eprimer32 Wiki The master copies of EMBOSS documentation are available at http://emboss.open-bio.org/wiki/Appdocs on the EMBOSS Wiki. Please help by correcting and extending the Wiki pages. Function Picks PCR primers and hybridization oligos Description eprimer32 is an interface to the 'primer3' program (2.x series) from the Whitehead Institute. The Whitehead program must be set up and on the path in order for eprimer32 to find and run it. The Whitehead executable must be renamed to primer32_core (or a link added) to avoid conflict with the eprimer3 application. Primer3 picks primers for PCR reactions, considering as criteria: * oligonucleotide melting temperature, size, GC content, and primer-dimer possibilities * PCR product size * positional constraints within the source sequence * miscellaneous other constraints. All of these criteria are user-specifiable as constraints. eprimer32 can also pick hybridisation oligos that are internal to the product. Advice for picking primers We suggest referring to: Wojciech Rychlik, "Selection of Primers for Polymerase Chain Reaction" in BA White, Ed., "Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications", 1993, pp 31-40, Humana Press, Totowa NJ Cautions Some of the most important issues in primer picking can be addressed only before using eprimer32. These are sequence quality (including making sure the sequence is not vector and not chimeric) and avoiding repetitive elements. Techniques for avoiding problems include a thorough understanding of possible vector contaminants and cloning artifacts coupled with database searches using blast, fasta, or other similarity searching program to screen for vector contaminants and possible repeats. Repbase (J. Jurka, A.F.A. Smit, C. Pethiyagoda, and others, 1995-1996, ftp://ncbi.nlm.nih.gov/repository/repbase) is an excellent source of repeat sequences and pointers to the literature. eprimer32 now allows you to screen candidate oligos against a Mispriming Library (or a Mishyb Library in the case of internal oligos). Sequence quality can be controlled by manual trace viewing and quality clipping or automatic quality clipping programs. Low- quality bases should be changed to N's or can be made part of Excluded Regions. The beginning of a sequencing read is often problematic because of primer peaks, and the end of the read often contains many low-quality or even meaningless called bases. Therefore when picking primers from single-pass sequence it is often best to use the INCLUDED_REGION parameter to ensure that eprimer32 chooses primers in the high quality region of the read. In addition, eprimer32 takes as input a Sequence Quality list for use with those base calling programs (e.g. Phred, Bass/Grace, Trout) that output this information. What to do if eprimer32 cannot find a primers? Try relaxing various parameters, including the self-complementarity parameters and max and min oligo melting temperatures. For example, for very A-T-rich regions you might have to increase maximum primer size or decrease minimum melting temperature. It is usually unwise to reduce the minimum primer size if your template is complex (e.g. a mammalian genome), since small primers are more likely to be non-specific. Make sure that there are adequate stretches of non-Ns in the regions in which you wish to pick primers. If necessary you can also allow an N in your primer and use an oligo mixture containing all four bases at that position. Try setting the '-explainflag' option. Usage Here is a sample session with eprimer32 % eprimer32 tembl:x65921 x65921.eprimer32 -explain Picks PCR primers and hybridization oligos Go to the input files for this example Go to the output files for this example Command line arguments Picks PCR primers and hybridization oligos Version: EMBOSS:6.4.0.0 Standard (Mandatory) qualifiers: [-sequence] seqall The sequence from which to choose primers. The sequence must be presented 5' to 