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---
layout: default
title: Manual
---
<div id="main">
<h2>CONTENTS</h2><p>
<a href="#copyrightLicense">1. COPYRIGHT AND LICENSE</a><br>
<a href="#introduction">2. INTRODUCTION</a><br>
<a href="#citationRequest">3. CITING PRIMER3</a><br>
<a href="#licenseExplain">4. FAIR USE OF PRIMER3</a><br>
<a href="#differencePrimer3vsPrimer3Plus">5. DIFFERENCE BETWEEN PRIMER3 AND PRIMER3PLUS</a><br>
<a href="#changesFrom2.2.3">6. CHANGES FROM VERSION 2.2.3</a><br>
<a href="#installLinux">7. INSTALLATION INSTRUCTIONS - UNIX/LINUX</a><br>
<a href="#installMac">8. INSTALLATION INSTRUCTIONS - Mac OSX</a><br>
<a href="#installWindows">9. INSTALLATION INSTRUCTIONS - WINDOWS</a><br>
<a href="#systemRequirements">10. SYSTEM REQUIREMENTS</a><br>
<a href="#invokingPrimer3">11. INVOKING primer3_core</a><br>
<a href="#commandLineTags">12. COMMAND LINE ARGUMENTS</a><br>
<a href="#inputOutputConventions">13. INPUT AND OUTPUT CONVENTIONS</a><br>
<a href="#example">14. AN EXAMPLE</a><br>
<a href="#migrateTags">15. HOW TO MIGRATE TAGS TO IO VERSION 4</a><br>
<a href="#sequenceTags">16. "SEQUENCE" INPUT TAGS</a><br>
<a href="#globalTags">17. "GLOBAL" INPUT TAGS</a><br>
<a href="#programTags">18. "PROGRAM" INPUT TAGS</a><br>
<a href="#calculatePenalties">19. HOW PRIMER3 CALCULATES THE PENALTY VALUE</a><br>
<a href="#fileFormat">20. PRIMER3 SETTINGS FILE FORMAT</a><br>
<a href="#outputTags">21. OUTPUT TAGS</a><br>
<a href="#exampleOutput">22. EXAMPLE OUTPUT</a><br>
<a href="#pickAdvice">23. ADVICE FOR PICKING PRIMERS</a><br>
<a href="#primerBinding">24. GENERAL THOUGHTS ON PRIMER BINDING</a><br>
<a href="#cautions">25. CAUTIONS</a><br>
<a href="#findNoPrimers">26. WHAT TO DO IF PRIMER3 CANNOT FIND ANY PRIMERS?</a><br>
<a href="#earlierVersions">27. DIFFERENCES FROM EARLIER VERSIONS</a><br>
<a href="#exitStatusCodes">28. EXIT STATUS CODES</a><br>
<a href="#webInterface">29. PRIMER3 WWW INTERFACES</a><br>
<a href="#acknowledgments">30. ACKNOWLEDGMENTS</a><br>
</p>
<h2><a id="copyrightLicense">1. COPYRIGHT AND LICENSE</a></h2>
<pre>Copyright (c) 1996 - 2022
Whitehead Institute for Biomedical Research, Steve Rozen
(<a href="http://purl.com/STEVEROZEN/">http://purl.com/STEVEROZEN/</a>), Helen Skaletsky, Triinu Koressaar,
Maido Remm and Andreas Untergasser. All rights reserved.
This file is part of the Primer3 suite and libraries.
The Primer3 suite and libraries are free software;
you can redistribute them and/or modify them under the terms
of the GNU General Public License as published by the Free
Software Foundation; either version 2 of the License, or (at
your option) any later version.
This software is distributed in the hope that it will be useful,
but WITHOUT ANY WARRANTY; without even the implied warranty of
MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
GNU General Public License for more details.
You should have received a copy of the GNU General Public License
along with this software (file gpl-2.0.txt in the source
distribution); if not, write to the Free Software
Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA
THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS
"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 COPYRIGHT
OWNERS OR CONTRIBUTORS 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.
</pre>
<h2><a id="introduction">2. INTRODUCTION</a></h2>
<p>Primer3 picks primers for PCR reactions, considering as criteria:
<br>
o oligonucleotide melting temperature, size, GC content,
and primer-dimer possibilities,<br>
<br>
o PCR product size,<br>
<br>
o positional constraints within the source (template) sequence, and<br>
<br>
o possibilities for ectopic priming (amplifying the wrong sequence)<br>
<br>
o many other constraints.<br>
<br>
All of these criteria are user-specifiable as constraints, and
some are specifiable as terms in an objective function that
characterizes an optimal primer pair.<br>
<h2><a id="citationRequest">3. CITING PRIMER3</a></h2>
<p>We request but do not require that use of this software be cited in
publications as<br>
<br>
* Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M and Rozen SG.<br>
Primer3--new capabilities and interfaces.<br>
Nucleic Acids Res. 2012 Aug 1;40(15):e115.<br>
The paper is available at
<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424584/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424584/</a><br>
<br>
and <br>
<br>
* Koressaar T and Remm M.<br>
Enhancements and modifications of primer design program Primer3.<br>
Bioinformatics 2007;23(10):1289-1291.<br>
The paper is available at
<a href="https://www.ncbi.nlm.nih.gov/pubmed/17379693">https://www.ncbi.nlm.nih.gov/pubmed/17379693</a><br>
<br>
If you use masker function, please cite:<br>
* Koressaar T, Lepamets M, Kaplinski L, Raime K, Andreson R and Remm M.<br>
Primer3_masker: integrating masking of template sequence with primer design software.<br>
Bioinformatics 2018;34(11):1937-1938.<br>
The paper is available at
<a href="https://www.ncbi.nlm.nih.gov/pubmed/29360956">https://www.ncbi.nlm.nih.gov/pubmed/29360956</a><br>
<br>
Source code available at <a href="https://github.com/primer3-org/primer3">https://github.com/primer3-org/primer3</a>.</p>
<h2><a id="licenseExplain">4. FAIR USE OF PRIMER3</a></h2>
<p>
The development of Primer3 is promoted by a small group of
enthusiastic scientists mainly in their free time.They do not gain
any financial profit with Primer3.<br>
<br>
There are two groups of Primer3 users: end users, who run
Primer3 to pick their primers and programmers, who use Primer3
in their scripts or software packages. We encourage both to use
Primer3.
<br>
<br>
If you are an end user, we request but do not
require that use of this software be cited in publications
as listed above under CITING PRIMER3.
<br>
<br>
If you are a programmer, you will see that Primer3 is now
distributed under the GNU General Public License, version 2 or
(at your option) any later version of the License (GPL2).
As we understand it, if you include parts of the Primer3 source
code in your source code or link to Primer3 binary libraries in
your executable, you have to release your software also under
GPL2. If you only call Primer3 from your software and interpret
its output, you can use any license you want for your software.
If you modify Primer3 and then release your modified software,
you have to release your modifications in source code under
GPL2 as well.
