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release 2.4.0 2017-11-03 ===============================================
This release adds a new feature to primer3_core and related C code.
PCR PRIMER DESIGN TEMPLATE MASKING BEFORE PRIMER DESIGN. This feature
helps to prevent designing primers to template regions that are repetitive.
Primers with more binding sites tend to have higher failure rates. The
masking is based on statistical model (see Documentation for details).
NEW INPUT TAGS AND DEFAULT VALUES:
PRIMER_MASK_TEMPLATE=0
PRIMER_FAILURE_RATE=0.1
PRIMER_MASK_5P_DIRECTION=1
PRIMER_MASK_3P_DIRECTION=1
PRIMER_MASKING_LIST_PREFIX=homo_sapiens
PRIMER_WT_FAILURE_RATE=0.0
PRIMER_KMER_LISTS_PATH=../kmer_lists/
See the documentation for details. The tag PRIMER_MASK_TEMPLATE directs
primer3_core to premask template sequence for primer design.
NEW EXECUTABLE: primer3_masker
masker masks repetitive regions in INPUTFILE (given as fasta and/or multiple
fasta format) based on statistical approach.
IMPORTANT: because PRIMER_MASK_TEMPLATE=1,
PRIMER_KMER_LISTS_PATH must point to the right location.
This tag specifies the path to the directory that contains kmer list files
used by the template masking approach. The default location: ../kmer_lists/ If the
list files are not in that location, be sure to set
PRIMER_KMER_LISTS_PATH
By default, directory ../kmer_lists/ contains example kmers. Software for
making kmers can be downloaded http://bioinfo.ut.ee/FastGT/index.php?r=site/index
Premade list files can be downloaded from: http://primer3.ut.ee/lists.htm
In 2016:
Major correction:
1. The long primer3_core comand line options with single hyphen (-myargs)
are depreciated. Use double hyphens (--myargs) instead.
2. The PRIMER_TASK pick_discriminative_primers was modified. Now the primer
border the provided SEQUENCE_TARGET and do not overlap by one basepair.
Minor fixes:
1. The test scripts were updated to use the default valgrind path and to
use the --type comand line arguments instead of -type.
2. In thal.c the function finite() was replaced by the c99 isfinite().
3. In libprimer3 all abort() functions were removed and error handling
implemented.
release 2.3.7 2016-02-18 ===============================================
Major correction:
In some cases, ntthal resulted with two different dimer structures depending
on the order of the two oligo sequences were given by the user.
For example:
ntthal -s1 ACTGGAAAGAGGAAGGACTGGT -s2 TGGGACATCTCTATACCTTCCTCT
Calculated thermodynamical parameters for dimer: dS = -207.817 dH = -73000 dG = -8545.56 t = 26.0559
SEQ ACTGGAAA ACTGGT---------
SEQ GAGGAAGG
STR CTCCTTCC
STR T ATATCTCTACAGGGT
If the order of two oligo sequences change places, ntthal resulted with
other structure and other values of thermodynamic parameters.
ntthal -s2 ACTGGAAAGAGGAAGGACTGGT -s1 TGGGACATCTCTATACCTTCCTCT
Calculated thermodynamical parameters for dimer: dS = -290.416 dH = -93600 dG = -3527.63 t = 13.4587
SEQ TGGGA TCTCTATA T-------
SEQ CA CCTTCCTC
STR GT GGAAGGAG
STR TG CA------ AAAGGTCA
What was modified in the code:
1. calculation of melting temperature values were replaced with calculation
of dG values as calculating Tm values cannot result always the most stabile
dimer as dimers melting temperature depends significantly on salt correction
2. Dimers 5` end stability was calculated only when the most stabile dimer
was found. Now 5` ends stability is also considered at each dynamic
programming stage.
3. Some values of thermodynamic table were changed as they were not
symmetrical
4. Although AT dangling ends are more stable than AT pairing, ntthal now
prefers AT pairing to AT dangling end.
