*|***************************************************************************|*
*|                                                                           |*
*|   Program: phred                                                          |*
*|   Version: 0.020425.c                                                     |*
*|                                                                           |*
*|   Copyright (C) 1993-2002 by Phil Green and Brent Ewing.                  |*
*|   All rights reserved.                                                    |*
*|                                                                           |*
*|   This software is a beta-test version of the phred package.              |*
*|   It should not be redistributed or used for any commercial               |*
*|   purpose, including commercially funded sequencing, without              |*
*|   written permission from the author and the University of                |*
*|   Washington.                                                             |*
*|                                                                           |*
*|   This software is provided ``AS IS'' and any express or                  |*
*|   implied warranties, including, but not limited to, the                  |*
*|   implied warranties of merchantability and fitness for a                 |*
*|   particular purpose, are disclaimed.  In no event shall                  |*
*|   the authors or the University of Washington be liable for               |*
*|   any direct, indirect, incidental, special, exemplary, or                |*
*|   consequential damages (including, but not limited to,                   |*
*|   procurement of substitute goods or services; loss of use,               |*
*|   data, or profits; or business interruption) however caused              |*
*|   and on any theory of liability, whether in contract, strict             |*
*|   liability, or tort (including negligence or otherwise)                  |*
*|   arising in any way out of the use of this software, even                |*
*|   if advised of the possibility of such damage.                           |*
*|                                                                           |*
*|   Portions of the code benefit from ideas due to Dave Ficenec,            |*
*|   LaDeana Hillier, Mike Wendl, and Tim Gleeson.  These are                |*
*|   indicated in the relevant source files.                                 |*
*|                                                                           |*
*|***************************************************************************|*


PHRED Documentation
-------------------

1.  Introduction.

    Phred reads DNA sequencer trace data, calls bases, assigns quality
    values to the bases, and writes the base calls and quality values to
    output files. Phred can read trace data from chromatogram files in
    the SCF, ABI, and ESD formats. It automatically determines the file
    format, and whether the chromatogram file was compressed using gzip,
    bzip2, or UNIX compress.  After calling bases, phred writes the
    sequences to files in either FASTA format, the format suitable for
    XBAP, PHD format, or the SCF format.  Quality values for the bases
    are written to FASTA format files or PHD files, which can be used by
    the phrap sequence assembly program in order to increase the accuracy
    of the assembled sequence.

    I have tested phred base calling and quality value accuracies for
    data from the following sequencing machines.

      ABI models 373, 377, and 3700
      Molecular Dynamics MegaBACE
      LI-COR 4000

    I have tested the phred base calling accuracy only for data from
    the following sequencing machines.

      ABI model 3100
      Beckman CEQ

    Significant differences in this release

      -  quality value lookup table for ABI 3700 dye terminator chemistry
         data (phred still uses the quality value lookup table for
         ABI 373/377 dye primer data when it processes ABI 3700 dye primer
         chromatograms. I have insufficient dye primer data for this
         calibration)

      -  quality value lookup table for MegaBACE dye terminator data
         processed with the Cimarron version 3.0012 base caller (phred
         still uses the quality value lookup tables for Cimarron version
         1.53 processed dye primer data when it processes Cimarron
         version 3.0012 dye primer data. I have insufficient dye primer
         data for this calibration)

      -  phred reads the trace processor software version from the
         ABI, ESD, and SCF format chromatograms in order to select
         the best base processing parameter and quality value lookup
         table for the Cimarron processed MegaBACE data. Note that
         only phred writes the trace processing software version
         string in the SCF file comments, identifying it with the new
         'label' TPSW. 

      -  sequencing machine and chemistry specific basecalling
         parameters improve base calling accuracy.

         This change makes the phred base calling depend on correct
         identification of the chromatogram 'source', which means that
         phred must match the primer ID string in the chromatogram with
         a string in the (included) 'phredpar.dat file' in order for it
         to process the chromatogram correctly.

         The '-exit_nomatch' option forces phred to exit immediately if
         it cannot match the chromatogram primer ID string with a
         'phredpar.dat' entry.

         The '-process_nomatch' option allows phred to process a
         chromatogram with a non-matching primer ID string if the
         'phredpar.dat' file contains an entry for
         "__no_matching_string__".

         phred's 'error' messages reflect these changes. This document
         includes a summary of the general program flow and the
         consequent messages phred writes to 'stderr', which is your
         terminal normally, and the log file when you use the '-log'
         option.

         The 'peak prediction' has increased 'resolution' for detecting
         peak spacing changes (which causes phred to run about 2 times
         slower).

      -  trace 'noise' value calculated and stored in the phd file header.
         Phred calculates the ratio of the total uncalled-base peak area
         to the total called-base peak area within the high quality base
         segment of the read. It stores this value in the phd file header
         with the label 'TRACE_PEAK_AREA_RATIO'. The high quality base
         segment of the read is determine using the modified Mott trimming
         algorithm described below.

      -  phred checks the SCF file private data block for the Beckman
         CEQ 'fingerprint'

      -  phred base calling is tuned for Beckman CEQ; however, the
         quality value lookup table is not designed for the Beckman CEQ
         because I have insufficient data for quality value calibration.
         (Phred uses the ABI 3700 quality value lookup tables for the
         Beckman CEQ data.)

      -  processes ABI and SCF format files with no bases stored in them.

      -  phred exits if the PHRED_PARAMETER_FILE environment variable is
         not set or it cannot read the 'phredpar.dat' file successfully.

      -  earlier phred versions interpreted a chromatogram primer ID
         string consisting entirely of non-printing characters as an empty
         string. Now phred does no interpretation.