<br>
<br>
We chose GPL2 because we wanted Primer3 to evolve and for the
improvements to find their way back into the main distribution.
If you are programming a new web interface which runs Primer3,
please include in the about page of the tool the sentence
"<your software name> uses Primer3 version ...".
Please consider releasing your software under GPL2 as well,
especially if you do not want to maintain it in the future.
<br>
<br>
There is no need to ask us for permission to include Primer3
in your tools.</p>
<h2><a id="differencePrimer3vsPrimer3Plus">5. DIFFERENCE BETWEEN PRIMER3 AND PRIMER3PLUS</a></h2>
<p>Primer3Plus is a web interface to Primer3, so if you pick
primers with Primer3Plus, it will collect and reformat your input, run
the command line tool Primer3, collet and reformat it's output and
display it to you.<br>
In principle, both tools would give you the same output. In practice,
the default settings ob both tools differ. While Primer3 default
settings are usually kept for backward compatibility, the Primer3Plus
default settings are adapted for regular wetlab use.</p>
<h4>Primer3 Values:</h4>
<pre>
PRIMER_PRODUCT_SIZE_RANGE=100-300
PRIMER_SECONDARY_STRUCTURE_ALIGNMENT=0
PRIMER_NUM_RETURN=5
PRIMER_MAX_HAIRPIN_TH=47.0
PRIMER_INTERNAL_MAX_HAIRPIN_TH=47.0
PRIMER_MAX_END_STABILITY=100.0
PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE=-1
PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE=-1
PRIMER_EXPLAIN_FLAG=0
PRIMER_LIBERAL_BASE=0
PRIMER_FIRST_BASE_INDEX=0
PRIMER_MAX_TEMPLATE_MISPRIMING=-1.00
PRIMER_MAX_TEMPLATE_MISPRIMING_TH=-1.00
PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING=-1.00
PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=-1.00
</pre>
<h4>Primer3Plus Values:</h4>
<pre>
PRIMER_PRODUCT_SIZE_RANGE=501-600 601-700 401-500 701-850 851-1000 1001-1500
1501-3000 3001-5000 401-500 301-400 201-300
101-200 5001-7000 7001-10000 10001-20000
PRIMER_SECONDARY_STRUCTURE_ALIGNMENT=1
PRIMER_NUM_RETURN=10
PRIMER_MAX_HAIRPIN_TH=47.00
PRIMER_INTERNAL_MAX_HAIRPIN_TH=47.00
PRIMER_MAX_END_STABILITY=9.0
PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE=3
PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE=3
PRIMER_EXPLAIN_FLAG=1
PRIMER_LIBERAL_BASE=1
PRIMER_FIRST_BASE_INDEX=1
PRIMER_MAX_TEMPLATE_MISPRIMING=12.00
PRIMER_MAX_TEMPLATE_MISPRIMING_TH=47.00
PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING=24.00
PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=47.00
</pre>
<h2><a id="changesFrom2.2.3">6. CHANGES FROM VERSION 2.2.3</a></h2>
<p>
1. The tag PRIMER_THERMODYNAMIC_ALIGNMENT was replaced by two new tags:
<br><br>
- <a href="#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT">PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT</a> 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.
<br><br>
- <a href="#PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT">PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT</a> which governs the
oligo-template interactions and, when set to 1 (default is 0), will
cause Primer3 to use the thermodynamic alignment against templates.
<br><br>
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).
<br><br>
2. We changed the following default values:<br>
<br>
2.1. Changing default oligo temperature calculations
<br><br>
<a href="#PRIMER_TM_FORMULA">PRIMER_TM_FORMULA</a>=1 (was 0)<br>
<a href="#PRIMER_SALT_CORRECTIONS">PRIMER_SALT_CORRECTIONS</a>=1 (was 0)<br>
<br>
2.2. Making thermodynamic secondary structure calculations for oligos the default:<br>
<br>
<a href="#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT">PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT</a>=1 (was 0)<br>
<br>
2.3. The following need reasonable values to support the changes in 2.1
and 2.2 above:<br>
<br>
<a href="#PRIMER_SALT_DIVALENT">PRIMER_SALT_DIVALENT</a>=1.5 (was 0.0)<br>
<a href="#PRIMER_DNTP_CONC">PRIMER_DNTP_CONC</a>=0.6 (was 0.0)<br>
<br>
2.4. 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.<br>
<br>
2.5. IMPORTANT: because <a href="#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT">PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT</a>=1,
<a href="#PRIMER_THERMODYNAMIC_PARAMETERS_PATH">PRIMER_THERMODYNAMIC_PARAMETERS_PATH</a> 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 Linux, there
are two *default* locations that are tested if this tag is not
defined: ./primer3_config/ and /opt/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
<a href="#PRIMER_THERMODYNAMIC_PARAMETERS_PATH">PRIMER_THERMODYNAMIC_PARAMETERS_PATH</a>.<br>
<br>
2.6. Changed default for <br>
<br>
<a href="#PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS">PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS</a>=0 (was 1)<br>
<br>
(0 is almost always the the behavior one wants.)<br>
<br>
2.7. To get the behavior of -default_version=1 when -default_version=2
set the following:<br>
<br>
<a href="#PRIMER_TM_FORMULA">PRIMER_TM_FORMULA</a>=0<br>
<a href="#PRIMER_SALT_CORRECTIONS">PRIMER_SALT_CORRECTIONS</a>=0<br>
<a href="#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT">PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT</a>=0<br>
<a href="#PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT">PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT</a>=0<br>
<a href="#PRIMER_SALT_DIVALENT">PRIMER_SALT_DIVALENT</a>=0.0<br>
<a href="#PRIMER_DNTP_CONC">PRIMER_DNTP_CONC</a>=0.0<br>
<a href="#PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS">PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS</a>=1<br>
<br>
2.8. To get the behavior of -default_version=2 when -default_version=1
set the following:<br>
<br>
<a href="#PRIMER_TM_FORMULA">PRIMER_TM_FORMULA</a>=1<br>
<a href="#PRIMER_SALT_CORRECTIONS">PRIMER_SALT_CORRECTIONS</a>=1<br>
<a href="#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT">PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT</a>=1<br>
<a href="#PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT">PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT</a>=0<br>
<a href="#PRIMER_SALT_DIVALENT">PRIMER_SALT_DIVALENT</a>=1.5<br>
<a href="#PRIMER_DNTP_CONC">PRIMER_DNTP_CONC</a>=0.6<br>
<a href="#PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS">PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS</a>=0<br>
<br>
3. We changed the NULL value for SEQUENCE_FORCE_{LEFT,RIGHT}_START_END to
-1000000, and made it an error to select <a href="#PRIMER_FIRST_BASE_INDEX">PRIMER_FIRST_BASE_INDEX</a> <=
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 <a href="#PRIMER_FIRST_BASE_INDEX">PRIMER_FIRST_BASE_INDEX</a> 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).<br>
