Update: November 11, 2016: To accommodate the new Arrow consensus caller, Dazzler databases have been upgraded to hold either the information required by Quiver or the information required by Arrow, (or neither but never both), creating the distinction of Arrow-DBs versus Quiver-DBs versus Sequence-DBs (A-, Q-, and S-DBs). New programs arrow2DB and DB2arrow allow adding and recreating Arrow information from a database, and a new program DBwipe removes any Quiver or Arrow information stored in a database reverting a Q- or A-DB to an S-DB.
Update: May 21, 2016: fasta2DAM now can operate incrementally. fasta2DB, quiva2DB, and fasta2DAM can now all import entries from a redirection of the standard input. The data from multiple SMRT cells in a given file is also supported.
Update: April 21, 2016: The simulator now samples reads from a genome reference in a .dam file and can operate over a circular genome with the -C option. A new program rangen now generates a random genome sequence to feed to the simulator.
The latest source code can be found on Github here.
Before listing the UNIX commands and their description for this module, we first give the design goals for a Dazzler data base:
- The database stores the source Pacbio read information in such a way that it can recreate the original input data, thus permitting a user to remove the (effectively redundant) source files. This avoids duplicating the same data, once in the source file and once in the database.
- The data base can be built up incrementally, that is new sequence data can be added to the data base over time.
- The data base flexibly allows one to store any meta-data desired for reads. This is accomplished with the concept of tracks that implementers can add as they need them.
- The data is held in a compressed form equivalent to the .dexta and .dexqv/.dexar files of the data extraction module.
- Quiver or Arrow information can be added separately from the sequence information and later on if desired, but a database can only hold either Quiver or Arrow information, but not both. The Arrow or Quiver information can be removed from the database at any time leaving a database just containing sequence information.
- To facilitate job parallel, cluster operation of the phases of our assembler, the data base has a concept of a current partitioning in which all the reads that are over a given length and optionally unique to a well, are divided up into blocks containing roughly a given number of bases, except possibly the last block which may have a short count. Often programs can be run on blocks or pairs of blocks and each such job is reasonably well balanced as the blocks are all the same size. One must be careful about changing the partition during an assembly as doing so can void the structural validity of any interim block-based results.
A DB con contain the information needed by Quiver, or by Arrow, or neither, but not both. A DB containing neither Quiver or Arrow information is termed a Sequence-DB (S-DB). A DB with Quiver information is a Quiver-DB (Q-DB) and a DB with Arrow information is an Arrow-DB (A-DB). All commands are aware of the state of a DB and respond to options according to their type. The diagram below shows how the various commands to be described below affect the state of a DB.
A Dazzler DB consists of one named, visible file, e.g. FOO.db, and several invisible secondary files encoding various elements of the DB. The secondary files are “invisible” to the UNIX OS in the sense that they begin with a “.” and hence are not listed by “ls” unless one specifies the -a flag. We chose to do this so that when a user lists the contents of a directory they just see a single name, e.g. FOO.db, that is used to refer to the DB in commands. The files associated with a database named, say FOO, are as follows:
a text file containing (i) the list of input files added to the database so far, and (ii) how to partition the database into blocks (if the partition parameters have been set).
a binary “index” of all the meta-data about each read allowing, for example, one to randomly access a read’s sequence (in the store .FOO.bps). It is 32N + 100 bytes in size where N is the number of reads in the database.
a binary compressed “store” of all the DNA sequences. It is M/4 bytes in size where M is the total number of base pairs in the database.
a binary compressed “store” of the 5 Pacbio quality value streams for the reads. Its size is roughly 5/3M bytes depending on the compression achieved. This file only exists if Quiver information has been added to the database.
a binary compressed “store” of the clipped pulse width stream for the reads. Its size is roughly M/4 bytes. This file only exists if Arrow information has been added to the database.