Now primer3 results:
ntthal -s1 ACTGGAAAGAGGAAGGACTGGT -s2 TGGGACATCTCTATACCTTCCTCT
Calculated thermodynamical parameters for dimer: dS = -198.019 dH = -69700 dG = -8284.27 t = 24.4823
SEQ ACTGGAA ACTGGT---------
SEQ AGAGGAAGG
STR TCTCCTTCC
STR ATATCTCTACAGGGT
ntthal -s2 ACTGGAAAGAGGAAGGACTGGT -s1 TGGGACATCTCTATACCTTCCTCT
Calculated thermodynamical parameters for dimer: dS = -198.019 dH = -69700 dG = -8284.27 t = 24.4823
SEQ TGGGACATCTCTATA -------
SEQ CCTTCCTCT
STR GGAAGGAGA
STR TGGTCA AAGGTCA
We have also added tests for this bug.
Minor fixes:
1. updated oligotm executable to accept float values as input for the
concentration of monovalent cations and for concentration of DNA strands.
release 2.3.6 2013-10-22 ===============================================
Important changes:
1. Four new input tags were added:
- PRIMER_MUST_MATCH_THREE_PRIME
- PRIMER_MUST_MATCH_FIVE_PRIME
- PRIMER_INTERNAL_MUST_MATCH_THREE_PRIME
- PRIMER_INTERNAL_MUST_MATCH_FIVE_PRIME
The MUST_MATCH tags allow to define 5 nt patterns which the first and
the last five bases of a primer or oligo have to match. For example,
the pattern "NNGTN" would require the 3rd and 2nd last bases to be G and
T respectively.
Minor fixes:
1. Fixed two bugs in ntdpal and oligotm that caused segmentation fault
whenever these binaries were called without specifying a given
option value.
release 2.3.5 2013-01-03 ===============================================
Important changes:
1. The tag PRIMER_THERMODYNAMIC_ALIGNMENT was replaced by two new tags:
- PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT which governs hairpin
and oligo-oligo interactions. Therefore, whenever it is set to 1
(the default) thermodynamic alignments will be used for oligo-
oligo interactions and hairpins.
- PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT which governs the
oligo-template interactions and, when set to 1 (default is 0), will
cause primer3 to use the thermodynamic alignment against templates.
The reason for this change is to provide the option of using the
old alignment for oligo-template interactions when the thermodynamic
alignment is too slow or the template is too long (there is a hard
limit THAL_MAX_SEQ=10k on the length of sequences involved in
thermodynamic alignments).
1.1 Added corresponding functions to libprimer3.c and libprimer3.h:
p3_set_gs_primer_thermodynamic_oligo_alignment
p3_set_gs_primer_thermodynamic_template_alignment
2. Modified the error handling of non-memory related errors that
can occur during the thermodynamic alignment. They are no longer
fatal errors.
3. Made a performance improvement for the case when thermodynamic
alignment is used for templates: the alignment is done only once
for each oligo and the result is kept. This way we avoid performing
the same expensive alignment more than once.
4. Removed io_version 3, which was only kept for backward compatibility
with very old versions of primer3.
5. Fixed an error in which PRIMER_DNTP_CONC > PRIMER_SALT_DIVALENT causes
Primer3 to crash.
6. Fixed a bug that made Primer3 not generate results in some cases
when PRIMER_FIRST_BASE_INDEX is negative.
Minor corrections:
1. Added function p3_set_max_end_gc (sets max_end_gc in the
primer_global_settings struct).
2. Removed intentional compile time division by zero in thal.c if
macro INFINITY is defined.
3. Updated text in error messages from function thal() to make them
more informative.
release 2.3.4 2012-05-30 ===============================================
Major correction:
1. Fixed error in which PRIMER_..._..._TEMPLATE_MISPRIMING_TH was
calculated incorrectly in libprimer3.c
release 2.3.3 2012-05-18 ===============================================
Major correction:
1. Fixed error in which non-default values of monovalent and divalent
salt concentrations and non-default values of dNTP and DNA
concentrations were being ignored in thermodynamic calculations.
release 2.3.2 2012-03-15 ===============================================
Minor corrections:
1. _Removed_ warning if PRIMER_INTERNAL_SALT_DIVALENT > 0.0 but
PRIMER_INTERNAL_DNTP_CONC <= 0.0. (dNTPs not used in hybridizations.)
release 2.3.1 2012-02-27 ===============================================
Minor corrections:
1. Added warnings if PRIMER_SALT_DIVALENT > 0.0 but PRIMER_DNTP_CONC
<= 0.0 or PRIMER_INTERNAL_SALT_DIVALENT > 0.0 but
PRIMER_INTERNAL_DNTP_CONC <= 0.0.