3' [-outfile] outfile [*.eprimer32] Whitehead primer3_core program output file Additional (Optional) qualifiers (* if not always prompted): -[no]primer toggle [Y] Tell Eprimer32 to pick primer(s) * -task menu [1] Tell Eprimer32 what task to perform. Legal values are 1: 'Pick PCR primers', 2: 'Pick forward primer only', 3: 'Pick reverse primer only', 4: 'No primers needed'. (Values: 1 (Pick PCR primers); 2 (Pick forward primer only); 3 (Pick reverse primer only); 4 (No primers needed)) -hybridprobe toggle [N] An 'internal oligo' is intended to be used as a hybridization probe (hyb probe) to detect the PCR product after amplification. * -mishyblibraryfile infile Similar to MISPRIMING-LIBRARY, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library rather than priming from them. The file must be in (a slightly restricted) FASTA format (W. B. Pearson and D.J. Lipman, PNAS 85:8 pp 2444-2448 [1988]); we briefly discuss the organization of this file below. If this parameter is specified then Eprimer32 locally aligns each candidate oligo against each library sequence and rejects those primers for which the local alignment score times a specified weight (see below) exceeds INTERNAL-OLIGO-MAX-MISHYB. (The maximum value of the weight is arbitrarily set to 12.0.) Each sequence entry in the FASTA-format file must begin with an 'id line' that starts with '>'. The contents of the id line is 'slightly restricted' in that Eprimer32 parses everything after any optional asterisk ('*') as a floating point number to use as the weight mentioned above. If the id line contains no asterisk then the weight defaults to 1.0. The alignment scoring system used is the same as for calculating complementarity among oligos (e.g. SELF-ANY). The remainder of an entry contains the sequence as lines following the id line up until a line starting with '>' or the end of the file. Whitespace and newlines are ignored. Characters 'A', 'T', 'G', 'C', 'a', 't', 'g', 'c' are retained and any other character is converted to 'N' (with the consequence that any IUB / IUPAC codes for ambiguous bases are converted to 'N'). There are no restrictions on line length. An empty value for this parameter indicates that no library should be used. -numreturn integer [5] The maximum number of primer pairs to return. Primer pairs returned are sorted by their 'quality', in other words by the value of the objective function (where a lower number indicates a better primer pair). Caution: setting this parameter to a large value will increase running time. (Integer 0 or more) -includedregion range [(full sequence)] A sub-region of the given sequence in which to pick primers. For example, often the first dozen or so bases of a sequence are vector, and should be excluded from consideration. The value for this parameter has the form (start),(end) where (start) is the index of the first base to consider, and (end) is the last in the primer-picking region. -targetregion range [(full sequence)] If one or more Targets is specified then a legal primer pair must flank at least one of them. A Target might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair polymorphism. The value should be a space-separated list of (start),(end) pairs where (start) is the index of the first base of a Target, and (end) is the last E.g. 50,51 requires primers to surround the 2 bases at positions 50 and 51. -excludedregion range [(full sequence)] Primer oligos may not overlap any region specified in this tag. The associated value must be a space-separated list of (start),(end) pairs where (start) is the index of the first base of the excluded region, and and (end) is the last. This tag is useful for tasks such as excluding regions of low sequence quality or for excluding regions containing repetitive elements such as ALUs or LINEs. E.g. 401,407 68,70 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68. -forwardinput string The sequence of a forward primer to check and