      -  when phred runs with the '-id <chromat_dir> option and the
         <chromat_dir> contains subdirectories, phred no longer tries to
         process the subdirectories, so it will not warn of an 'unknown
         file type' for the subdirectories.

      -  more sensitive compression motif detection for ABI 3700 dye primer
         data

      -  the '-v 1' option causes phred to write the command line and
         the time phred starts running to both stdout and stderr.


2.  Acknowledgements.

    Phred benefits from ideas developed by LaDeana Hillier, Mike Wendl,
    Dave Ficenec, Tim Gleeson, Alan Blanchard, and Richard Mott.
   

3.  Algorithms.

    Phred uses simple Fourier methods to examine the four base traces in
    the region surrounding each point in the data set in order to predict
    a series of evenly spaced predicted locations.  That is, it determines
    where the peaks would be centered if there were no compressions,
    dropouts, or other factors shifting the peaks from their "true"
    locations.

    Next phred examines each trace to find the centers of the actual, or
    observed, peaks and the areas of these peaks relative to their neighbors.
    The peaks are detected independently along each of the four traces so
    many peaks overlap.  A dynamic programming algorithm is used to match
    the observed peaks detected in the second step with the predicted peak
    locations found in the first step.

    Phred evaluates the trace surrounding each called base using four or
    five quality value parameters to quantify the trace quality.  It
    uses a quality value lookup table to assign the corresponding quality
    value.  The quality value is related to the base call error probability
    by the formula

      QV = - 10 * log_10( P_e )

      where P_e is the probability that the base call is an error.

    Phred uses data from a chemistry parameter file called 'phredpar.dat'
    in order to identify dye primer data.  For dye primer data, phred
    identifies loop/stem sequence motifs that tend to result in
    CC and GG merged peak compressions.  It reduces the quality values
    of potential merged peaks and splits those peaks that have certain
    trace characteristics indicative of merged CC and GG peaks.  In
    addition, the chemistry and dye information are passed to phrap.


4.  Building and installing.

    The INSTALL file describes the steps for building and installing
    phred.
 
    Copy the phred parameter file, called 'phredpar.dat', to a
    directory that is accessible by phred users and set the environment
    variable 'PHRED_PARAMETER_FILE' to the full path name of the file.
    For example, if you copy 'phredpar.dat' to '/usr/local/etc/PhredPar'
    and you are using the C shell then issue the command

      % setenv PHRED_PARAMETER_FILE /usr/local/etc/PhredPar/phredpar.dat

    It is most convenient to set the environment variable in the system-
    wide shell startup (cshrc or equivalent) file.

    You can rename the phred parameter file but the PHRED_PARAMETER_FILE
    environment variable must reflect the new name.

    With Windows NT you give the command

      % set PHRED_PARAMETER_FILE=\usr\local\etc\PhredPar\phredpar.dat

    in the DOS command window in which you will run phred.

    Note: if you compile phred on a SUN Solaris OS using the BSD C
          compiler in the directory '/usr/ucb', you will find that
          the '-id' command line option fails (phred reports that it
          cannot read files, and it prints the name of each file it
          fails to read; however, the name it prints lacks the first
          few characters of the true name of the file). If this occurs,
          recompile phred using either the optional C compiler in the
          directory /opt/SUNWspro/bin or the GNU C compiler.


5.  Running phred.

    Phred uses command line options to control input, processing, and
    output.  The command line options are delimited by a dash, "-".


    The command line options are

   Input Options
   -------------
   
   -id <directory name>		Read and process files in <directory name>

   -if <file name>		Read and process files listed in the file
                                <file name>  Each line in <file name> must
                                specify a valid path to a single input file.

   -zd <directory name>         Location of compression program.  If -zd is
                                omitted, phred uses the current path to search
                                for the compression program.

   -zt <directory name>         Directory where chromat is uncompressed. If
                                -zd is omitted, phred uses /usr/tmp.  When
                                phred processes a compressed file, it 
                                uncompresses the chromat into this temporary
                                directory before it reads the file.  It
                                subsequently deletes the uncompressed file in
                                the temporary directory.


   Processing Options
   ------------------

   -nocall			Disable phred base calling and set the
                                current sequence to the ABI base calls
                                that are read from the input file.  By
                                default, the current sequence is set
                                to the phred base calls.  This affects
                                the base trimming and output options.

   -trim_alt <enzyme sequence>  Perform sequence trimming on the current
                                sequence.  Bases are trimmed from the start
                                and end of the sequence on the basis of
                                trace quality.  Specifically, for each base,
                                the phred error probability is subtracted
                                from the default value of 0.05 (or the value
                                set using the '-trim_cutoff' option), and the
                                resulting values are summed to find the
                                maximum scoring subsequence.  Furthermore,
                                the subsequence must have a minimum number
                                of bases.  In addition, <enzyme sequence>
                                specifies a short base sequence (typically
                                the recognition sequence of the restriction
                                enzyme sequence used for subcloning) that
                                is used to trim bases off the start of the
                                current sequence. You can specify a NULL
                                enzyme sequence using empty double quotes,
                                "". (We recommend that you use '-trim_alt'
                                rather than '-trim' option described below
                                because we believe that '-trim' trims off
                                too many good bases).

   -trim_cutoff <value>         Set trimming error probability for the
                                '-trim_alt' option and the trimming points
                                written in the phd files. The default value
                                is 0.05.

   -trim_fasta                  Trim sequences written to sequence and
                                quality value FASTA files. Set trimming
                                information in the FASTA headers to reflect
                                the high quality of the sequence, and append
                                the string 'trimmed' to the header.