<br>
4. We changed the <a href="#PRIMER_TASK">PRIMER_TASK</a> called 'pick_detection_primers' to
'generic' while retaining 'pick_detection_primers' as an alias for
backward compatibility.<br>
<br>
5. 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.<br>
<br>
6. We removed PRIMER_PAIR_MAX_HAIRPIN_TH (which was ignored previously).<br>
<br>
7. Primer3 now requires the user to set <a href="#SEQUENCE_TARGET">SEQUENCE_TARGET</a>, not
<a href="#SEQUENCE_INCLUDED_REGION">SEQUENCE_INCLUDED_REGION</a> when <a href="#PRIMER_TASK">PRIMER_TASK</a>=pick_discriminative_primers<br>
<br>
8. When <a href="#PRIMER_TASK">PRIMER_TASK</a>=pick_discriminative_primers or
<a href="#PRIMER_TASK">PRIMER_TASK</a>=pick_cloning_primers the value of <a href="#SEQUENCE_INCLUDED_REGION">SEQUENCE_INCLUDED_REGION</a>
is no longer changed to the entire input sequence.<br>
<br>
9. The handling of divalent cations when <a href="#PRIMER_SALT_CORRECTIONS">PRIMER_SALT_CORRECTIONS</a>=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 below in the manual.<br>
<br>
10. We modified the error handling of non-memory related errors that
can occur during the thermodynamic alignment. They are no longer
fatal errors.<br>
<br>
11. We removed io_version 3, which was only kept for backward compatibility
with very old versions of Primer3.<br>
<br>
12. In addition, several error corrections:<br>
<br>
12.1. Corrected a short, fixed-size buffer for the file/path names specified
by the command line arguments -output, -error and for the settings
file.<br>
<br>
12.2. 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.<br>
<br>
12.3. Primer3 now detects and handles the situation in which user-supplied
primers (<a href="#SEQUENCE_PRIMER">SEQUENCE_PRIMER</a> and <a href="#SEQUENCE_PRIMER_REVCOMP">SEQUENCE_PRIMER_REVCOMP</a>) 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.<br></p>
<h2><a id="installLinux">7. INSTALLATION INSTRUCTIONS - UNIX/LINUX</a></h2>
<p>Unzip and untar the distribution.
DO NOT do this on a PC if you are going to
compile and/or test on a different operation system.
Primer3_core will not compile
and various tests will fail if pc
newlines get inserted into the code and test files. Instead, move the
distribution (primer3-<release>.tar.gz) to Unix/Linux, and then<br>
<br>
$ unzip primer3-<release>.tar.gz<br>
$ tar xvf primer3-<release>.tar<br>
$ cd primer3-<release>/src<br>
<br>
If you do not use gcc, then you will probably have
to modify the makefile to
use your (ANSI) C compiler with the compile
and link flags it understands.<br>
<br>
$ make all<br>
<br>
# You should have created executables primer3_core, ntdpal,<br>
# olgotm, and long_seq_tm_test<br>
<br>
$ make test<br>
<br>
# You should not see 'FAILED' during the tests.<br>
<br>
If your perl command is not called perl (for example, if it is
called perl5) you will have to modify the
Makefile in the test/ directory.<br>
<br>
ntdpal (NucleoTide Dynamic Programming ALignment) is a
stand-alone program that provides Primer3's alignment
functionality (local, a.k.a. Smith-Waterman, global,
a.k.a. Needleman-Wunsch, plus "half global"). It is provided
strictly as is; for further documentation please see the code.</p>
<h2><a id="installMac">8. INSTALLATION INSTRUCTIONS - Mac OSX</a></h2>
<p>How to install this software<br>
============================<br>
<br>
1. Double click on the .tar.gz file to extract the archive.<br>
<br>
2. The binary files are located in the 'bin' [for 'binary']
folder<br>
<br>
3. (Optional) To run the tests, cd to the new directory and
then the test folder<br>
<br>
4. (Optional) Within this folder run:
a. 'perl -w p3test.pl'<br>
<br>
5. (Optional) You should not see 'FAILED' during the tests.<br>
<br>
6. (Optional) *NOTE*: If your perl command is not called
perl (for example, if it is called perl5) you will have
to modify the internals of the test scripts).<br>
<br>
7. Copy the following files to a location of your choice:<br>
a. bin/long_seq_tm_test<br>
b. bin/ntdpal<br>
c. bin/oligotm<br>
d. bin/primer3_core<br>
<br>
8. (Optional) Make sure this location is within your $PATH
(see below)<br>
<br>
<br>
Where to put the binary files<br>
=============================<br>
<br>
A good place to put these is within ~/bin/ (this means in
your home folder, within a folder named `bin` [for 'binary']).<br>
<br>
You can also just drag the 'bin' folder to a location within
your home directory.<br>
<br>
You can certainly also copy the files within 'bin' to
/usr/local/bin (if you are an administrator) or another
similar location.<br>
<br>
You may need to adjust the permissions on the binaries if
you get fancy.<br>
<br>
<br>
Add the location to your $PATH<br>
==============================<br>
<br>
This is an optional step, but it will allow you to run
Primer3 in any directory on your machine as your user just
by typing its name (primer3_core).<br>
<br>
*** You should be very careful when altering your $PATH as
things can go very wrong. See below for an alternate
method. *** <br>
<br>
If you added the binaries to /usr/local/bin, then you do
not need to do this.<br>
<br>
If you added the binaries to a local directory (let's say
~/bin/), do the following:<br>
<br>
1. Edit your ~/.bash_profile. You can edit this on
the command line (Terminal) with:<br>
<br>
nano ~/.bash_profile<br>
<br>
2. Add the following line if it is not present
(replacing '~/bin' if you used another directory):<br>
<br>
PATH=$PATH:~/bin/<br>
<br>
3. If a PATH line *is* present, ensure you add a colon
to the end of what is there and then the directory,
so if you have something like:<br>
<br>
a) PATH=$PATH:/usr/local/genome/bin:/sw/bin<br>
<br>
make it look like:<br>
<br>
b) PATH=$PATH:/usr/local/genome/bin:/sw/bin:~/bin<br>
<br>
4. Quit and restart terminal for the changes to take
effect.<br>
<br>
If you don't add the location to your $PATH<br>
===========================================<br>
<br>
Assuming you don't want to modify your $PATH, you can still
run the binaries. Let's assume you put the files in
'~/bin/. You may run primer3_core by doing either of the
following:<br>
<br>
1. ~/bin/primer3_core < yourInputFile<br>
2. /Users/<your username>/bin/primer3_core < yourInputFile<br>
<br>
The first option is just a shortcut to the second.</p>
<h2><a id="installWindows">9. INSTALLATION INSTRUCTIONS - WINDOWS</a></h2>
<p>Functionality warning<br>
=====================<br>
<br>
The windows version does not support the masker functionality.