(f) .FOO.<track>.anno, .FOO.<track>.data
a track called <track> containing customized meta-data for each read. For example, the DBdust command annotates low complexity intervals of reads and records the intervals for each read in the two files .FOO.dust.anno and .FOO.dust.data that are together called the “dust” track. Any kind of information about a read can be recorded, such as micro-sat intervals, repeat intervals, corrected sequence, etc. Specific tracks will be described as phases that produce them are created and released.
If one does not like the convention of the secondary files being invisible, then un-defining the constant HIDE_FILES in DB.h before compiling the library, creates commands that do not place a prefixing “.” before secondary file names, e.g. FOO.idx instead of .FOO.idx. One then sees all the files realizing a DB when listing the contents of a directory with
All programs add suffixes (e.g. .db, .fasta) as needed. The -v option universally turns on a verbose reporting mode for the relevant programs. The formal UNIX command line descriptions and options for the Dazzler database commands are as follows:
Builds an initial data base, or adds to an existing database, either (a) the list of .fasta files following the database name argument, or (b) the list of .fasta files in <file> if the -f option is used, or (c) entries piped from the standard input if the -i option is used. If the DB is being created it is established as a Sequence-DB (S-DB) otherwise its type is unchanged. If a faux file name, <name>, follows the -i option then all the input received is considered to have come from a file by the name of <name>.fasta by DB2fasta, otherwise it will be sent to the standard output by DB2fasta. The SMRT cells in a given named input (i.e. all sources other than -i without a name) can only be added consecutively to the DB (this is checked by the command). The .fasta headers must be in the “Pacbio” format (i.e. the output of the Pacbio tools or our dextract program) and the well, pulse interval, and read quality are extracted from the header and kept with each read record. If the files are being added to an existing database, and the partition settings of the DB have already been set (see DBsplit below), then the partitioning of the database is updated to include the new data. A file may contain the data from multiple SMRT cells provided the reads for each SMRT cell are consecutive in the file.
The set of .fasta files for the given DB are recreated from the DB exactly as they were input. That is, this is a perfect inversion, including the reconstitution of the proper .fasta headers. Because of this property, one can, if desired, delete the .fasta source files once they are in the DB as they can always be recreated from it. Entries imported from the standard input will be place in the faux file name given on import, or to the standard output if no name was given. By default the output sequences are in lower case and 80 chars per line. The -U option specifies upper case should be used, and the characters per line, or line width, can be set to any positive value with the -w option.
Adds .quiva streams to an existing DB “path”. The DB must either be an S-DB or a Q-DB and upon completion the DB is a Q-DB. The data comes from (a) the given .quiva files on the command line, or (b) those in the file specified by the -f option, or (c) the standard input if the -i option is given. The input files can be added incrementally but must be added in the same order as the .fasta files were and have the same root names, e.g. FOO.fasta and FOO.quiva. This is enforced by the program. With the -l option set the compression scheme is a bit lossy to get more compression (see the description of dexqv in the DEXTRACTOR module here).
The set of .quiva files for the given Q-DB are recreated from the DB exactly as they were input. That is, this is a perfect inversion, including the reconstitution of the proper .quiva headers. Because of this property, one can, if desired, delete the .quiva source files once they are in the DB as they can always be recreated from it. Entries imported from the standard input will be place in the faux file name given on import, or to the standard output if no name was given. By .fastq convention each QV vector is output as a line without intervening new-lines, and by default the Deletion Tag vector is in lower case letters. The -U option specifies upper case letters should instead be used for said vector.
Adds .arrow streams to an existing DB “path”. The DB must either be an S-DB or an A-DB and upon completion the DB is an A-DB. The data comes from (a) the given .arrow files on the command line, or (b) those in the file specified by the -f option, or (c) the standard input if the -i option is given. The input files can be added incrementally but must be added in the same order as the .fasta files were and have the same root names, e.g. FOO.fasta and FOO.arrow. This is enforced by the program.
The set of .arrow files for the given A-DB are recreated from the DB exactly as they were input. That is, this is a perfect inversion, including the reconstitution of the proper .arrow headers. Because of this property, one can, if desired, delete the .arrow source files once they are in the DB as they can always be recreated from it. Entries imported from the standard input will be place in the faux file name given on import, or to the standard output if no name was given. By default the output sequences are formatted 80 chars per line, but the characters per line, or line width, can be set to any positive value with the -w option.