2. No longer an error if a settings file has
PRIMER_THERMODYNAMIC_ALIGNMENT=1 but does not have a valid
PRIMER_THERMODYNAMIC_PARAMETERS_PATH.
(PRIMER_THERMODYNAMIC_PARAMETERS_PATH can then be set in the regular
boulder IO input stream.)
3. Updates to documetation on salt corrections.
4. Corrections to primer3web_v3_0_0_default_settings.txt (removed
non-existent tags and removed the setting of
PRIMER_THERMODYNAMIC_PARAMETERS_PATH)
5. Removed un-needed files from distribution .tar.gz file.
6. Removed the -i option from the oligotm executable, as the
necessary information is in the Primer3 manual.
7. Updated PRIMER_PRODUCT_SIZE_RANGE in
primer3web_v3_0_0_default_settings to same value as in previous
version of primer3web.
release 2.3.0 2012-02-03 ===============================================
1. *** We changed the following default values:
1.1 Changing default oligo temperature calculations
PRIMER_TM_FORMULA=1 (was 0)
PRIMER_SALT_CORRECTIONS=1 (was 0)
1.2 Making thermodynamic secondary structure calculations the default:
PRIMER_THERMODYNAMIC_ALIGNMENT=1 (was 0)
1.3 The following need reasonable values to support the changes in 1.1
and 1.2 above:
PRIMER_SALT_DIVALENT=1.5 (was 0.0)
PRIMER_DNTP_CONC=0.6 (was 0.0)
1.4 To make old defaults easily accessible, we added a command line
argument, --default_version=1, which directs primer3_core to use the
old defaults. --default_version=2 directs primer3_core to use the new
defaults. The default is --default_version=2.
1.5 IMPORTANT: because PRIMER_THERMODYNAMIC_ALIGNMENT=1,
PRIMER_THERMODYNAMIC_PARAMETERS_PATH must point to the right location.
This tag specifies the path to the directory that contains all the
parameter files used by the thermodynamic approach. In Debian, there
are two *default* locations that are tested if this tag is not
defined: ./primer3_config/ and /etc/primer3_config/. For Windows,
there is only one default location: .\primer3_config\. If the the
parameter files are not in one these locations, be sure to set
PRIMER_THERMODYNAMIC_PARAMETERS_PATH
1.6 Changed default for
PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0 (was 1)
(0 is almost always the the behavior one wants.)
1.7 To get the behavior of -default_version=1 when -default_version=2
set the following:
PRIMER_TM_FORMULA=0
PRIMER_SALT_CORRECTIONS=0
PRIMER_THERMODYNAMIC_ALIGNMENT=0
PRIMER_SALT_DIVALENT=0.0
PRIMER_DNTP_CONC=0.0
PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=1
1.8 To get the behavior of -default_version=2 when -default_version=1
set the following:
PRIMER_TM_FORMULA=1
PRIMER_SALT_CORRECTIONS=1
PRIMER_THERMODYNAMIC_ALIGNMENT=1
PRIMER_SALT_DIVALENT=1.5
PRIMER_DNTP_CONC=0.6
PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0
2. We changed the NULL value for SEQUENCE_FORCE_{LEFT,RIGHT}_START_END to
-1000000, and made it an error to select PRIMER_FIRST_BASE_INDEX <=
this value. This is to correct an error when
SEQUENCE_FORCE_{LEFT,RIGHT}_START_END was -1 (value previously used to
indicate a NULL) but PRIMER_FIRST_BASE_INDEX was < 0, which caused the
intended NULL value (-1) to be treated as a constraint on primer
location (a constraint that was not possible to satisfy).
3. We changed the PRIMER_TASK called 'pick_detection_primers' to
'generic' while retaining 'pick_detection_primers' as an alias for
backward compatibility.