around which to design reverse primers and optional internal oligos. Must be a substring of SEQUENCE. (Any string) -reverseinput string The sequence of a reverse primer to check and around which to design forward primers and optional internal oligos. Must be a substring of the reverse strand of SEQUENCE. (Any string) * -gcclamp integer [0] Require the specified number of consecutive Gs and Cs at the 3' end of both the forward and reverse primer. (This parameter has no effect on the internal oligo if one is requested.) (Integer 0 or more) * -osize integer [20] Optimum length (in bases) of a primer oligo. Eprimer32 will attempt to pick primers close to this length. (Integer 0 or more) * -minsize integer [18] Minimum acceptable length of a primer. Must be greater than 0 and less than or equal to MAX-SIZE. (Integer 1 or more) * -maxsize integer [27] Maximum acceptable length (in bases) of a primer. Currently this parameter cannot be larger than 35. This limit is governed by the maximum oligo size for which Eprimer32's melting-temperature is valid. (Integer up to 35) * -otm float [60.0] Optimum melting temperature(Celsius) for a primer oligo. Eprimer32 will try to pick primers with melting temperatures are close to this temperature. The oligo melting temperature formula in Eprimer32 is that given in Rychlik, Spencer and Rhoads, Nucleic Acids Research, vol 18, num 21, pp 6409-6412 and Breslauer, Frank, Bloecker and Marky, Proc. Natl. Acad. Sci. USA, vol 83, pp 3746-3750. Please refer to the former paper for background discussion. (Any numeric value) * -mintm float [57.0] Minimum acceptable melting temperature(Celsius) for a primer oligo. (Any numeric value) * -maxtm float [63.0] Maximum acceptable melting temperature(Celsius) for a primer oligo. (Any numeric value) * -maxdifftm float [100.0] Maximum acceptable (unsigned) difference between the melting temperatures of the forward and reverse primers. (Any numeric value) * -ogcpercent float [50.0] Primer optimum GC percent. (Any numeric value) * -mingc float [20.0] Minimum allowable percentage of Gs and Cs in any primer. (Any numeric value) * -maxgc float [80.0] Maximum allowable percentage of Gs and Cs in any primer generated by Primer. (Any numeric value) * -saltconc float [50.0] The millimolar concentration of salt (usually KCl) in the PCR. Eprimer32 uses this argument to calculate oligo melting temperatures. (Any numeric value) * -dnaconc float [50.0] The nanomolar concentration of annealing oligos in the PCR. Eprimer32 uses this argument to calculate oligo melting temperatures. The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research--0.5 microliters of 20 micromolar concentration for each primer oligo in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius. This parameter corresponds to 'c' in Rychlik, Spencer and Rhoads' equation (ii) (Nucleic Acids Research, vol 18, num 21) where a suitable value (for a lower initial concentration of template) is 'empirically determined'. The value of this parameter is less than the actual concentration of oligos in the reaction because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures. See ADVICE FOR PICKING PRIMERS. (Any numeric value) * -maxpolyx integer [5] The maximum allowable length of a mononucleotide repeat in a primer, for example AAAAAA. (Integer 0 or more) * -psizeopt integer [200] The optimum size for the PCR product. 0 indicates that there is no optimum product size. (Integer 0 or more) * -prange range [100-300] The associated values specify the lengths of the product that the user wants the primers to create, and is a space separated list of elements of the form (x)-(y) where an (x)-(y) pair is a legal range of lengths for the product. For example, if one wants PCR products to be between 100 to 150 bases (inclusive) then one would set this parameter to 100-150. If one desires PCR products in either the range from 100 to 150 bases or in the range from 200 to 250 bases then one would set this parameter to 100-150 200-250. Eprimer32 favours ranges to the left side of the parameter string. Eprimer32 will return legal primers pairs in the first range regardless the value of the objective function for these pairs. Only if there are an insufficient number of primers in the first range will Eprimer32 return primers in a subsequent range. * -ptmopt float [0.0] The optimum melting temperature for the PCR product. 