Builds an initial map DB or DAM, or adds to an existing DAM, either (a) the list of .fasta files following the database name argument, or (b) the list of .fasta files in <file> if the -f option is used, or (c) entries piped from the standard input if the -i option is used. If a faux file name, <name>, follows the -i option then all the input received is considered to have come from a file by the name of <name>.fasta by DAM2fasta, otherwise it will be sent to the standard output by DAM2fasta. Any .fasta entry that has a run of N’s in it will be split into separate “contig” entries and the interval of the contig in the original entry recorded. The header for each .fasta entry is saved with the contigs created from it.
The set of .fasta files for the given map DB or DAM are recreated from the DAM exactly as they were input. That is, this is a perfect inversion, including the reconstitution of the proper .fasta headers and the concatenation of contigs with the proper number of N’s between them to recreate scaffolds. Entries imported from the standard input will be place in the faux file name given on import, or to the standard output if no name was given. By default the output sequences are in lower case and 80 chars per line. The -U option specifies upper case should be used, and the characters per line, or line width, can be set to any positive value with the -w option.
Divide the database <path>.db or <path>.dam conceptually into a series of blocks referable to on the command line as <path>.1, <path>.2, … If the -x option is set then all reads less than the given length are ignored, and if the -a option is not set then secondary reads from a given well are also ignored. The remaining reads, constituting what we call the trimmed DB, are split among the blocks so that each block is of size -s * 1Mbp except for the last which necessarily contains a smaller residual. The default value for -s is 200Mbp because blocks of this size can be compared by our “overlapper” dalign in roughly 16Gb of memory. The blocks are very space efficient in that their sub-index of the master .idx is computed on the fly when loaded, and the .bps and .qvs files (if a .db) of base pairs and quality values, respectively, is shared with the master DB. Any relevant portions of tracks associated with the DB are also computed on the fly when loading a database block. If the -f option is set, the split is forced regardless of whether or not the DB in question has previously bin split, i.e. one is not interactively asked if they wish to proceed.
DBtrim [-af] [-x<int>] <path:db|dam>
Exactly like DBsplit except that it only resets the trimming parameters (and not the split partition itself).
Runs the symmetric DUST algorithm over the reads in the untrimmed DB <path>.db or <path>.dam, producing a track .<path>.dust[.anno,.data] that marks all intervals of low complexity sequence, where the scan window is of size -w, the threshold for being a low-complexity interval is -t, and only perfect intervals of size greater than -m are recorded. If the -b option is set then the definition of low complexity takes into account the frequency of a given base. The command is incremental if given a DB to which new data has been added since it was last run on the DB, then it will extend the track to include the new reads. It is important to set this flag for genomes with a strong AT/GC bias, albeit the code is a tad slower. The dust track, if present, is understood and used by DBshow, DBstats, and dalign.
DBdust can also be run over an untrimmed DB block in which case it outputs a track encoding where the trace file names contain the block number, e.g. .FOO.3.dust.anno and .FOO.3.dust.data, given FOO.3 on the command line. This permits job parallelism in block-sized chunks, and the resulting sequence of block tracks can then be merged into a track for the entire untrimmed DB with Catrack.
Find all block tracks of the form .<path>.#.<track>… and merge them into a single track, .<path>.<track>…, for the given DB (or DAM). The block track files must all encode the same kind of track data (this is checked), and the files must exist for block 1, 2, 3, … up to the last block number. If the -f option is set, then the concatenation takes place regardless of whether or not the single, combined track exists or not.
Displays the requested reads in the database <path>.db or <path>.dam. By default the command applies to the trimmed database, but if -u is set then the entire DB is used. If no read arguments are given then every read in the database or database block is displayed. Otherwise the input file or the list of supplied integer ranges give the ordinal positions in the actively loaded portion of the db. In the case of a file, it should simply contain a read index, one per line. In the other case, a read range is either a lone integer or the symbol $, in which case the read range consists of just that read (the last read in the database if $). One may also give two positive integers separated by a dash to indicate a range of integers, where again a $ represents the index of the last read in the actively loaded db. For example, 1 3-5 $ displays reads 1, 3, 4, 5, and the last read in the active db. As another example, 1-$ displays every read in the active db (the default).