4. The code now uses 'end' alignments when assessing template mispriming
using thermodynamic alignments. This is consistent with the approach
taken with the previous alignment algorithm and with checking for
mispriming against repeat libraries.
4. We removed PRIMER_PAIR_MAX_HAIRPIN_TH (which was ignored previously).
5. Primer3 now requires the user to set SEQUENCE_TARGET, not
SEQUENCE_INCLUDED_REGION when PRIMER_TASK=pick_discriminative_primers
5. When PRIMER_TASK=pick_discriminative_primers or
PRIMER_TASK=pick_cloning_primers the value of SEQUENCE_INCLUDED_REGION
is no longer changed to the entire input sequence.
6. The handling of divalent cations when PRIMER_SALT_CORRECTIONS=2
(not the default and not the recommended value) has been updated. The
rationale is that, when divalent cations are present, the formula by
Owczarzy et al., 2004 (used previously by Primer3) can be improved
upon as described in (Ahsen et al., 2010; Owczarzy et al.,
2008). Therefore we have updated the melting temperature calculation
to follow the scheme in Figure 9 of (Owczarzy et al., 2008). Please
find references to these papers in the Primer3 manual.
7. In addition, several error corrections:
7.1 Corrected a short, fixed-size buffer for the file/path names specified
by the command line arguments -output, -error and for the settings
file.
7.2 Two other corrected errors were in p3_set_gs_primer_self_end and
p3_set_gs_primer_internal_oligo_self_end, which erroneously multiplied
their 'val' arguments by 100.
7.3 Primer3 now detects and handles the situation in which user-supplied
primers (SEQUENCE_PRIMER and SEQUENCE_PRIMER_REVCOMP) have the left
primer to the right of the right primer. Also issue a warning if
user-supplied primers occur in more than one location in the template.
release 2.2.3 2010-07-08 ===============================================
Minor error corrections, minor cleanup.
This is a stable release.
release 2.2.2 beta 2010-03-04 ==========================================
This release adds a performance enhancement for inputs involving
SEQUENCE_PRIMER_PAIR_OK_REGION_LIST.
This release adds two new tags, PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE
and PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE
release 2.2.1 alpha 2009-10-28 ==========================================
This release adds several performance enhancements.
release 2.2.0 alpha 2009-10-23 ==========================================
This release adds multiple new features to primer3_core and related
C code.
The enhancements fall under several categories:
1. PRIMER/OLIGO SECONDARY STRUCTURE AND PRIMER-DIMER AND TEMPLATE
MISPRIMING PREDICTION
We added capabilities for predicting secondary structure,
primer-dimer, and template mispriming based on a thermodynamic model.
INPUT/OUTPUT TAGS:
Added tags PRIMER_THERMODYNAMIC_ALIGNMENT,
PRIMER_INTERNAL_MAX_{HAIRPIN,SELF_ANY_TH,SELF_END_TH,TEMPLATE_MISHYB_TH},
PRIMER_INTERNAL_WT_{HAIRPIN,SELF_ANY_TH,SELF_END_TH,TEMPLATE_MISHYB_TH},
PRIMER_MAX_{HAIRPIN,SELF_ANY_TH,SELF_END_TH,TEMPLATE_MISPRIMING_TH},
PRIMER_PAIR_MAX_{COMPL_ANY_TH,COMPL_END_TH,HAIRPIN,TEMPLATE_MISPRIMING_TH},
PRIMER_PAIR_WT_{COMPL_ANY_TH,COMPL_END_TH,HAIRPIN,TEMPLATE_MISPRIMING_TH},
PRIMER_WT_{HAIRPIN,SELF_ANY_TH,SELF_END_TH,TEMPLATE_MISPRIMING_TH}
See the documentation for details. The tag
PRIMER_THERMODYNAMICAL_ALIGNMENT=1 directs primer3_core to use the
thermodynamic alignments.
NEW EXECUTABLE: ntthal
ntthal is analogous to ntdpal. Between two sequences, ntthal finds
alignment/sec structure, that has the highest melting
temperature. Ntthal is based on nearest-neighbor thermodynamical
approach. For input parameters to ntthal, execute: ./ntthal (without
arguments).