0 indicates that there is no optimum temperature. (Any numeric value) * -ptmmin float [-1000000.0] The minimum allowed melting temperature of the amplicon. Please see the documentation on the maximum melting temperature of the product for details. (Any numeric value) * -ptmmax float [1000000.0] The maximum allowed melting temperature of the amplicon. Product Tm is calculated using the formula from Bolton and McCarthy, PNAS 84:1390 (1962) as presented in Sambrook, Fritsch and Maniatis, Molecular Cloning, p 11.46 (1989, CSHL Press). Tm = 81.5 + 16.6(log10([Na+])) + .41*(%GC) - 600/length Where [Na+} is the molar sodium concentration, (%GC) is the percent of Gs and Cs in the sequence, and length is the length of the sequence. A similar formula is used by the prime primer selection program in GCG, which instead uses 675.0/length in the last term (after F. Baldino, Jr, M.-F. Chesselet, and M.E. Lewis, Methods in Enzymology 168:766 (1989) eqn (1) on page 766 without the mismatch and formamide terms). The formulas here and in Baldino et al. assume Na+ rather than K+. According to J.G. Wetmur, Critical Reviews in BioChem. and Mol. Bio. 26:227 (1991) 50 mM K+ should be equivalent in these formulae to .2 M Na+. Eprimer32 uses the same salt concentration value for calculating both the primer melting temperature and the oligo melting temperature. If you are planning to use the PCR product for hybridization later this behavior will not give you the Tm under hybridization conditions. (Any numeric value) * -oexcludedregion range [(full sequence)] Middle oligos may not overlap any region specified by this tag. The associated value must be a space-separated list of (start),(end) pairs, where (start) is the index of the first base of an excluded region, and (end) is the last. Often one would make Target regions excluded regions for internal oligos. * -oligoinput string The sequence of an internal oligo to check and around which to design forward and reverse primers. Must be a substring of SEQUENCE. (Any string) * -osizeopt integer [20] Optimum length (in bases) of an internal oligo. Eprimer32 will attempt to pick primers close to this length. (Integer 0 or more) * -ominsize integer [18] Minimum acceptable length of an internal oligo. Must be greater than 0 and less than or equal to INTERNAL-OLIGO-MAX-SIZE. (Integer 0 or more) * -omaxsize integer [27] Maximum acceptable length (in bases) of an internal oligo. Currently this parameter cannot be larger than 35. This limit is governed by maximum oligo size for which Eprimer32's melting-temperature is valid. (Integer up to 35) * -otmopt float [60.0] Optimum melting temperature (Celsius) for an internal oligo. Eprimer32 will try to pick oligos with melting temperatures that are close to this temperature. The oligo melting temperature formula in Eprimer32 is that given in Rychlik, Spencer and Rhoads, Nucleic Acids Research, vol 18, num 21, pp 6409-6412 and Breslauer, Frank, Bloecker and Marky, Proc. Natl. Acad. Sci. USA, vol 83, pp 3746-3750. Please refer to the former paper for background discussion. (Any numeric value) * -otmmin float [57.0] Minimum acceptable melting temperature(Celsius) for an internal oligo. (Any numeric value) * -otmmax float [63.0] Maximum acceptable melting temperature (Celsius) for an internal oligo. (Any numeric value) * -ogcopt float [50.0] Internal oligo optimum GC percent. (Any numeric value) * -ogcmin float [20.0] Minimum allowable percentage of Gs and Cs in an internal oligo. (Any