</p>
<p>How to install this software<br>
============================<br>
<br>
Get your copy of the source code<br>
--------------------------------<br>
1. Install Git for Windows from<br>
https://git-scm.com/download/win<br>
2. Open Git for Windows and run<br>
git clone https://github.com/primer3-org/primer3.git<br>
cd primer3<br>
3. Use git commands to obtain tagged version<br>
git tag<br>
git checkout tags/v2.3.7<br>
or
1. Download and unpack latest from<br>
https://github.com/primer3-org/primer3/releases<br>
Compile Primer3<br>
---------------<br>
<br>
1. Download and install TDM-GCC MinGW Compiler from<br>
https://sourceforge.net/projects/tdm-gcc/
2. Open the windows command line and change to the src folder in
Primer3<br>
3. Run: mingw32-make TESTOPTS=--windows<br>
<br>
Running the tests<br>
=================<br>
You must install a perl distribution to run the
windows tests.<br>
<br>
We *strongly* recommend you install ActiveState perl
(http://www.activestate.com/products/activeperl/)
as this was used to test our Primer3 builds, and it is
known to work.<br>
<br>
1. Click on 'Start > Run...'<br>
2. Type 'cmd' into the space provided<br>
3. Hit enter (or select 'OK')<br>
4. Navigate to the location of the tests:<br>
<br>
A. if you put it in <br>
C:/Documents and Settings/YourName/primer3-<release>/test/,<br>
you would type <br>
'cd c:/Documents and Settings/YourName/primer3-<release>/test/'<br>
<br>
B. you can also type 'cd ' (don't forget the space
after cd) and drag the primer3-<release> folder onto the
command-line window from windows explorer, this will
fill in the location for you<br>
<br>
5. On the command line, run 'mingw32-make TESTOPTS=--windows' in this
directory. You can also run the tests individually,
for example 'perl p3test.pl --windows'.<br>
6. You should see [OK] for the majority of the tests. The masker
functionality is not supported on Windows and coresponding tests
will fail.<br>
<br>
Running the software<br>
====================<br>
<br>
To run the program, you must do so from the MS-DOS
command-line. The intricacies of the DOS command line are
beyond the scope of this document. Google for more
information, if needed. Here is a quick summary:<br>
<br>
1. Click on 'Start > Run...'<br>
2. Type 'cmd' into the space provided<br>
3. Hit enter (or select 'OK')<br>
4. Navigate to the location of the binary:<br>
<br>
A. if you put it in<br>
C:/Documents and Settings/YourName/Temp,<br>
you would type <br>
'cd c:/Documents and Settings/YourName/Temp'<br>
<br>
B. you can also type 'cd ' (don't forget the space
after cd) and drag the Primer3 folder onto the
command-line window from windows explorer, this will
fill in the location for you<br>
<br>
5. Run the example file by typing:<br>
<br>
primer3_core.exe < example<br>
<br>
Other files may be run in a similar fashion. If your input
filename is 'MyData.txt' you can run Primer3 using this
file (in the correct format; see README) with:<br>
<br>
primer3_core.exe < MyData.txt<br>
<br>
If your file is not in the folder containing
primer3_core.exe, you could run the program from the
primer3_core folder using:<br>
<br>
primer3_core.exe < c:/someOtherFolder/someOtherFolder/MyData.txt<br>
<br>
Finally, if you want to run the program without going to
its folder, assuming primer3_core.exe is in c:/Temp, you
could run:<br>
<br>
c:/Temp/primer3_core.exe < c:/someOtherFolder/someOtherFolder/MyData.txt</p>
<h2><a id="systemRequirements">10. SYSTEM REQUIREMENTS</a></h2>
<p>Please see <a href="https://github.com/primer3-org/primer3">https://github.com/primer3-org/primer3</a> for up-to-date
information. Primer3 should compile on any system using with an
ANSI C compiler like Linux/Unix, MacOS 10 or MS Windows. The
Makefile will probably need to be modified for compilation with
C compilers other than gcc.
</p>
<h2><a id="invokingPrimer3">11. INVOKING primer3_core</a></h2>
<p>By default, the executable program produced by the Makefile is
called primer3_core. This is the C program that does the heavy
lifting of primer picking. There are also two, more user-friendly,
web interfaces (distributed separately).<br>
<br>
The command line for Primer3 is:<br>
<br>
primer3_core [ --format_output ] [ --default_version=1|--default_version=2 ] [ --io_version=4 ] [ --p3_settings_file=<file_path> ] [ --echo_settings_file ] [ --strict_tags ] [ --output=<file_path> ] [ --error=<file_path> ] [ input_file ] <br>
<br>
A complete list of valid command line arguments can be found in
COMMAND LINE ARGUMENTS below.<br>
<br>
If no input file is specified, primer3_core will read its input
from stdin.</p>
<h2><a id="commandLineTags">12. COMMAND LINE ARGUMENTS</a></h2>
<p>This section presents the list of arguments that may given in
the command line. Any unique abbreviation of the arguments is allowed
(e.g. -ab instead of -about) and each argument can be preceded by
either one or two dashes. In the case of arguments that receive
values, the '=' can be replaced
by a space.</p>
<h3>--about</h3>
<p>With this argument Primer3 generates one line of output
indicating the release number and then exits. This allows scripts to
query Primer3 for its version.</p>
<h3>--default_version=n</h3>
<p><i>n</i>=2 is the default, and directs primer3_core
to use the latest default values (the ones documented here).
<i>n</i>=1 directs primer3_core to use defaults from version
2.2.3 and before.
</p>
<h3>--format_output</h3>
<p>This argument indicates that primer3_core should generate
user-oriented (rather than program-oriented) output.</p>
<h3>--strict_tags</h3>
<p>This argument indicates that primer3_core should generate
a fatal error if there is any tag in the input that
it does not recognize. This tag also applies to the settings file (see
documentation for the --p3_settings_file argument),
if any, but with limitations: lines that do not begin with
PRIMER_ or P3_FILE_ID are always ignored in the settings file.</p>
<h3>--p3_settings_file=file_path</h3>
<p>This argument specifies a settings file,
<i>file_path</i>. Primer3 reads the global
("PRIMER_...") parameters from this file first.
Tags appearing in the settings file override
default Primer3 settings. The values set in
the settings files can be also overridden
by tags in the input file. See
Primer3 file documentation for details on the file
format. <strong>WARNING: the --strict_tags flag applies only to
tags beginning with the string "PRIMER_"; lines that do
not begin with the string "PRIMER_" are ignored.</strong></p>
<h3>--echo_settings_file</h3>
<p>This argument indicates that primer3_core
should print the input tags specified
in the given settings file. If no settings file is
provided or if the --format_output option is given,
this argument will have no effect.</p>
<h3>--io_version=n</h3>
<p>This argument is provided for backward compatibility.