By default a .fasta file of the selected read sequences is displayed. If the -q option is set and the DB is a Q-DB, then the QV streams are also displayed in a non-standard modification of the .fasta format. Similarly, if the -a option is set and the DB is an A-DB, then the pulse width stream is also displayed in a non-standard format. If the -n option is set then the DNA sequence is not displayed. If the -Q option is set then a .quiva file of the selected reads is displayed and all other options except -u and -U are ignored. If the -A option is set then a .arrow file of the selected reads is displayed and all other options except -u and -w are ignored.
If one or more masks are set with the -m option then the track intervals are also displayed in an additional header and the bases within an interval are displayed in the case opposite that used for all other bases. By default the output sequences are in lower case and 80 characters per line. The -U option specifies upper case should be used, and the characters per line, or line width, can be set to any positive value with the -w option.
The .fasta, .quiva, and .arrow files that are output can be used to build a new DB with fasta2DB, quiva2DB, and arrow2DB, giving one a simple way to make a DB of a subset of the reads for testing purposes.
Like DBshow, DBdump allows one to display a subset of the reads in the DB and select which information to show about them including any mask tracks. The difference is that the information is written in a very simple 1-code ASCII format that makes it easy for one to read and parse the information for further use. easy for one to read and parse the information for further use. The option flags determine which items of information are output as follows:
- -r requests that each read number be displayed in an R-line (see below, useful if only a subset of reads is requested).
- -h request the header information be output as the source file name on an H-line, the well # and pulse range on an L-line, and optionally the quality of the read if present on a Q-line.
- -s requests the sequence be output on an S-line.
- -a requests the Arrow information be output as a pulse-width string on an A-line and the 4 SNR channel values (on an N-line),
- -q requests that the 5 Quiver quality streams be output on d-, c-, i-, m-, and s-lines.
- -i requests that the intrinsic quality values be output (if available) on an I-line.
- -p requests the repeat profile be output (if available) on a P-line
- -m\<track\> requests that mask <track> be output on a T-line.
Set -u if you want data from the untrimmed database (the default is trimmed) and et -U if you’d like upper-case letter used in the DNA sequence strings.
The format is very simple. A requested unit of information occurs on a line. The first character of every line is a 1-code character that tells you what information to expect on the line. The rest of the line contains the information where each item is separated by a single blank space. Strings are output as first an integer giving the length of the string, a blank space, and then the string terminated by a new-line. Intrinsic quality values are between 0 and 50, inclusive, and a vector of said are displayed as an alphabetic string where ‘a’ is 0, ‘b’ is ‘1’, … ‘z’ is 25, ‘A’ is 26, ‘B’ is 27, … and ‘Y’ is 50. Repeat profiles are also displayed as strings where ‘_’ denotes 0 repetitions, and then ‘a’ through ‘N’ denote the values 1 through 40, respectively. The set of all possible lines is as follows:
R # - read number
H # string - original file name string (header)
L # # # - location: well, pulse start, pulse end
Q # - quality of read (#/1000)
N # # # # - SNR of ACGT channels (#/100)
Tx #n (#b #e)^#n - x’th track on command line, #n intervals all on same line
S # string - sequence string
A # string - arrow pulse-width string
I # string - intrinsic quality vector (as an ASCII string)
P # string - repeat profile vector (as an ASCII string)
d # string - Quiva deletion values (as an ASCII string)
c # string - Quiva deletion character string
i # string - Quiva insertion value string
m # string - Quiva merge value string
s # string - Quiva substitution value string
+ X # - Total amount of X (X = H or S or I or P or R or M or T#)
@ X # - Maximum amount of X (X = H or S or I or P or T#)
1-code lines that begin with
@ are always the first lines in the output. They give size information about what is contained in the output. That is ‘
+ X #‘ gives the number of reads (
X=R), the number of masks (
X=M), or the total number of characters in all headers (
X=H), sequences (
X=S), intrinsic quality vectors (
X=I), repeat profile vectors (
X=P), or mask (
X=T#). And ‘
@ X #‘ gives the maximum number of characters in any header (
X=H), sequence (
X=S), intrinsic quality vector (
X=I), repeat profile vectors (
X=P), or mask (
X=T#). The size numbers for the Quiva strings are identical to that for the sequence as they are all of the same length for any given entry.