numeric value) * -ogcmax float [80.0] Maximum allowable percentage of Gs and Cs in any internal oligo generated by Primer. (Any numeric value) * -osaltconc float [50.0] The millimolar concentration of salt (usually KCl) in the hybridization. Eprimer32 uses this argument to calculate internal oligo melting temperatures. (Any numeric value) * -odnaconc float [50.0] The nanomolar concentration of annealing internal oligo in the hybridization. (Any numeric value) * -oanyself float [12.00] The maximum allowable local alignment score when testing an internal oligo for (local) self-complementarity. Local self-complementarity is taken to predict the tendency of oligos to anneal to themselves The scoring system gives 1.00 for complementary bases, -0.25 for a match of any base (or N) with an N, -1.00 for a mismatch, and -2.00 for a gap. Only single-base-pair gaps are allowed. For example, the alignment 5' ATCGNA 3' || | | 3' TA-CGT 5' is allowed (and yields a score of 1.75), but the alignment 5' ATCCGNA 3' || | | 3' TA--CGT 5' is not considered. Scores are non-negative, and a score of 0.00 indicates that there is no reasonable local alignment between two oligos. (Number up to 9999.990) * -oendself float [12.00] The maximum allowable 3'-anchored global alignment score when testing a single oligo for self-complementarity. The scoring system is as for the Maximum Complementarity argument. In the examples above the scores are 7.00 and 6.00 respectively. Scores are non-negative, and a score of 0.00 indicates that there is no reasonable 3'-anchored global alignment between two oligos. In order to estimate 3'-anchored global alignments for candidate oligos, Primer assumes that the sequence from which to choose oligos is presented 5' to 3'. INTERNAL-OLIGO-SELF-END is meaningless when applied to internal oligos used for hybridization-based detection, since primer-dimer will not occur. We recommend that INTERNAL-OLIGO-SELF-END be set at least as high as INTERNAL-OLIGO-SELF-ANY. (Number up to 9999.990) * -opolyxmax integer [5] The maximum allowable length of an internal oligo mononucleotide repeat, for example AAAAAA. (Integer 0 or more) * -omishybmax float [12.0] Similar to MAX-MISPRIMING except that this parameter applies to the similarity of candidate internal oligos to the library specified in INTERNAL-OLIGO-MISHYB-LIBRARY. (Number up to 9999.990) Advanced (Unprompted) qualifiers: -mispriminglibraryfile infile The name of a file containing a nucleotide sequence library of sequences to avoid amplifying (for example repetitive sequences, or possibly the sequences of genes in a gene family that should not be amplified.) The file must be in (a slightly restricted) FASTA format (W. B. Pearson and D.J. Lipman, PNAS 85:8 pp 2444-2448 [1988]); we briefly discuss the organization of this file below. If this parameter is specified then Eprimer32 locally aligns each candidate primer against each library sequence and rejects those primers for which the local alignment score times a specified weight (see below) exceeds MAX-MISPRIMING. (The maximum value of the weight is arbitrarily set to 100.0.) Each sequence entry in the FASTA-format file must begin with an 'id line' that starts with '>'. The contents of the id line is 'slightly restricted' in that Eprimer32 parses everything after any optional asterisk ('*') as a floating point number to use as the weight mentioned above. If the id line contains no asterisk then the weight defaults to 1.0. The alignment scoring system used is the same as for calculating complementarity among oligos (e.g. SELF-ANY). The remainder of an entry contains the sequence as lines following the id line up until a line starting with '>' or the end of the file. Whitespace and newlines are ignored. Characters 'A', 'T', 'G', 'C', 'a', 't', 'g', 'c' are retained and any other character is converted to 'N' (with the consequence that any IUB / IUPAC codes for ambiguous bases are converted