--io_version=4 <strong>is the only legal value and the default
</strong>.</p>
<h3>--output=file_path</h3>
<p>This argument specifies the file where the output
should be written. If omitted, stdout is used. </p>
<h3>--error=file_path</h3>
<p>This argument specifies the file where the error
messages should be written. If omitted, stderr is used.</p>
<h2><a id="inputOutputConventions">13. INPUT AND OUTPUT CONVENTIONS</a></h2>
<p>By default, Primer3 accepts input in Boulder-IO format, a
pre-XML, pre-RDF, text-based input/output format for
program-to-program data interchange. By default, Primer3 also
produces output in the same format.<br>
<br>
When run with the --format_output command-line flag, Primer3
prints a more user-oriented report for each sequence.<br>
<br>
Primer3 exits with 0 status if it operates correctly. See EXIT
STATUS CODES below for additional information.<br>
<br>
The syntax of the version of Boulder-IO recognized by Primer3 is
as follows:</p>
<pre>
o Input consists of a sequence of RECORDs.
o A RECORD consists of a sequence of (TAG,VALUE) pairs, each terminated
by a newline character (\n). A RECORD is terminated by '='
appearing by itself on a line.
o A (TAG,VALUE) pair has the following requirements:
o the TAG must be immediately (without spaces)
followed by '='.
</pre>
<p>An example of a legal (TAG,VALUE) pair is<br>
<br>
<a href="#SEQUENCE_ID">SEQUENCE_ID</a>=my_marker<br>
<br>
and an example of a Boulder-IO record is<br>
<br>
<a href="#SEQUENCE_ID">SEQUENCE_ID</a>=test1<br>
<a href="#SEQUENCE_TEMPLATE">SEQUENCE_TEMPLATE</a>=GACTGATCGATGCTAGCTACGATCGATCGATGCATGCTAGCTAGCTAGCTGCTAGC<br>
=<br>
<br>
Many records can be sent, one after another. Below is an example
of three different records which might be passed through a
Boulder-IO stream:<br>
<br>
<a href="#SEQUENCE_ID">SEQUENCE_ID</a>=test1<br>
<a href="#SEQUENCE_TEMPLATE">SEQUENCE_TEMPLATE</a>=GACTGATCGATGCTAGCTACGATCGATCGATGCATGCTAGCTAGCTAGCTGCTAGC<br>
=<br>
<a href="#SEQUENCE_ID">SEQUENCE_ID</a>=test2<br>
<a href="#SEQUENCE_TEMPLATE">SEQUENCE_TEMPLATE</a>=CATCATCATCATCGATGCTAGCATCNNACGTACGANCANATGCATCGATCGT<br>
=<br>
<a href="#SEQUENCE_ID">SEQUENCE_ID</a>=test3<br>
<a href="#SEQUENCE_TEMPLATE">SEQUENCE_TEMPLATE</a>=NACGTAGCTAGCATGCACNACTCGACNACGATGCACNACAGCTGCATCGATGC<br>
=<br>
<br>
Primer3 reads Boulder-IO on stdin and echos its input and returns
results in Boulder-IO format on stdout. Primer3 indicates many
user-correctable errors by a value in the output tag <a href="#PRIMER_ERROR">PRIMER_ERROR</a>
(see below). Primer3 indicates other errors, including system configuration
errors, resource errors (such out-of-memory errors), and detected
programming errors by a message on stderr and a non-zero exit
status.<br>
<br>
This document lists the input tags that Primer3 recognizes.
Primer3 echos and ignores any tags it does not recognize, unless
the --strict_tags flag is set on the command line, in which case
Primer3 prints an error in the <a href="#PRIMER_ERROR">PRIMER_ERROR</a> output tag (see
below), and prints additional information on stdout; this option
can be useful for debugging systems that incorporate Primer3.<br>
<br>
Except for tags with the type "interval list" each tag should only
appear ONCE in any given input record. This restriction is not
systematically checked, and if a tag appears more than once, the
new value silently over-writes the previous value.
</p>
<h2><a id="example">14. AN EXAMPLE</a></h2>
<p>One might be interested in performing PCR on an STS with a CA
repeat in the middle of it. Primers need to be chosen based on
the criteria of the experiment.<br>
<br>
We need to create a Boulder-IO record to send to Primer3 via
stdin. There are lots of ways to accomplish this. We could save
the record into a text file called 'example', and then type the
Unix command 'primer3_core < example'.<br>
<br>
Let's look at the input record itself:
<br>
<a href="#SEQUENCE_ID">SEQUENCE_ID</a>=example<br>
<a href="#SEQUENCE_TEMPLATE">SEQUENCE_TEMPLATE</a>=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG<br>
<a href="#SEQUENCE_TARGET">SEQUENCE_TARGET</a>=37,21<br>
<a href="#PRIMER_TASK">PRIMER_TASK</a>=generic<br>
<a href="#PRIMER_PICK_LEFT_PRIMER">PRIMER_PICK_LEFT_PRIMER</a>=1<br>
<a href="#PRIMER_PICK_INTERNAL_OLIGO">PRIMER_PICK_INTERNAL_OLIGO</a>=1<br>
<a href="#PRIMER_PICK_RIGHT_PRIMER">PRIMER_PICK_RIGHT_PRIMER</a>=1<br>
<a href="#PRIMER_OPT_SIZE">PRIMER_OPT_SIZE</a>=18<br>
<a href="#PRIMER_MIN_SIZE">PRIMER_MIN_SIZE</a>=15<br>
<a href="#PRIMER_MAX_SIZE">PRIMER_MAX_SIZE</a>=21<br>
<a href="#PRIMER_MAX_NS_ACCEPTED">PRIMER_MAX_NS_ACCEPTED</a>=1<br>
<a href="#PRIMER_PRODUCT_SIZE_RANGE">PRIMER_PRODUCT_SIZE_RANGE</a>=75-100<br>
<a href="#P3_FILE_FLAG">P3_FILE_FLAG</a>=1<br>
<a href="#SEQUENCE_INTERNAL_EXCLUDED_REGION">SEQUENCE_INTERNAL_EXCLUDED_REGION</a>=37,21<br>
<a href="#PRIMER_EXPLAIN_FLAG">PRIMER_EXPLAIN_FLAG</a>=1<br>
=<br>
<br>
A breakdown of the reasoning behind each of the TAG=VALUE pairs
is below:<br>
<br>
<a href="#SEQUENCE_ID">SEQUENCE_ID</a>=example<br>
<br>
The main intent of this tag is to provide an identifier for the
sequence that is meaningful to the user, for example when Primer3
processes multiple records, and by default this tag is optional.
However, this tag is _required_ when <a href="#P3_FILE_FLAG">P3_FILE_FLAG</a> is non-0
Because it provides the names of the files that contain lists
of oligos that Primer3 considered.<br>
<br>
<a href="#SEQUENCE_TEMPLATE">SEQUENCE_TEMPLATE</a>=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG<br>
<br>
The <a href="#SEQUENCE_TEMPLATE">SEQUENCE_TEMPLATE</a> provides the sequence
from which Primer3 will design primers.
Note that there is no newline until the sequence terminates completely.<br>
<br>
<a href="#SEQUENCE_TARGET">SEQUENCE_TARGET</a>=37,21<br>
<br>
There is a simple sequence repeat in this sequence.