DBstats [-nu] [-b<int(1000)] [-m<track>]+ <path:db|dam>
Show overview statistics for all the reads in the trimmed data base <path>.db or <path>.dam, including a histogram of read lengths where the bucket size can be controlled with the -b option (default 1000). If the -u option is given then the untrimmed database is summarized. If the -n option is given then the histogram of read lengths is not displayed. Any track such as a “dust” track that gives a series of intervals along the read can be specified with the -m option in which case a summary and a histogram of the interval lengths is displayed.
Delete all the files for the given data bases. Do not use
rm to remove a database, as there are at least two and often several secondary files for each DB including track files, and all of these are removed by DBrm.
Delete any Arrow or Quiver data from the given databases. This removes the .arw or .qvs file and resets information in the .idx file containing information for Arrow or Quiver. Basically, converts an A-DB or Q-DB back to a simple S-DB.
simulator <genome:dam> [-CU] [-m<int(10000)>] [-s<int(2000)>] [-e<double(.15)]
[-c<double(50.)>] [-f<double(.5)>] [-x<int(4000)>]
[-w<int(80)>] [-r<int>] [-M<file>]
In addition to the DB commands we include here, somewhat tangentially, a simple simulator that generates synthetic reads over a given genome reference contained in a supplied .dam DB. The simulator first reconstitutes the scaffolds of the reference genome and fills in their gaps (a run of N’s in .fasta format indicating the estimate gap length) with a random sequence that follows the base distribution of the contigs. It will then sample reads from these scaffold sequences.
The simulator generates sample reads of mean length -m from a log-normal length distribution with standard deviation -s, but ignores reads of length less than -x. It collects enough reads to cover the genome -c times and Introduces -e fraction errors into each read where the ratio of insertions, deletions, and substitutions are set by defined constants INS_RATE (default 73%) and DEL_RATE (default 20%) within generate.c. One can control the rate at which reads are picked from the forward and reverse strands with the -f option. The -r option seeds the random number generator for the generation process so that one can reproducibly generate the same dataset. If this parameter is missing, then the job id of the invocation seeds the random number generator effectively guaranteeing a different sampling with each invocation.
The output is sent to the standard output (i.e. it is a UNIX pipe). The output is in Pacbio .fasta format suitable as input to fasta2DB. Uppercase letters are used if the -U option is given, and the width of each line can be controlled with the -w option.
Finally, the -M option requests that the scaffold and coordinates within said scaffold from which each read has been sampled are written to the indicated file, one line per read, ASCII encoded. This “map” file essential tells one where every read belongs in an assembly and is very useful for debugging and testing purposes. If the map line for a read is say ‘
s b e‘ then if b < e the read is a perturbed copy of s[b,e] in the forward direction, and a perturbed copy s[e,b] in the reverse direction otherwise.
rangen <genlen:double> [-U] [-b<double(.5)>] [-w<int(80)>] [-r<int>]
Generate a random DNA sequence of length genlen*1Mbp that has an AT-bias of -b. Output the sequence to the standard output in .fasta format. Use uppercase letters if -U is set and -w base pairs per line (default 80). The result can then be converted into a .dam DB and given to the simulator to create a read database over a random synthetic sequence. The -r option seeds the random number generator for the generation process so that one can reproducibly generate the same sequence. If this parameter is missing, then the job id of the invocation seeds the random number generator effectively guaranteeing a different sequence with each invocation.