*|***************************************************************************|*
*| |*
*| 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

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)

o use '-exit_nomatch' command line option

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

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

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.