The repeat starts
at base 37, and has a length of 21 bases. We instruct Primer3 to
choose primers that flank the repeat site (because
we want to use the PCR product for
determining the length of the repeat, which is likely to be polymorphic).
<br>
<br>
<a href="#PRIMER_TASK">PRIMER_TASK</a>=generic<br>
<br>
The <a href="#PRIMER_TASK">PRIMER_TASK</a> tells Primer3 which
type of primers to pick. You can select typical primers for PCR
detection, primers for cloning or for sequencing.<br>
<br>
<a href="#PRIMER_PICK_LEFT_PRIMER">PRIMER_PICK_LEFT_PRIMER</a>=1<br>
<a href="#PRIMER_PICK_INTERNAL_OLIGO">PRIMER_PICK_INTERNAL_OLIGO</a>=1<br>
<a href="#PRIMER_PICK_RIGHT_PRIMER">PRIMER_PICK_RIGHT_PRIMER</a>=1<br>
<br>
We would like to pick a left primer, an internal oligo and a
right primer, so we set these flags to 1 (true). In combination
with the <a href="#PRIMER_TASK">PRIMER_TASK</a> this tags control which primers are
picked.<br>
<br>
<a href="#PRIMER_OPT_SIZE">PRIMER_OPT_SIZE</a>=18<br>
<br>
Since our sequence length is rather small (only 108 bases long),
we lower the <a href="#PRIMER_OPT_SIZE">PRIMER_OPT_SIZE</a> from 20 to 18. It is
more likely that Primer3 will succeed if it aims for smaller
primers.<br>
<br>
<a href="#PRIMER_MIN_SIZE">PRIMER_MIN_SIZE</a>=15<br>
<a href="#PRIMER_MAX_SIZE">PRIMER_MAX_SIZE</a>=21<br>
<br>
With the lowering of optimal primer size, it's good to lower
the minimum and maximum sizes as well.<br>
<br>
<a href="#PRIMER_MAX_NS_ACCEPTED">PRIMER_MAX_NS_ACCEPTED</a>=1<br>
<br>
Since the sequence is short with a
non-negligible amount of unknown bases (N's) in it, we
make Primer3's job easier by allowing it to pick primers that have
at most 1 unknown base.<br>
<br>
<a href="#PRIMER_PRODUCT_SIZE_RANGE">PRIMER_PRODUCT_SIZE_RANGE</a>=75-100<br>
<br>
We reduce the product size range from the default of 100-300
because our source sequence is only 108 base pairs long. If we
insisted on a product size of 100 base pairs Primer3 would have
few possibilities to choose from.<br>
<br>
<a href="#P3_FILE_FLAG">P3_FILE_FLAG</a>=1<br>
<br>
Because we have a short sequence with many Ns and
with a simple sequence repeat that we must amplify,
Primer3 might fail to
pick and primers. Therefore, we want to get the list of primers it
considered, so that we could manually pick primers
ourselves if Primer3 fails to do so. Setting this flag to 1
asks Primer3 to output the primers it considered to a
forwar primers and reverse primers to output files.<br>
<br>
<a href="#SEQUENCE_INTERNAL_EXCLUDED_REGION">SEQUENCE_INTERNAL_EXCLUDED_REGION</a>=37,21<br>
<br>
Normally CA-repeats make poor hybridization probes (because they
not specific enough). Therefore, we exclude the CA repeat (which
is the TARGET) from consideration for the internal oligo.<br>
<br>
<a href="#PRIMER_EXPLAIN_FLAG">PRIMER_EXPLAIN_FLAG</a>=1<br>
<br>
We want to see statistics about the oligos and oligo triples
(left primer, internal oligo, right primer) that Primer3
examined.<br>
<br>
=<br>
<br>
The '=' character terminates the record.<br>
<br>
There were many Boulder-IO input tags that were not
specified,
which is legal. For the tags with default
values, those defaults will be used in the analysis. For the
tags with NO default values (for example <a href="#SEQUENCE_TARGET">SEQUENCE_TARGET</a>), the
functionality requested by the those tags will be absent.
Also, please note
that it is not the case that we need to surround a simple sequence
repeat every time we want to pick primers!</p>
<h2><a id="migrateTags">15. HOW TO MIGRATE TAGS TO IO VERSION 4</a></h2>
<p>With Primer3 release 2.0, many Boulder-IO tags were
modified and new tags were introduced. The new Primer3 tags are
designed with the idea in mind that computer scripts and other
programs use primer3_core. The modifications make it easier for
programs to read and write Primer3 input and output.<br>
<br>
Furthermore the Primer3 default values and the use of
<a href="#PRIMER_WT_TEMPLATE_MISPRIMING">PRIMER_WT_TEMPLATE_MISPRIMING</a> and <a href="#PRIMER_PAIR_WT_TEMPLATE_MISPRIMING">PRIMER_PAIR_WT_TEMPLATE_MISPRIMING</a>
have changed in version 2.0.
<br>
<br>
There are three classes of input: "Sequence" input tags describe
a particular input sequence to Primer3, and are reset after
every Boulder record (now starting with SEQUENCE_). "Global"
input tags describe the general parameters that Primer3 should
use in its searches, and the values of these tags persist
between input Boulder records until or unless they are
explicitly reset (now starting with PRIMER_). "Program"