   -trim_scf                    Trim sequence, quality values, and base
                                locations written to SCF file. Append the
                                string 'trimmed' to the comments.

   -trim_phd                    Trim sequence, quality values, and base
                                locations written to PHD files. Also set the
                                first and last high quality base locations
                                specified in the 'TRIM' comment field to
                                the numbers of the first and last bases of
                                the trimmed sequence (the first base in the
                                sequence is base number zero). Finally set
                                the error probability cutoff value in the
                                'TRIM' comment field to -1.00 to indicate that
                                the sequence is trimmed, and that the trim
                                points may be unrelated to the error
                                probability cutoff value.

   -trim_out                    Trim information in the FASTA, SCF, and
                                PHD output files. This is equivalent to
                                specifying '-trim_fasta', '-trim_scf',
                                and '-trim_phd' on the command line.

   -trim <enzyme sequence>      Perform sequence trimming on the current
                                sequence.  Bases are trimmed from the start
                                and end of the sequence on the basis of
                                trace quality. In addition, <enzyme sequence>
                                specifies a short base sequence (typically
                                the recognition sequence of the restriction
                                enzyme sequence used for subcloning) that 
                                is used to trim bases off the start of the 
                                current sequence. You can specify a NULL 
                                enzyme sequence using empty double quotes, 
                                "". We recommend against using this option
                                because we consider it to be too
                                conservative. See the note below on the
                                effect of using the trim option.

   -nonorm                      Disable phred trace normalization.  This
                                option is not recommended unless the base
                                caller fails due to huge noise peaks
                                extending over a large region at the start
                                of the trace, as is characteristic of some
                                dye terminator reactions.

   -nosplit                     Disable compressed peak splitting.  By
                                default, phred identifies and splits
                                C and G peaks that may be a merged pair
                                of peaks.  Phred searches for compression
                                prone loop/stem sequence motifs and
                                attempts to confirm a compression using
                                characteristics of the trace, primarily
                                the size of the candidate peak.

   -nocmpqv                     Force phred to use the four parameter quality
                                values.  By default, phred uses five parameter
                                quality values for dye primer data (only) in
                                order to reduce the quality values of merged
                                CC and GG peaks.  (Phred uses the four
                                parameter quality values for dye terminator
                                chemistry data automatically.  If phred cannot
                                determine the chemistry, it uses the four
                                parameter quality values.)

   -ceilqv <ceil_qv>            Specifies a maximum quality value assigned
                                to bases.  Bases with quality value parameters
                                that correspond to quality values greater
                                than <ceil_qv> are assigned the value
                                <ceil_qv>

   -beg_pred <trace_point>      Specifies the trace point at which to begin the
                                peak prediction. This point should be in a
                                region of 'good' trace where the peak spacing
                                is even and representative of the peak spacing
                                throughout the trace. In addition the peaks
                                should be large and the noise low in the
                                region, and the value of <trace_point> must not
                                be within 100 points of the trace ends.

   -exit_nomatch                When unable to match a chromatogram primer ID
                                string with a 'phredpar.dat' file entry, exit
                                immediately.

   -process_nomatch             When unable to match a chromatogram primer ID
                                string to a 'phredpar.dat' file entry, use
                                the "__no_matching_string__" entry in the
                                'phredpar.dat' file to identify the chromatogram
                                chemistry/dye/machine type. If you use this
                                option and the 'phredpar.dat' file lacks the
                                "__no_matching_string__" entry, phred exits
                                immediately when it encounters a chromatogram
                                with an unmatchable primer ID string. Use this
                                option only when processing chromatograms from
                                one type of sequencing machine running one
                                type of sequencing chemistry. See the section
                                describing the Phred parameter file below for
                                more information before using this option.


   Output Options
   --------------

   -st fasta                    Set the output sequence file format
                                to FASTA. (Default.) Trimming options
                                affect the FASTA file; see the Notes
                                below for more information.

   -st xbap                     Set the output sequence file format
                                to XBAP.

   -s                           Write sequence output files with the
                                names obtained by appending ".seq" to
                                the names of the input files, and store
                                them in the directory where phred is
                                running.

   -s <file name>               Write a sequence output file with the
                                name <file name>
                                This option is valid for a single input
                                file only.

   -sd <directory name>         Write sequence output files with the
                                names obtained by appending ".seq" to
                                the names of the input files, and write
                                them in the directory <directory name>

   -sa <file name>              Write a sequence output file in FASTA
                                format with the name <file name>  The
                                file contains the base calls of all the
                                reads processed in this run of phred.

   -qt fasta                    Set the output quality file format
                                to FASTA. (Default.) Trimming options
                                affect the FASTA file; see the Notes
                                below for more information.

   -qt xbap                     Set the output quality file format
                                to XBAP.  Trimmed off base quality
                                values are omitted.

   -qt mix                      Set the output quality file format
                                to FASTA. Base quality values for
                                all bases are written (including those
                                for trimmed off bases).

   -q                           Write quality output files with the
                                names obtained by appending ".qual" to
                                the names of the input files, and store
                                them in the directory where phred is
                                running.
                                This option is valid for FASTA format
                                output files only.

   -q <file name>               Write a quality output file with the
                                name <file name>
                                This option is valid for a single input
                                file and a FASTA format output file only.

   -qd <directory name>         Write quality output files with the
                                names obtained by appending ".qual" to
                                the names of the input files, and store
                                them in the directory <directory name>

   -qa <file name>              Write a quality output file in FASTA
                                format with the name <file name>  The
                                file contains the quality values of all the
                                reads processed in this run of phred.

