 215:403–10.

Altschul, SF, Madden, TL, Schäffer, AA, Zhang, J, Zhang, Z, Miller, W, and DJ Lipman (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25(17):3389–402.

Claverie, JM, and DJ States (1993). Information enhancement methods for large scale sequence analysis. Computers in Chemistry 17:191–201.

Collins, JF, and AF Coulson (1990). Significance of protein sequence similarities. Methods Enzymol. 183:474–7.

Dembo, A, and S Karlin (1991). Strong limit theorems of empirical functionals for large exceedances of partial sums of i.i.d. variables. Ann. Probab. 19:1737–55.

Dembo, A, and S Karlin (1992). Limit distributions of maximal segmental score among Markov dependent partial sums. Adv. Appl. Probab. 24:113–40.

Gish, W, and DJ States (1993). Identification of protein coding regions by database similarity search. Nat. Genet. 3:266–72.

Hancock, JM, and JS Armstrong (1994). SIMPLE34: an improved and enhanced implementation for VAX and Sun computers of the SIMPLE algorithm for analysis of clustered repetitive motifs in nucleotide sequences. Comput. Appl. Biosci. 10:67–70.

Karlin, S, and SF Altschul (1990). Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc. Natl. Acad. Sci. USA 87:2264–8.

Karlin, S, and SF Altschul (1993). Applications and statistics for multiple high-scoring segments in molecular sequences. Proc. Natl. Acad. Sci. USA 90:5873–7.

Karlin, S, Dembo, A, and T Kawabata (1990). Statistical composition of high scoring segments from molecular sequences. Ann. Stat. 18:571–81.

RF Mott (1992). Maximum-likelihood estimation of the statistical distribution of Smith-Waterman local sequence similarity scores. Bull. Math. Biol. 54:59–75.

Smith, TF, and MS Waterman (1981). Identification of common molecular subsequences. J. Mol. Biol. 147:195–7.

States, DJ, and W Gish (1994). Combined use of sequence similarity and codon bias for coding region identification. J. Comp. Biol. 1:39–50.

Waterman, MS, and M Vingron (1994). Rapid and accurate estimates of statistical significance for sequence data base searches. Proc. Natl. Acad. Sci. USA 91:4625–8.

Wootton, JC, and S Federhen (1993). Statistics of local complexity in amino acid sequences and sequence databases. Computers in Chemistry 17:149–63.

Wootton, JC, and S Federhen (1996). Analysis of compositionally biased regions in sequence databases. ed. R. Doolittle. Methods Enzymol. 266:554–71.

Zhang, Z, Schäffer, AA, Miller, W, Madden, TL, Lipman, DJ, Koonin, EV, and SF Altschul (1998). Protein sequence similarity searches using patterns as seeds. Nucl. Acids Res. 26:3986–90.


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