parameters that deal with the behavior of the Primer3 program
itself (now starting with P3_). See below for a list of the
modified tags.<br>
<br>
The handling of PRIMER_TASK changed completely. In the past we
used it to tell Primer3 what task to perform. Now the task is
complemented with <a href="#PRIMER_PICK_RIGHT_PRIMER">PRIMER_PICK_RIGHT_PRIMER</a>,
<a href="#PRIMER_PICK_INTERNAL_OLIGO">PRIMER_PICK_INTERNAL_OLIGO</a> and <a href="#PRIMER_PICK_LEFT_PRIMER">PRIMER_PICK_LEFT_PRIMER</a>, which
specify which primers are to be picked.<br>
<br>
These Tags are modified:<br>
<br>
The "per sequence" tags:</p>
<pre>
NEW VERSION - OLD VERSION
----------------------------------------------------------------------------------------
<a href="#SEQUENCE_ID">SEQUENCE_ID</a> - PRIMER_SEQUENCE_ID
<a href="#SEQUENCE_TEMPLATE">SEQUENCE_TEMPLATE</a> - SEQUENCE
<a href="#SEQUENCE_QUALITY">SEQUENCE_QUALITY</a> - PRIMER_SEQUENCE_QUALITY
<a href="#SEQUENCE_INCLUDED_REGION">SEQUENCE_INCLUDED_REGION</a> - INCLUDED_REGION
<a href="#SEQUENCE_TARGET">SEQUENCE_TARGET</a> - TARGET
<a href="#SEQUENCE_EXCLUDED_REGION">SEQUENCE_EXCLUDED_REGION</a> - EXCLUDED_REGION
<a href="#SEQUENCE_START_CODON_POSITION">SEQUENCE_START_CODON_POSITION</a> - PRIMER_START_CODON_POSITION
<a href="#SEQUENCE_PRIMER">SEQUENCE_PRIMER</a> - PRIMER_LEFT_INPUT
<a href="#SEQUENCE_PRIMER_REVCOMP">SEQUENCE_PRIMER_REVCOMP</a> - PRIMER_RIGHT_INPUT
<a href="#SEQUENCE_INTERNAL_OLIGO">SEQUENCE_INTERNAL_OLIGO</a> - PRIMER_INTERNAL_OLIGO_INPUT
<a href="#SEQUENCE_INTERNAL_EXCLUDED_REGION">SEQUENCE_INTERNAL_EXCLUDED_REGION</a> - PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION
--------------------------------------------------------------------------------
The "global" tags:
NEW VERSION - OLD VERSION
<a href="#PRIMER_TASK">PRIMER_TASK</a> - PRIMER_TASK (modified use)
<a href="#PRIMER_PICK_RIGHT_PRIMER">PRIMER_PICK_RIGHT_PRIMER</a> - --- did not exist
<a href="#PRIMER_PICK_INTERNAL_OLIGO">PRIMER_PICK_INTERNAL_OLIGO</a> - PRIMER_PICK_INTERNAL_OLIGO (modified use)
<a href="#PRIMER_PICK_LEFT_PRIMER">PRIMER_PICK_LEFT_PRIMER</a> - --- did not exist
<a href="#PRIMER_PAIR_WT_TEMPLATE_MISPRIMING">PRIMER_PAIR_WT_TEMPLATE_MISPRIMING</a>- PRIMER_PAIR_WT_TEMPLATE_MISPRIMING (modified use)
<a href="#PRIMER_WT_TEMPLATE_MISPRIMING">PRIMER_WT_TEMPLATE_MISPRIMING</a> - PRIMER_WT_TEMPLATE_MISPRIMING (modified use)
<a href="#PRIMER_MAX_LIBRARY_MISPRIMING">PRIMER_MAX_LIBRARY_MISPRIMING</a> - PRIMER_MAX_MISPRIMING
<a href="#PRIMER_INTERNAL_MAX_LIBRARY_MISHYB">PRIMER_INTERNAL_MAX_LIBRARY_MISHYB</a>- PRIMER_INTERNAL_OLIGO_MAX_MISHYB
<a href="#PRIMER_PAIR_MAX_LIBRARY_MISPRIMING">PRIMER_PAIR_MAX_LIBRARY_MISPRIMING</a>- PRIMER_PAIR_MAX_MISPRIMING
<a href="#PRIMER_WT_LIBRARY_MISPRIMING">PRIMER_WT_LIBRARY_MISPRIMING</a> - PRIMER_WT_REP_SIM
<a href="#PRIMER_INTERNAL_WT_LIBRARY_MISHYB">PRIMER_INTERNAL_WT_LIBRARY_MISHYB</a> - PRIMER_INTERNAL_WT_REP_SIM
<a href="#PRIMER_PAIR_WT_LIBRARY_MISPRIMING">PRIMER_PAIR_WT_LIBRARY_MISPRIMING</a> - PRIMER_PAIR_WT_REP_SIM
<a href="#PRIMER_MAX_NS_ACCEPTED">PRIMER_MAX_NS_ACCEPTED</a> - PRIMER_NUM_NS_ACCEPTED
<a href="#PRIMER_PAIR_MAX_DIFF_TM">PRIMER_PAIR_MAX_DIFF_TM</a> - PRIMER_MAX_DIFF_TM
<a href="#PRIMER_SALT_MONOVALENT">PRIMER_SALT_MONOVALENT</a> - PRIMER_SALT_CONC
<a href="#PRIMER_SALT_DIVALENT">PRIMER_SALT_DIVALENT</a> - PRIMER_DIVALENT_CONC
<a href="#PRIMER_TM_FORMULA">PRIMER_TM_FORMULA</a> - PRIMER_TM_SANTALUCIA
<a href="#PRIMER_MAX_SELF_ANY">PRIMER_MAX_SELF_ANY</a> - PRIMER_SELF_ANY
<a href="#PRIMER_MAX_SELF_END">PRIMER_MAX_SELF_END</a> - PRIMER_SELF_END
<a href="#PRIMER_WT_SELF_ANY">PRIMER_WT_SELF_ANY</a> - PRIMER_WT_COMPL_ANY
<a href="#PRIMER_WT_SELF_END">PRIMER_WT_SELF_END</a> - PRIMER_WT_COMPL_END
<a href="#PRIMER_PAIR_MAX_COMPL_ANY">PRIMER_PAIR_MAX_COMPL_ANY</a> - PRIMER_PAIR_ANY
<a href="#PRIMER_PAIR_MAX_COMPL_END">PRIMER_PAIR_MAX_COMPL_END</a> - PRIMER_PAIR_END
<a href="#P3_FILE_FLAG">P3_FILE_FLAG</a> - PRIMER_FILE_FLAG
<a href="#P3_COMMENT">P3_COMMENT</a> - PRIMER_COMMENT
<a href="#PRIMER_INTERNAL_SALT_MONOVALENT">PRIMER_INTERNAL_SALT_MONOVALENT</a> - PRIMER_INTERNAL_OLIGO_SALT_CONC
<a href="#PRIMER_INTERNAL_SALT_DIVALENT">PRIMER_INTERNAL_SALT_DIVALENT</a> - PRIMER_INTERNAL_OLIGO_DIVALENT_CONC
<a href="#PRIMER_INTERNAL_WT_SELF_ANY">PRIMER_INTERNAL_WT_SELF_ANY</a> - PRIMER_IO_WT_COMPL_ANY
<a href="#PRIMER_INTERNAL_WT_SELF_END">PRIMER_INTERNAL_WT_SELF_END</a> - PRIMER_IO_WT_COMPL_END
<a href="#PRIMER_INTERNAL_MAX_NS_ACCEPTED">PRIMER_INTERNAL_MAX_NS_ACCEPTED</a> - PRIMER_INTERNAL_OLIGO_NUM_NS
<a href="#PRIMER_INTERNAL_MAX_SELF_ANY">PRIMER_INTERNAL_MAX_SELF_ANY</a> - PRIMER_INTERNAL_OLIGO_SELF_ANY