   -qr <file name>              Write a histogram of the number of high
                                quality bases per read.  This is meaning-
                                ful when phred processes more than one
                                read.

   -c                           Write SCF files with the trace data,
                                the base calls of the current sequences,
                                and the positions of the base calls.  The
                                SCF files have the names of the input
                                files (phred will refuse to write the SCF
                                file if you ask it to write the SCF file
                                in the directory in which the input file
                                resides).

   -c <file name>               Write an SCF file with the trace data,
                                the base calls of the current sequence,
                                and the positions of the base calls.
                                The SCF file has the name <file name>
                                This option is valid for a single input
                                file only.

   -cd <directory name>         Write SCF files with the trace data,
                                the base calls of the current sequences,
                                and the positions of the base calls.
                                The SCF files are written in the directory
                                <directory name> and have the same names
                                as the input files.

   -cp <number of bytes>        Store SCF trace data as 1 or 2 byte values.
                                Defaults to 1 when the maximum trace value is
                                less than 256, or to 2 when the maximum
                                trace value is greater than or equal to 256.
                                This is the trace precision.

   -cv <version number>         Write SCF output file in SCF format version 2
                                or SCF format version 3. The default is
                                version 2.

   -cs                          Always scale traces before writing them to
                                an SCF output file. This ensures that the
                                largest trace value has the largest value
                                that can be stored in the SCF file. When the
                                file trace precision is '1', the maximum
                                value is 255, and when the precision is 2,
                                the maximum value is 65535. Without this
                                option, phred does not scale the trace unless
                                (a) the trace was read from an ESD file or
                                (b) the maximum trace value exceeds the value
                                that can be stored in the SCF file at the
                                precision used. Trace scaling ensures the
                                maximum digital resolution for a given
                                storage precision but it will make a
                                uniformly low level trace appear to be a
                                high level.

   -p                           Write a PHD file, which is used by the
                                consed editor to display bases.  A PHD
                                file contains a set of comments used by
                                consed for maintaining consistency between
                                the chromat file, the .ace file and
                                the PHD file, and it contains base data
                                as triples consisting of the base call,
                                quality, and position.  Phred always
                                writes the first version of the PHD
                                file for a read, which has the name
                                <filename>phd.1.  When a read is edited
                                using consed, a new version of the phd is
                                written by consed, for example, the second
                                version has the name <filename>phd.2.  With
                                the -p option, <filename> is the name of the
                                input file.

   -p <filename>                Write a PHD file with the name <filename>phd.1.
                                This option is valid for processing a single
                                input file.

   -pd <directory name>         Write PHD files in directory <directory name>
                                The PHD files have the names <filename>phd.1
                                where <filename> is the name of the input file.

   -d                           Write a data file that is used for detecting
                                polymorphic bases.  The file has the
                                name <filename>poly where <filename> is the
                                name of the input file.  The first line of
                                the file consists of the sequence name, the
                                smallest amplitude normalization factor, and
                                the amplitude normalization factors for the
                                A, C, G, and T traces.  One line for each
                                called base follows the header line.  The
                                information on each line consists of the
                                called base, the position of the called base,
                                the area of the called peak, the relative area
                                of the called peak, the uncalled base, the
                                position of the uncalled base, the area of the
                                uncalled base, the relative area of the
                                uncalled base, and the amplitudes of the four
                                traces at the position of the called base.

   -dd <dirname>                Write polymorphism data files in directory
                                <directory name>  The files have the names
                                <filename>poly where <filename> is the name
                                of the input file.
              
   -raw <sequence name>         Write <sequence name> in the header of
                                the sequence output file and the quality
                                output file.
                                By default, the name of the input file
                                is written in the headers of these files.
                                This option is valid for a single input
                                file only.

   -log                         Make phred append a log entry describing
                                the processing run in the file "phred.log".



   Miscellaneous
   -------------

   -v  <n>                      Verbose operation. You can control the level of
                                verbosity with <n> which ranges from 1 to 63.
                                The value '1' cause phred to write the command
                                line and the time it starts running to both the
                                stdout and stderr.

   -tags                        Label common output with tags in order to
                                facilitate output parsing.

   -h, -help                    Display a command line option summary.

   -doc                         Display phred documentation.

   -V                           Display phred version.                                


    Examples
    --------

    If you plan to use phred base calls and base quality information as
    input to the phrap assembly program and to the consed finishing
    program, we encourage you to use the phredPhrap Perl script that is
    part of the consed distribution. Please follow the documentation
    supplied with  consed and then type:

    phredPhrap
   
    (with no arguments)

    If you intend to use consed, you *MUST* use this perl script.  Failure
    to use this script will result in many consed features not working
    correctly, including consed's autofinish function, user-defined
    consensus tags, tagging ALU and other repeats, and tagging vector
    sequence.  Use the phredPhrap perl script.

    An outline of the important processing steps performed by the script
    follows.

    Let us say you want to call bases from the chromat files in
    subdirectory "chromat_dir", use phrap to assemble the contigs, and
    run consed to edit/examine the contigs.  In this case you must ask
    phred to create "phd" output files, which are required by consed.

