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.