<a href="#PRIMER_INTERNAL_MAX_SELF_END">PRIMER_INTERNAL_MAX_SELF_END</a> - PRIMER_INTERNAL_OLIGO_SELF_END
The following tags INTERNAL_OLIGO is replaced by INTERNAL:
<a href="#PRIMER_INTERNAL_OPT_SIZE">PRIMER_INTERNAL_OPT_SIZE</a> - PRIMER_INTERNAL_OLIGO_OPT_SIZE
<a href="#PRIMER_INTERNAL_MIN_SIZE">PRIMER_INTERNAL_MIN_SIZE</a> - PRIMER_INTERNAL_OLIGO_MIN_SIZE
<a href="#PRIMER_INTERNAL_MAX_SIZE">PRIMER_INTERNAL_MAX_SIZE</a> - PRIMER_INTERNAL_OLIGO_MAX_SIZE
<a href="#PRIMER_INTERNAL_OPT_TM">PRIMER_INTERNAL_OPT_TM</a> - PRIMER_INTERNAL_OLIGO_OPT_TM
<a href="#PRIMER_INTERNAL_MIN_TM">PRIMER_INTERNAL_MIN_TM</a> - PRIMER_INTERNAL_OLIGO_MIN_TM
<a href="#PRIMER_INTERNAL_MAX_TM">PRIMER_INTERNAL_MAX_TM</a> - PRIMER_INTERNAL_OLIGO_MAX_TM
<a href="#PRIMER_INTERNAL_MIN_GC">PRIMER_INTERNAL_MIN_GC</a> - PRIMER_INTERNAL_OLIGO_MIN_GC
<a href="#PRIMER_INTERNAL_OPT_GC_PERCENT">PRIMER_INTERNAL_OPT_GC_PERCENT</a> - PRIMER_INTERNAL_OLIGO_OPT_GC_PERCENT
<a href="#PRIMER_INTERNAL_MAX_GC">PRIMER_INTERNAL_MAX_GC</a> - PRIMER_INTERNAL_OLIGO_MAX_GC
<a href="#PRIMER_INTERNAL_DNTP_CONC">PRIMER_INTERNAL_DNTP_CONC</a> - PRIMER_INTERNAL_OLIGO_DNTP_CONC
<a href="#PRIMER_INTERNAL_DNA_CONC">PRIMER_INTERNAL_DNA_CONC</a> - PRIMER_INTERNAL_OLIGO_DNA_CONC
<a href="#PRIMER_INTERNAL_MAX_POLY_X">PRIMER_INTERNAL_MAX_POLY_X</a> - PRIMER_INTERNAL_OLIGO_MAX_POLY_X
<a href="#PRIMER_INTERNAL_MISHYB_LIBRARY">PRIMER_INTERNAL_MISHYB_LIBRARY</a> - PRIMER_INTERNAL_OLIGO_MISHYB_LIBRARY
<a href="#PRIMER_INTERNAL_MIN_QUALITY">PRIMER_INTERNAL_MIN_QUALITY</a> - PRIMER_INTERNAL_OLIGO_MIN_QUALITY
The following tags IO is replaced by INTERNAL:
<a href="#PRIMER_INTERNAL_WT_TM_GT">PRIMER_INTERNAL_WT_TM_GT</a> - PRIMER_IO_WT_TM_GT
<a href="#PRIMER_INTERNAL_WT_TM_LT">PRIMER_INTERNAL_WT_TM_LT</a> - PRIMER_IO_WT_TM_LT
<a href="#PRIMER_INTERNAL_WT_SIZE_LT">PRIMER_INTERNAL_WT_SIZE_LT</a> - PRIMER_IO_WT_SIZE_LT
<a href="#PRIMER_INTERNAL_WT_SIZE_GT">PRIMER_INTERNAL_WT_SIZE_GT</a> - PRIMER_IO_WT_SIZE_GT
<a href="#PRIMER_INTERNAL_WT_GC_PERCENT_LT">PRIMER_INTERNAL_WT_GC_PERCENT_LT</a> - PRIMER_IO_WT_GC_PERCENT_LT
<a href="#PRIMER_INTERNAL_WT_GC_PERCENT_GT">PRIMER_INTERNAL_WT_GC_PERCENT_GT</a> - PRIMER_IO_WT_GC_PERCENT_GT
<a href="#PRIMER_INTERNAL_WT_NUM_NS">PRIMER_INTERNAL_WT_NUM_NS</a> - PRIMER_IO_WT_NUM_NS
<a href="#PRIMER_INTERNAL_WT_SEQ_QUAL">PRIMER_INTERNAL_WT_SEQ_QUAL</a> - PRIMER_IO_WT_SEQ_QUAL
</pre>
<p>--------------------------------------------------------------------------------<br>
OUTPUT TAGS:<br>
<br>
There are three big changes on the output:<br>
- INTERNAL_OLIGO is now replaced by INTERNAL.<br>
- The first version is numbered 0.<br>
- The "PRODUCT" tags are renamed<br>
- The errors are modified<br>
- Errors caused by a specific primer are given as <a href="#PRIMER_LEFT_4_PROBLEMS">PRIMER_LEFT_4_PROBLEMS</a><br>
<br>
Now all primer related output follows the rule:
PRIMER_{LEFT,RIGHT,INTERNAL,PAIR}_<j>_<tag_name>.
where <j> is an integer from 0 to n, where n is at most the
value of <a href="#PRIMER_NUM_RETURN">PRIMER_NUM_RETURN</a> - 1.<br>
<br>
This allows easy scripting by using the underscores _ to split
the name. The first part is PRIMER, the second the type of oligo
or pair parameters, the third is always a number, starting at
0 and the rest is used by the tags.<br>
<br>
That affects also (shown for output number 4):</p>
<pre>
NEW VERSION - OLD VERSION
------------------------------------------------------------------------------------------------------
<a href="#PRIMER_PAIR_4_PENALTY">PRIMER_PAIR_4_PENALTY</a> - PRIMER_PAIR_PENALTY_4 (number moved behind PAIR)
<a href="#PRIMER_PAIR_4_PRODUCT_SIZE">PRIMER_PAIR_4_PRODUCT_SIZE</a> - PRIMER_PRODUCT_SIZE_4 (grouped with PAIR)
<a href="#PRIMER_PAIR_4_PRODUCT_TM">PRIMER_PAIR_4_PRODUCT_TM</a> - PRIMER_PRODUCT_TM_4 (grouped with PAIR)
<a href="#PRIMER_PAIR_4_PRODUCT_TM_OLIGO_TM_DIFF">PRIMER_PAIR_4_PRODUCT_TM_OLIGO_TM_DIFF</a> - PRIMER_PRODUCT_TM_OLIGO_TM_DIFF_4 (grouped with PAIR)
<a href="#PRIMER_INTERNAL_EXPLAIN">PRIMER_INTERNAL_EXPLAIN</a> - PRIMER_INTERNAL_OLIGO_EXPLAIN
PRIMER_LEFT_4_LIBRARY_MISPRIMING - PRIMER_LEFT_4_MISPRIMING_SCORE
PRIMER_INTERNAL_4_LIBRARY_MISHYB - PRIMER_INTERNAL_OLIGO_4_MISHYB_SCORE
PRIMER_RIGHT_4_LIBRARY_MISPRIMING - PRIMER_RIGHT_4_MISPRIMING_SCORE
PRIMER_PAIR_4_LIBRARY_MISPRIMING - PRIMER_PAIR_4_MISPRIMING_SCORE
</pre>
<h2><a id="sequenceTags">16. "SEQUENCE" INPUT TAGS</a></h2>
<p>"Sequence" input tags start with SEQUENCE_... and describe a
particular input sequence to Primer3. They are reset after every
Boulder record. Errors in "Sequence" input tags invalidate the