2. NEW COMMAND LINE FLAGS AND ARGUMENTS
primer3_core now has a more conventional command line. For example,
you can type
primer3_core -output my_output -error my_error my_input
and primer3_core will take its input from my_input and put
its outputs in the files my_output and my_error. There
is also a command line flag -echo_settings_file to cause
the settings file contents to be echoed to output.
3. NEW INPUT TAGS TO CONTROL PRIMER LOCATION
See the documentation for SEQUENCE_PRIMER_PAIR_OK_REGION_LIST. Tags
SEQUENCE_OVERLAP_JUNCTION_LIST,
PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION,
PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION have _replaced_
SEQUENCE_PRIMER_OVERLAP_POS and PRIMER_POS_OVERLAP_TO_END_DIST. See
the documentation.
4. PERFORMANCE IMPROVEMENTS
release 2.0.0 alpha 2008-12-15 ==========================================
This is major release, involving enhancements to functionality,
software architecture, and input and output conventions.
INPUT/OUTPUT CHANGES:
* A new command line flag, -io_version=3 provides backward compatible
behavior for boulder IO input, and boulder IO output that is
backward compatible except for error messages, warnings, and the
text of PRIMER...EXPLAIN tags.
* -io_version=3 IS NOT the default -- you must specify it on the
command line.
For the new default IO, we renamed many Boulder IO INPUT/OUTPUT TAGS
for clarification and easier integration into scripts
NEW OR IMPROVED FUNCTIONALITY:
Primer3 'settings' files were added. A settings file can be read in
at program start up to set global parameters. Our intent is that
parameter values suitable for particular primer design objectives
can be easily stored and re-used.
We added new "tasks" such as selection of primer lists, sequencing
primers, or cloning primers, or the checking of primers or primer pairs
in the absence of a target sequence. See tag PRIMER_TASK.
It is now possible to request only unique primers when multiple
primer pairs are requested. (In the past, each _pair_ was unique,
but the same primer might occur in more than one pair). The user
also has some control over what constitutes a 'unique' primer. See
tag PRIMER_MIN_THREE_PRIME_DISTANCE.
When an illegal primer pair is specified for checking primer3, the
program now assess it. In the past, primer3 assessed individual
primers if they violated a constraint, but in some cases not pairs
that violated constraints on pairs (such as product size).
We added new functionality with tags
SEQUENCE_PRIMER_OVERLAP_POS, PRIMER_POS_OVERLAP_TO_END_DIST,
PRIMER_MAX_END_GC. Please see the documentation.
We added tags PRIMER_PAIR_MAX_COMPL_END and
PRIMER_PAIR_MAX_COMPL_ANY, which previously took the same values as
PRIMER_SELF_{END,ANY}
OTHER CHANGES TO BEHAVIOR:
We corrected a few errors and a mis-feature in the ways in which
primer3 searches for primer pairs. Consequently, the latest version
of primer3 will return different sets of primer pairs in a few
instances.
INTERNAL CHANGES:
It is now possible to use the main primer3 functionality from a
single C function. We cleanly separated the functionality of reading
in Boulder IO records from the primer choosing / designing
functionality. We also substantially simplified the organization
and documentation of the main primer picking code, now in
libprimer3.[ch]. The file libprimer3.h specifies the external
interface, although it is not separately documented. It is also
possible to create a binary library file that provides this
functionality (libprimer3.a or libprimer3.so in Unix/Linux). All C
code is ANSI C.
release 1.1.2 2007-06-10 ================================================
Updates to dpal.c so that no error forces an exit.
Adaptation in primer3_main.c and ntdpal_main.c to
updated error handling in dpal().
Arranged for primer3_main.c align() to check for
and circumvent local alignments to "targets" < nucleotides
long, and added tests for this condition.