    It runs phred with the options

      % phred -id chromat_dir -pd phd_dir

    which causes phred to read the chromat files in "chromat_dir" and
    write the "phd" files to "phd_dir".  Next it makes FASTA files
    from the "phd" files by running the phd2fasta program.
    For example,

      % phd2fasta -id phd_dir -os seqs_fasta -oq seqs_fasta.screen.qual

    Subsequently it screens out the vector in the sequences in
    "seqs_fasta" using cross_match:

      % cross_match seqs_fasta vector.seq -minmatch 12 -minscore 20 -screen > screen.out

    which generates the screened sequence file "seqs_fasta.screen",

    It runs phrap to perform the sequence assembly as follows:

      % phrap seqs_fasta.screen -new_ace > phrap.out

    Phrap writes the the assembled contigs to the file
    "seqs_fasta.screen.contigs", and creates a .ace file that can be
    used for importing the assembly to xbap, consed, or ace-mbly for
    editing.

    As another example, again you want to process the chromat files
    in subdirectory "chromat_dir",  but now you want phred to write
    the base calls to a FASTA file named "seqs_fasta" and the base
    quality values to "seqs_fasta.qual".  In this case you run phred
    with the options

      % phred -id chromat_dir -sa seqs_fasta -qa seqs_fasta.qual

    We recommend that you not use the trim option.  Inaccurate bases
    called near the ends of the traces will not interfere with proper
    phrap assembly.

    Refer to the file "phrap.doc", which is part of the phrap
    distribution, for information on cross_match and phrap.


    Return values
    -------------

    Phred returns 0 for successful processing and for non-fatal errors.
    It returns -1 for 'fatal errors'. 'Fatal errors' include memory
    allocation failure and file write (usually due to no disk
    space) failure.


6.  Phred parameter file

    Phred reads the 'primer ID' string in the chromatogram and tries to
    find the same name in the phred parameter file, which is mentioned in
    the 'Building and installing' section above.  If it succeeds, the
    'phredpar.dat' entry for the 'primer ID' identifies the sequencing
    reaction chemistry (primer or terminator), the dye type, and the
    sequencing machine type.

    If phred cannot read the 'phredpar.dat' file, it exits immediately.
    The reasons that phred may not read the 'phredpar.dat' file include

      o  the PHRED_PARAMETER_FILE environment variable is unset

      o  the PHRED_PARAMETER_FILE environment variable is not set
         to a valid 'phredpar.dat' file

    If phred cannot match the primer ID string to a 'phredpar.dat'
    entry, its operation depends on the command line options
    '-exit_nomatch' and '-process_nomatch'. The possible results
    are

      o  neither '-exit_nomatch' nor '-process_nomatch' is used

           phred skips to the next chromatogram without writing
           to an output file

      o  '-exit_nomatch' is used

           phred exits immediately when it finds a chromatogram
           with an unmatchable primer ID string
           reports

      o  '-process_nomatch' is used

           phred looks for a "__no_matching_string__" entry in
           'phredpar.dat'. If it finds this entry, it uses the entry
           to process the chromatogram. That is, the
           "__no_matching_string__" entry becomes the default
           machine/chemistry/dye type. The "__no_matching_string__"
           entry is commented out in the included 'phredpar.dat' file
           so, if you want to use the '-process_nomatch' option, you
           must remove the comment character (#) at the start of this
           line, and change the chemistry, dye, and machine types to
           the correct values. Use this option only if you use phred
           to process chromatograms from one type of sequencing machine
           running one type of sequencing chemistry. If phred cannot
           find the "__no_matching_string__" entry in the 'phedpar.dat'
           file, it exits immediately.

    Additionally, when phred  cannot find the 'primer ID' name in the
    'phredpar.dat'file, it provides the information

      unknown chemistry (xxxx) in chromat yyyy
      add a line of the form
      "xxxx"    <chemistry>      <dye type>      <machine type>
      to the file zzzz
      type 'phred -doc' for more information

    where xxxx is the 'primer ID', yyyy is the chromatogram name, and
    zzzz is the 'phredpar.dat' file. In order to add the correct entry
    to 'phredpar.dat', you will need to know the sequencing chemistry
    type (primer or terminator), the dye name, and the type of sequencing
    machine. 'Cut' the entry template phred provides, 'paste' it into the
    'phredpar'dat file, and add the correct chemistry, dye, and sequencing
    machine values in the indicate fields. You will find additional
    information about the acceptable form of entries in the header of the
    'phredpar.dat' file.

    The fields in the 'phredpar.dat' file are

    field       value name
    -----       ----------
    1           primer identification string
    2           chemistry
    3           dye
    4           sequencing machine type

    where the field values are separated by spaces or horizontal tabs.

    The values phred recognizes are

    value name                 values
    ----------                 ------
    primer ID string           primer name enclosed in double quotes
    chemistry                  primer, terminator, unknown
    dye                        rhodamine, d-rhodamine, big-dye,
                               energy-transfer, bodipy, unknown
    sequencing machine type    ABI_373_377, ABI_3100, ABI_3700,
                               Beckman_CEQ_2000, LI-COR_4000, and
                               MolDyn_MegaBACE


    NOTES:

      o  phred treats the 'unknown' chemistry type the same as the
         'terminator' chemistry type for base calling and quality
         value assignment; and it sets the chemistry type in the phd
         file header to 'unknown' (the chemistry type information in
         the phd file header is written in the FASTA sequence headers
         by the phd2fasta program in order to pass the information to
         phrap).

      o  phred does not use the dye type information for base calling
         or quality values but it writes the information in the
         phd file header (the dye type information in the phd file
         header is written in the FASTA sequence headers by the
         phd2fasta program in order to pass the information to phrap).

      o  SCF files created by the Beckman CEQ sequencer have no
         primer ID string but they have a special identifier in
         the private data block. Phred checks the SCF private data
         block for the Beckman identifier when the primer ID string
         is empty. If it finds the identifier, it sets the primer ID
         string to "BeckmanCEQ", and subsequently looks in
         'phredpar.dat' for the corresponding entry.

      o  the 'MegaBACE Mobility File' entry in the phredpar.dat file
         specifies 'unknown' chemistry, rather than 'primer' or
         'terminator' because some early MegaBACE software wrote
         'MegaBACE Mobility File' for the primer ID string in both
         primer and terminator chemistry ABD files. You may want to
         change this value if you process exclusively primer or
         terminator chemistry MegaBACE data.

      o  phred considers a missing primer ID string to be an empty
         string so it will match it to the empty string entry in
         'phredpar.dat', if the entry exists.