to 'N'). There are no restrictions on line length. An empty value for this parameter indicates that no repeat library should be used. -explainflag boolean [N] If this flag is true, produce LEFT-EXPLAIN, RIGHT-EXPLAIN, and INTERNAL-OLIGO-EXPLAIN output tags, which are intended to provide information on the number of oligos and primer pairs that Eprimer32 examined, and statistics on the number discarded for various reasons. -fileflag boolean [N] If the associated value is true, then Eprimer32 creates two output files for each input SEQUENCE. File (sequence-id).for lists all acceptable forward primers for (sequence-id), and (sequence-id).rev lists all acceptable reverse primers for (sequence-id), where (sequence-id) is the value of the SEQUENCE-ID tag (which must be supplied). In addition, if the input tag TASK is 1 or 4, Eprimer32 produces a file (sequence-id).int, which lists all acceptable internal oligos. -firstbaseindex integer [1] This parameter is the index of the first base in the input sequence. For input and output using 1-based indexing (such as that used in GenBank and to which many users are accustomed) set this parameter to 1. For input and output using 0-based indexing set this parameter to 0. (This parameter also affects the indexes in the contents of the files produced when the primer file flag is set.) (Any integer value) -pickanyway boolean [N] If true pick a primer pair even if LEFT-INPUT, RIGHT-INPUT, or INTERNAL-OLIGO-INPUT violates specific constraints. -maxmispriming float [12.00] The maximum allowed weighted similarity with any sequence in MISPRIMING-LIBRARY. (Number up to 9999.990) -pairmaxmispriming float [24.00] The maximum allowed sum of weighted similarities of a primer pair (one similarity for each primer) with any single sequence in MISPRIMING-LIBRARY. (Number up to 9999.990) -numnsaccepted integer [0] Maximum number of unknown bases (N) allowable in any primer. (Integer 0 or more) -selfany float [8.00] The maximum allowable local alignment score when testing a single primer for (local) self-complementarity and the maximum allowable local alignment score when testing for complementarity between forward and reverse primers. Local self-complementarity is taken to predict the tendency of primers to anneal to each other without necessarily causing self-priming in the PCR. The scoring system gives 1.00 for complementary bases, -0.25 for a match of any base (or N) with an N, -1.00 for a mismatch, and -2.00 for a gap. Only single-base-pair gaps are allowed. For example, the alignment 5' ATCGNA 3' ...|| | | 3' TA-CGT 5' is allowed (and yields a score of 1.75), but the alignment 5' ATCCGNA 3' ...|| | | 3' TA--CGT 5' is not considered. Scores are non-negative, and a score of 0.00 indicates that there is no reasonable local alignment between two oligos. (Number from 0.000 to 9999.990) -selfend float [3.00] The maximum allowable 3'-anchored global alignment score when testing a single primer for self-complementarity, and the maximum allowable 3'-anchored global alignment score when testing for complementarity between forward and reverse primers. The 3'-anchored global alignment score is taken to predict the likelihood of PCR-priming primer-dimers, for example 5' ATGCCCTAGCTTCCGGATG 3' .............||| ||||| ..........3' AAGTCCTACATTTAGCCTAGT 5' or 5' AGGCTATGGGCCTCGCGA 3' ...............