Comment and code readability updates in dpal.h and dpal.c
release 1.1.1 3/37/2007 ==================================================
Minor corrections
release 1.1.0 1/17/2007 ==================================================
Added tags PRIMER_TM_SANTALUCIA, PRIMER_SALT_CORRECTIONS,
PRIMER_LOWERCASE_MASKING, PRIMER_DIVALENT_CONC,
and PRIMER_DNTP_CONC. See the README for
details of what these tags do. Briefly, the first two
tags allow the use of modern melting temperature
calculations. New tests for this functionality
were added to the tests/ subdirectory
NOTE: The oligotm executable now takes additional
arguments.
Cleaned up Makefile, and created Makefile for the test
directory with a target to clean up after tests
Most (but not all) temporary files created by the
tests now end in .tmp or .tmp2
Tried to create more informative test for
MAX_PRIMER_LENGTH > DPAL_MAX_ALIGN in primer3_main.c
NOTE: The oligtotm() function is now provided as
a library, liboligotm.
NOTE: non-backward compatible change: removed support of
flag -2x_compat.
release 1.0.1 11/06 ==================================================
Identical to 1.0b except for release number
release 1.0b 11/06 ==================================================
Corrected bug involving check for template mispriming
Minor code cleanup
release 1.0a 9/06 ==================================================
No new functionality (bug fixes and minor code cleanup only)
Bug fixes:
* Corrected calculation of ..._TEMPLATE_MISPRIMING when
INCLUDED_REGION was set (thanks to Tomoaki Nishiyama, Advanced
Sciences Research Center, Kanazawa University, Japan).
* Corrected error that caused crash when
PRIMER_WT_TEMPLATE_MISPRIMING was set (thanks to Peng Yu,
Functional Genomics Group II, Chinese National Human Genome
Center, Beijing).
* Added olgiotm_main.c (for executable oligotm)
release 1.0 6/04 ==================================================
Added ability to search source sequence (template) for
mispriming or mishybing sites.
Added _experimental_ facility to calculate Tm on long
oligos using a GC% based formula.
Miscellaneous fixes and cleanup, including:
* Avoiding closing NULL file handles when a mispriming or mishyb
library is missing
* Correction to long_seq_tm() in oligotm.c plus addition of
related tests.
* Gave user the ability to _not_ treat IUB/IUPAC ambiguity
codes as a consensus while matching against mispriming
or mishyb libraries. Inability to turn off this "feature"
caused severe problems when libraries contained strings
of N's: NNNNNNNNN, since if N is treated as a consensus,
any base matches.
release 0.9 9/98 ==================================================
Corrected errors in calculation of amplicon Tm.
release 0.8 6/98 ==================================================
Added flexible objective function. Various tags governing
the penalty weights are not fully documented in this
release.
Added much changed web interface.
Added ability to select only a single
left or right primer or hyb probe.
Made it possible to continue even if
PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_INPUT is
not legal.
Added PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_GC_PERCENT
output tags.
release 0.7 5/98 ==================================================
Fixed the 'case problem' for
PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_INPUT (and at the
same time a related bug).
Added experimental PRIMER_START_CODON_POSITION tag
and related outputs. This feature should still be
considered experimental and the user should scrutinize
the output.
Added PRIMER_PRODUCT_{MAX,MIN}_TM and related outputs.
release 0.6 ==================================================
Bug fixes (most introduced as part of 0.5 enhancements).
release 0.5 ==================================================
** Non-Backward compatible change:
Fixed inconsistency in output format;
PRIMER_INTERNAL_n_OLIGO_SEQUENCE
-> PRIMER_INTERNAL_OLIGO_n_SEQUENCE.
The old format can be preserved by
compiling with the precompiler
symbol USE_OLD_FORMAT_MISTAKE defined.
Added end-anchored local alignment option to dpal.
Removed some un-needed dpal implementations.
Added end-anchored local alignments for checking
mispriming libraries.
Added mispriming estimates for primer _pairs_
(see PRIMER_PAIR_MISPRIMING_LIBRARY),
with related new output tags. Trivial change
in maximum allowable library sequence weight.
Added an experimental primer position component to
the objective function (PRIMER_{IN,OUT}SIDE_PENALTY),
with related new output tags.
Added sequence quality scores as constraints
in primer picking (PRIMER_MIN_{,END_}_QUALITY,
PRIMER_QUALITY_RANGE_{MIN,MAX}) with related
new output tags.