7.  Sequence Trimming

    First, a warning: in general, do not trim sequences that phrap
    will assemble. We introduced trimming capabilities in phred to allow
    identification of the high quality region of reads, and to permit
    trimming off low quality segments of reads that are not destined for
    a phrap assembly. We emphatically recommend against trimming reads
    for shotgun (or similar) sequencing projects. (Trimming may make
    sense for single pass sequencing when the quality values will
    be unavailable for subsequent analyses.)

    Second, another warning: if you must trim sequences, we strongly
    recommend that you use the '-trim_alt' option rather than the
    '-trim' option because we believe that it generally preserves
    more high quality bases, and it allows you to fine tune the
    trimming using the '-trim_cutoff' option.

    Phred uses two different algorithms to calculate trimming values.
    The algorithm used and its effect depend on the trimming command line
    options and the output file type.

    The phd output file always contains trimming information in the
    header. Phred calculates this trimming information using a modified
    Mott algorithm (it does not trim off vector sequence so the trimming
    information identifies the entire high quality segment of the read,
    including high quality vector sequence). The trimming information
    appears in the phd file header in the form

    TRIM: <n1> <n2> <r1>
 
    where <n1> is the first high quality base (where the first base in
    the sequence is number zero) and <n2> is the last high quality base.
    <r1> is the error probability cutoff value used to calculate the
    trim points. The command line option '-trim_cutoff' affects the
    phd file trimming information by setting the error probability cutoff
    value used to calculate the base scores. If the sequence has fewer
    than 20 high quality bases, the values <n1> and <n2> are set to -1.
    If the '-trim_phd' or '-trim_out' option is used, <n1> and <n2>
    are set to the numbers of the first and last bases in the trimmed
    sequence (so <n1> is always zero), and <r1> is set to -1.00 to
    indicate that the sequence is trimmed and that the error probability
    cutoff value may be unrelated to the trim points.

    The sequence, quality value, SCF, and PHD output files can be
    affected by the trimming-related command line options. (Sequence
    and quality value files are those created using the -s, -sa, -sd,
    -q, -qa, and -qd options, SCF files are created using the -c and
    -cd options, and PHD files are created using the -p and -pd
    options). When phred runs without trimming-related options set,
    it does not calculate trimming values for the sequence, quality
    value, and SCF output files (and it does not 'trim' the values
    stored in them).

    The '-trim_alt' and '-trim' options select the trimming algorithm
    used to calculate the trimming information used in the sequence,
    quality value, and SCF output files. The algorithm used for the
    '-trim_alt' option is based on the modified Mott algorithm: it uses
    the base error probabilities calculated from the phred quality
    values and the error probability cutoff (the cutoff can be adjusted
    using the -trim_cutoff option). The algorithm used for the '-trim'
    option is based directly on characteristics of the trace. It predates
    phred and phred quality values. We believe that the '-trim' option
    tends to be conservative, 'trimming off' more bases, in comparison to
    the '-trim_alt' option. So we recommend using the '-trim_alt'
    algorithm. Both the '-trim_alt' and '-trim' options take an argument
    consisting of a restriction enzyme recognition sequence. If the
    argument is "" (null), phred finds the high quality segment of the
    read. If the argument is not null, and phred finds the beginning of
    the recognition sequence within the first 100 bases of the read, phred
    sets the left trim point to remove the sequence up to this point as
    well as low quality bases. Please note that the sequence must match
    the recognition sequence nearly exactly for phred to recognize it.
    Caution: this is not a substitute for vector masking. We recommend
    that you use cross_match to mask vector sequence in the reads. (The
    phredPhrap script automatically calls cross_match to mask vector in
    the reads.)

    Selecting either '-trim_alt' or '-trim' causes phred to determine
    trimming information and to modify the sequence, quality value, and
    SCF files as follows.

      The FASTA sequence header contains trimming information
      but the sequence is unaffected. The header has the form

      >chromat_name   1323     15    548  ABI

      where the sequence name immediately follows the header
      delimiter, which is "> the first integer is the number
      of bases called by phred, the second integer is the
      number of bases 'trimmed off' the beginning of the
      sequence, the third integer is the number of bases
      'remaining following trimming', and the string describes
      the type of input file.

      The XBAP-type of sequence header contains trimming
      information, and the low quality bases are commented out.

      For quality value file type option '-qt fasta' (default),
      the FASTA quality value header contains the same trimming
      information as in the FASTA sequence header and the
      quality values of the 'trimmed off' bases are set to zero.

      For quality value file type option '-qt xbap', phred
      writes a XBAP-type of sequence header with trimming
      information followed by the quality values of the bases
      remaining after trimming on subsequent lines.

      For quality value file type option '-qt mix', phred
      writes a FASTA quality value header with the same
      trimming information as in the FASTA sequence header
      followed by the quality values of all bases (without
      trimming).