|||||| ............3' AGCGCTCCGGGTATCGGA 5' The scoring system is as for the Maximum Complementarity argument. In the examples above the scores are 7.00 and 6.00 respectively. Scores are non-negative, and a score of 0.00 indicates that there is no reasonable 3'-anchored global alignment between two oligos. In order to estimate 3'-anchored global alignments for candidate primers and primer pairs, Primer assumes that the sequence from which to choose primers is presented 5' to 3'. It is nonsensical to provide a larger value for this parameter than for the Maximum (local) Complementarity parameter because the score of a local alignment will always be at least as great as the score of a global alignment. (Number 0.000 or more) -scorrection menu [1] Specifies the salt correction formula for the melting temperature calculation. (Values: 0 (Schildkraut & Lifson); 1 (SantaLucia); 2 (Owczarzy et al)) -tmformula menu [1] Specifies details of melting temperature calculation. (Values: 0 (Breslauer et al); 1 (SantaLucia)) -maxendstability float [9.0] The maximum stability for the five 3' bases of a forward or reverse primer. Bigger numbers mean more stable 3' ends. The value is the maximum delta G for duplex disruption for the five 3' bases as calculated using the nearest neighbor parameters published in Breslauer, Frank, Bloecker and Marky, Proc. Natl. Acad. Sci. USA, vol 83, pp 3746-3750. Eprimer32 uses a completely permissive default value for backward compatibility (which we may change in the next release). Rychlik recommends a maximum value of 9 (Wojciech Rychlik, 'Selection of Primers for Polymerase Chain Reaction' in BA White, Ed., 'Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications', 1993, pp 31-40, Humana Press, Totowa NJ). (Number up to 1000.000) Associated qualifiers: "-sequence" associated qualifiers -sbegin1 integer Start of each sequence to be used -send1 integer End of each sequence to be used -sreverse1 boolean Reverse (if DNA) -sask1 boolean Ask for begin/end/reverse -snucleotide1 boolean Sequence is nucleotide -sprotein1 boolean Sequence is protein -slower1 boolean Make lower case -supper1 boolean Make upper case -sformat1 string Input sequence format -sdbname1 string Database name -sid1 string Entryname -ufo1 string UFO features -fformat1 string Features format -fopenfile1 string Features file name "-outfile" associated qualifiers -odirectory2 string Output directory General qualifiers: -auto boolean Turn off prompts -stdout boolean Write first file to standard output -filter boolean Read first file from standard input, write first file to standard output -options boolean Prompt for standard and additional values -debug boolean Write debug output to program.dbg -verbose boolean Report some/full command line options -help boolean Report command line options and exit. More information on associated and general qualifiers can be found with -help -verbose -warning boolean Report warnings -error boolean Report errors -fatal boolean Report fatal errors -die boolean Report dying program messages -version boolean Report version number and exit Input file format eprimer32 reads one or more nucleotide sequences. The input is a standard EMBOSS sequence query (also known as a 'USA'). Major sequence database sources defined as standard in EMBOSS installations include srs:embl, srs:uniprot and ensembl Data can also be read from sequence output in any supported format written by an EMBOSS or third-party application. The input format can be specified by using the command-line qualifier -sformat xxx, where 'xxx' is replaced by the name of the required format. The available format names are: gff (gff3), gff2, embl (em), genbank (gb, refseq), ddbj, refseqp, pir (nbrf), swissprot (swiss, sw), dasgff and debug. See: http://emboss.sf.net/docs/themes/SequenceFormats.html for further information on sequence formats. Input files for usage example 'tembl:x65921' is a sequence entry in the example nucleic acid database 'tembl' Database entry: tembl:x65921 ID X65921; SV 1; linear; genomic DNA; STD; HUM; 2016 BP. XX AC X65921; S45242; XX DT 13-MAY-1992 (Rel. 31, Created) DT 14-NOV-2006 (Rel. 89, Last updated, Version 7) XX DE H.sapiens fau 1 gene XX KW fau 1 gene. XX OS Homo sapiens (human) OC Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; OC Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; OC Homo. XX RN [1] RP 1-2016 RA Kas K.; RT ; RL Submitted (29-APR-1992) to the EMBL/GenBank/DDBJ databases. RL K. Kas, University of Antwerp, Dept of Biochemistry T3.22, RL Universiteitsplein 1, 2610 Wilrijk, BELGIUM XX RN [2] RP 1-2016 RX DOI; 10.1016/0006-291X(92)91286-Y. RX PUBMED; 1326960. RA Kas K., Michiels L., Merregaert J.; RT "Genomic structure and expression of the human fau gene: encoding the RT ribosomal protein S30 fused to a ubiquitin-like protein"; RL Biochem. Biophys. Res. Commun. 