Added more error reporting to primer3.cgi when primer3
exits with a non-0 termination status.
Fixed bug when non-0, non-1 value was supplied
for pick internal oligos.
Added PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_INPUT
for checking or for designing around existing primers or
internal oligos.
Added error checking for duplicates of certain
tags, such as SEQUENCE.
Added PRIMER_MAX_END_STABILITY and related changes
to output.
release 0.4b ==================================================
Corrected bug in parsing of INCLUDED_REGION tag.
release 0.4 ==================================================
Added optional check of oligos against "mispriming" or
"mishyb" libraries with related changes to formated output
and to the contents of primer list files.
Modified search algorithm to compute complementarity/
similarity measures as late as possible.
Got rid of quotes (") around error and warning strings.
Fixed numerical overflow on input sequences longer than
MAX_SHRT. (Thanks to Pete Young for finding this one.)
release 0.3 ==================================================
Changes to dpal to make it substantially faster on the maxgap=1
case for local and end-anchored complementarity measures.
Removed restriction on maximum sequence length (by removing
ftar, fexcl, fexcl_int, fnn, fn, fgc arrays, which necessitated
internal changes to methods for calculating overlap with
excluded regions and targets and to calculate spanning of
targets.
Modified 'primer list' format (clean up and hopefully got rid
of spurious regression test discrepancy on some systems due to
differences in rounding).
Exit codes for some errors changed.
Text of error messages for PRIMER_PRODUCT_SIZE -> PRIMER_PRODUCT_SIZE_RANGE.
Some errors that should have been global (fatal) were not.
These were fixed and error handling rationalized somewhat.
An error in the display of the start position of targets, and
excluded regions in formated output was corrected.
An error in which overlapping product size ranges caused
primer3 to return duplicate primer pairs was corrected.
Made regression tests more complete.
Made the maximum number of primer pairs to return a
parameter (PRIMER_NUM_RETURN).
Added optional n-based indexing in input and output
(PRIMER_FIRST_BASE_INDEX=n). Has not been tested
for n < 0.
release 0.2b (10/11/96) ==================================================
Added PRIMER_WARNING tag.
Made it legal for excluded regions and targets to lie outside
of included region. Documentation fixes (especially to
PRIMER_DNA_CONC).
release 0.2a ==================================================
Tweaks to formated output to make it more complete
(included complementarity measures); adjusted tests.
release 0.2 ==================================================
Made 0-length excluded region legal, and adjusted tests.
Tweaked error reporting of negative-length excluded regions,
internal oligo excluded regions, and target.
==============================================================
==============================================================
Compared to non-primer3 progenitors:
Compared to primer 0.5
---------------
Completely different input format.
It has been reported the 0.5 deleted Ns when they occurred in
primers.
More stringent self-complementarity defaults.
Primer3 selects internal oligos on request (and produces .int
files if requested).
Compared to both primer 0.5 and primer v2
---------------------------
The format of the contents of .for, .rev (and .int) files is
different.
Primer3 returns a user-specifiable number of primer pairs (or
triples) sorted by "goodness".
Primer3 will find a primer pair if any acceptable pair exists.
Optional n-based indexing into source sequence.
Use of sequence quality and 3' stability as constraints in primer
picking. Optional positional component to objective function.
Compared to primer v2
-------------
Tag name changes. However, primer3 should understand most or
all Primer v2 input tags, and should produce v2-compatible output
tag names when the -v2_compat command-line switch is used.
The one exception is that the PRIMER_RECOMMEND tag is no longer
produced. Instead primer3 produces the PRIMER_x_EXPLAIN output
tags. The format of the data in this tags is different from the
data in v2's PRIMER_RECOMMEND output tag.
Numerous fixes.
Uses the PRIMER_SELF_ANY and PRIMER_SELF_END parameters to govern
maximum allowable complementarity between left and right primers,
as well as complementarity between copies of a single oligo or
within a single oligo. This behaviour is very close to that of
primer 0.5; self complementarity calculations in v2 were
unreliable.
Primer3 produces much more output information, including the TMs
and self complementarity measures of selected primers.
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