      The SCF file contains trimming information in the header,
      and the sequence, quality values, and trace locations of
      the called peaks are unaffected. The left clip is the
      number of bases to trim off the left end of the sequence
      and the right clip is the number of bases to trim off
      the right end.
     
    When the '-trim_fasta' or '-trim_out' option is used with the
    '-trim_alt' or '-trim' (and -s, -sa, -sd, -q, -qa, or -qd) option,
    phred writes the trimmed sequence to the sequence FASTA file and
    trimmed quality values to the quality value FASTA file; that is, it
    writes only the high quality bases and the corresponding quality
    values. In addition, it appends the string 'trimmed' to the FASTA
    headers and the trimming information in the header indicates that
    no (additional) bases are to be trimmed off. The option '-trim_fasta'
    is invalid with the '-qt xbap' and '-qt mix' options.

    When the '-trim_scf' or '-trim_out' option is used with the '-trim_alt'
    or '-trim' (and -c or -cd) option, phred writes the trimmed sequence,
    trimmed quality value, and trimmed called peak locations to the SCF
    output file. In addition, it appends the string 'trimmed' to the
    comment field and the left and right clip values are set to zero.

    When the '-trim_phd' or '-trim_out' option is used with the '-trim_alt'
    or '-trim' (and -p or -pd) option, phred writes the trimmed sequence,
    trimmed quality value, and trimmed called peak locations to the PHD
    output file. In addition, when it writes the 'TRIM' field in the
    comment block (at the beginning of the file), it sets the values for
    the first and last high quality bases to the numbers of the first and
    last bases of the trimmed sequence (where the first base is number
    zero), and it sets the error probability cutoff value to -1.00. Setting
    the cutoff value to -1.00 indicates that the sequence is trimmed, and
    that the trim points may be unrelated to the error probability cutoff
    value.

    The modified Mott trimming algorithm, which is used to calculate the
    trimming information for the '-trim_alt' option and the phd files,
    uses base error probabilities calculated from the phred quality
    values. For each base it subtracts the base error probability from an
    error probability cutoff value (0.05 by default, and changed using
    the '-trim_cutoff' option) to form the base score. Then it finds the
    highest scoring segment of the sequence where the segment score is
    the sum of the segment base scores (the score can have non-negative
    values only). The algorithm requires a minimum segment length, which
    is set to 20 bases.


8.  Trace noise calculation

    phred calculates a value related to the amount of 'noise' in the
    trace, and stores this value in the phd file header. The value
    is the ratio of the total uncalled-base peak area to the total
    called-base peak area within the high quality segment of the read.
    If the high quality region consists of fewer than 20 bases or the
    area of the called peaks is 0, the value is set to 100. The value
    appears in the phd file header with the label 'TRACE_PEAK_AREA_RATIO'.
    This value may be useful for identifying low quality traces due to
    low signal levels and due to template mixtures.


9.  Phred program flow and messages

    The following is a overview of the phred program flow and the
    most important associated messages. phred produces the shown
    messages when the '-tags' option is used, which I recommend for
    parsing the phred output with another program/script. phred
    produces similar messages when run without the '-tags' option.

    a. read PHRED_PARAMETER_FILE environment variable

         succeeds)

           MESSAGE:     none
           RESULT:      phred continues

         fails)

           MESSAGE:     FATAL_ERROR: PHRED_PARAMETER_FILE environment variable not set
           RESULT:      phred exits immediately


    b. read 'phredpar.dat' file

         succeeds)

           MESSAGE:     none
           RESULT:      phred continues

         fails)

           MESSAGE:     FATAL_ERROR: unable to read parameter file
           RESULT:      phred exits immediately


    c. memory allocation for chromatogram reading

         succeeds)

           MESSAGE:     none
           RESULT:      phred continues

         fails)

           MESSAGE:     FATAL_ERROR: <chromat_name> error while reading
           RESULT:      phred exits immediately


    d. chromat file type identification

         succeeds)

           MESSAGE:     none
           RESULT:      phred continues

         fails)

           MESSAGES:    FILE_ERROR: <file_name> file read error: unknown file type
           RESULT:      phred skips to next chromatogram (does not write phd
                        file)


    e. chromat reading

         succeeds)

           MESSAGE:     PROCESS: <chromat_name>
           RESULT:      phred continues

         fails)

           MESSAGE:     FILE_ERROR: <chromat_name> file read error: <error_description>
           RESULT:      phred continues processing chromatogram but does not
                        call bases **


    f. data checking (does chromat contain a nonzero trace?)

         succeeds)

           MESSAGE:      none
           RESULT:       phred continues

         fails)

           MESSAGES:    FILE_ERROR: <chromat_name> trace data missing

                        OR  

                        FILE_ERROR: <chromat_name> flat trace data
           RESULT:      phred continues but does not call bases **


    g. chromatogram identification (matching chromatogram primer ID string
       with an entry in phredpar.dat)
       Note: phred ignores match failures when '-nocall' option is used

         succeeds)

           MESSAGE:     none
           RESULT:      phred continues and calls bases

         fails)

           o  default: use neither '-process_nomatch' nor '-exit_nomatch'
              command line option

                MESSAGE:     FILE_SKIP_NOMATCH: <chromat_name> unable to match primer ID string
                RESULT:      phred skips to next chromatogram without
                             writing a phd file (or any other output file
                             entry)

              OR

           o  use '-exit_nomatch' command line option

                MESSAGE:     FATAL_ERROR: <chromat_name> unable to match primer ID string
                RESULT:      phred exits immediately

              OR

           o  use '-process_nomatch' option and '__no_matching_string__' entry
              exists in 'phredpar.dat' file

                MESSAGE:     FILE_PROCESS_NOMATCH: <chromat_name> using '__no_matching_string__'
                RESULT:      phred continues, using the chemistry, dye type,
                             and machine type given in the
                             '__no_matching_string__' entry

              OR

           o  use '-process_nomatch' option and no
              '__no_matching_string__' entry exists in 'phredpar.dat' file

                MESSAGE:     FATAL_ERROR: <chromat_name> unable to match primer ID string
                RESULT:      phred exits immediately



    ** the resulting phd file has no bases


10. Phd files

    Phred writes 'phd' files to store base calling information,
    including the sequence, quality values, and peak locations,
    when it is run with either the '-pd' or the '-p' options.
    Phred creates phd files with the name '<chromat_name>phd.1'
    where the '1' at the end of the name is the version number
    of the phd file for that chromatogram. It always writes
    version '1' phd files, whereas 'consed' writes phd files
    with higher version numbers (it increments the version 
    number each time it saves an edited read).

    The phd files phred creates begin with the line

      BEGIN_SEQUENCE <sequence_name>

    and end with the line

      END_SEQUENCE

    Enclosed between these lines phred writes a header data block,
    which is enclosed between lines with the labels 'BEGIN_COMMENT'
    and 'END_COMMENT', and a read data block, which is enclosed
    between lines with the labels 'BEGIN_DNA' and 'END_DNA'. Thus
    the overall file structure is (the lines are indented here)
    
      BEGIN_SEQUENCE <sequence_name>

      BEGIN_COMMENT

        [comment block]

      END_COMMENT

      BEGIN_DNA

        [read data block]

      END_DNA

      END_SEQUENCE

    The header data consists of a number of lines where each line begins
    with a label followed by a colon and one or more values.  Currently,
    the phd header has the following information

      header entry                 description
      ------------                 -----------
      CHROMAT_FILE: <string>       chromatogram file name
      ABI_THUMBPRINT: <n>          an integer assigned by the ABI software
      PHRED_VERSION: <string>      phred version used to create the file
      CALL_METHOD: <string>        <string>phred" unless run with '-nocall'
      QUALITY_LEVELS: <n>          maximum quality value permitted
      TIME: <string>               the time and date the file was created
      TRACE_ARRAY_MIN_INDEX: <n>   the index for the first trace point (always 0)
      TRACE_ARRAY_MAX_INDEX: <n>   the index for the last trace point (npoints-1)
      TRIM: <n1> <n2> <r>          read trim points. See (a) below.
      TRACE_PEAK_AREA_RATIO: <r>   trace noise level. See (b) below.
      CHEM: <string>               chromatogram sequencing chemistry type
      DYE: <string>                chromatogram sequencing dye type

    (a) the 'TRIM' values consist of the first and last bases in the high
        quality read segment (where the first base of the read is zero)
        and the error probability used to calculate the trim points. The
        modified Mott algorithm is used to calculate the the trim points.

    (b) the 'TRACE_PEAK_AREA_RATIO' is the ratio of the total uncalled-base
        peak area to the total called-base peak area within the high
        quality segment of the read. Thus this value indicates the level
        of the 'background' signal as a fraction of the called-base peak
        area. This value will tend to be relatively high for traces with
  
          o  little or no signal

          o  a mixture of inserts

    The read data block consists of one line for each read base. Each
    line has the three values

      o  the called base (a, c, g, t, or n)

      o  the quality value assigned to the base

      o  the location of the called-base peak
         in the trace

    The values are separated from each other by a single
    space.


11. Notes

    ESD Files
    ---------

    Phred reads processed MegaBACE ESD files.  It cannot read the raw
    ESD files.  It is important that you identify the dye chemistry
    correctly when you run the MegaBACE base caller so that phred can
    assign the right base to each trace. (This is important with ABI
    data as well.)

    In order to obtain the best phred quality value accuracy with
    MegaBACE data, phred must use the quality value lookup tables
    designed for this data.  Phred identifies the sequencing machine
    by reading the primer ID string in the chromatogram and matching
    it with an entry in the phredpar.dat file.  The matching entry
    lists the chemistry, dye, and sequencing machine types. For example,
    the primer ID string of the form 'ET Primer' identifies a
    chromatogram as ET dye primer data generated on a MegaBACE
    sequencing machine. You can check that phred interprets the
    primer ID string correctly by using the '-v 63' option to have
    phred write diagnostic information to the screen.


    LI-COR Data
    -----------

    Band Spread Ratio (BSR)

    Phred reads SCF files created by the LI-COR gel processing software
    and has quality value lookup tables calibrated for traces processed
    with Band Spread Ratio (BSR) of 2.2. The LI-COR software writes a
    primer ID string in the SCF file that indicates the BSR value
    used in the trace processing, which for BSR=2.2 is 
    'DyePrimer{LI-COR_IR_2.2}'. Accordingly, the phredpar.dat file in
    this distribution has an entry with this string, which enables
    phred to recognize LI-COR traces processed with BSR=2.2, and to
    use the quality value lookup table designed for this LI-COR data.
    Phred has a quality value lookup table for data processed with
    BSR=2.2 only so the quality values for LI-COR traces processed
    with other BSR values will have reduced accuracy.


12. References

    Brent Ewing, LaDeana Hillier, Michael C. Wendl, and Phil Green.
    Base-calling of automated sequencer traces using phred. I. Accuracy
    assessment. 1998. Genome Research 8:175-185.

    Brent Ewing and Phil Green
    Base-calling of automated sequencer traces using phred. II. Error
    probabilities. 1998. Genome Research 8:186-194.