187(2):927-933(1992). XX DR GDB; 191789. DR GDB; 191790. DR GDB; 354872. DR GDB; 4590236. XX FH Key Location/Qualifiers FH FT source 1..2016 FT /organism="Homo sapiens" FT /mol_type="genomic DNA" FT /clone_lib="CML cosmid" FT /clone="15.1" FT /db_xref="taxon:9606" FT mRNA join(408..504,774..856,951..1095,1557..1612,1787..>1912) FT /gene="fau 1" FT exon 408..504 FT /number=1 [Part of this file has been deleted for brevity] FT RAKRRMQYNRRFVNVVPTFGKKKGPNANS" FT intron 857..950 FT /number=2 FT exon 951..1095 FT /number=3 FT intron 1096..1556 FT /number=3 FT exon 1557..1612 FT /number=4 FT intron 1613..1786 FT /number=4 FT exon 1787..>1912 FT /number=5 FT polyA_signal 1938..1943 XX SQ Sequence 2016 BP; 421 A; 562 C; 538 G; 495 T; 0 other; ctaccatttt ccctctcgat tctatatgta cactcgggac aagttctcct gatcgaaaac 60 ggcaaaacta aggccccaag taggaatgcc ttagttttcg gggttaacaa tgattaacac 120 tgagcctcac acccacgcga tgccctcagc tcctcgctca gcgctctcac caacagccgt 180 agcccgcagc cccgctggac accggttctc catccccgca gcgtagcccg gaacatggta 240 gctgccatct ttacctgcta cgccagcctt ctgtgcgcgc aactgtctgg tcccgccccg 300 tcctgcgcga gctgctgccc aggcaggttc gccggtgcga gcgtaaaggg gcggagctag 360 gactgccttg ggcggtacaa atagcaggga accgcgcggt cgctcagcag tgacgtgaca 420 cgcagcccac ggtctgtact gacgcgccct cgcttcttcc tctttctcga ctccatcttc 480 gcggtagctg ggaccgccgt tcaggtaaga atggggcctt ggctggatcc gaagggcttg 540 tagcaggttg gctgcggggt cagaaggcgc ggggggaacc gaagaacggg gcctgctccg 600 tggccctgct ccagtcccta tccgaactcc ttgggaggca ctggccttcc gcacgtgagc 660 cgccgcgacc accatcccgt cgcgatcgtt tctggaccgc tttccactcc caaatctcct 720 ttatcccaga gcatttcttg gcttctctta caagccgtct tttctttact cagtcgccaa 780 tatgcagctc tttgtccgcg cccaggagct acacaccttc gaggtgaccg gccaggaaac 840 ggtcgcccag atcaaggtaa ggctgcttgg tgcgccctgg gttccatttt cttgtgctct 900 tcactctcgc ggcccgaggg aacgcttacg agccttatct ttccctgtag gctcatgtag 960 cctcactgga gggcattgcc ccggaagatc aagtcgtgct cctggcaggc gcgcccctgg 1020 aggatgaggc cactctgggc cagtgcgggg tggaggccct gactaccctg gaagtagcag 1080 gccgcatgct tggaggtgag tgagagagga atgttctttg aagtaccggt aagcgtctag 1140 tgagtgtggg gtgcatagtc ctgacagctg agtgtcacac ctatggtaat agagtacttc 1200 tcactgtctt cagttcagag tgattcttcc tgtttacatc cctcatgttg aacacagacg 1260 tccatgggag actgagccag agtgtagttg tatttcagtc acatcacgag atcctagtct 1320 ggttatcagc ttccacacta aaaattaggt cagaccaggc cccaaagtgc tctataaatt 1380 agaagctgga agatcctgaa atgaaactta agatttcaag gtcaaatatc tgcaactttg 1440 ttctcattac ctattgggcg cagcttctct ttaaaggctt gaattgagaa aagaggggtt 1500 ctgctgggtg gcaccttctt gctcttacct gctggtgcct tcctttccca ctacaggtaa 1560 agtccatggt tccctggccc gtgctggaaa agtgagaggt cagactccta aggtgagtga 1620 gagtattagt ggtcatggtg ttaggacttt ttttcctttc acagctaaac caagtccctg 1680 ggctcttact cggtttgcct tctccctccc tggagatgag cctgagggaa gggatgctag 1740 gtgtggaaga caggaaccag ggcctgatta accttccctt ctccaggtgg ccaaacagga 1800 gaagaagaag aagaagacag gtcgggctaa gcggcggatg cagtacaacc ggcgctttgt 1860 caacgttgtg cccacctttg gcaagaagaa gggccccaat gccaactctt aagtcttttg 1920 taattctggc tttctctaat aaaaaagcca cttagttcag tcatcgcatt gtttcatctt 1980 tacttgcaag gcctcaggga gaggtgtgct tctcgg 2016 // Output file format Output files for usage example File: x65921.eprimer32 # EPRIMER32 RESULTS FOR X65921 # FORWARD PRIMER STATISTICS: # considered 18855 # GC content failed 156 # low tm 9245 # high tm 3587 # high any compl 2 # high end compl 61 # long poly-x seq 45 # ok 5759 # REVERSE PRIMER STATISTICS: # considered 18706 # GC content failed 161 # low tm 9106 # high tm 3606 # high any compl 3 # high end compl 25 # long poly-x seq 66 # ok 5739 # PRIMER PAIR STATISTICS: # considered 4135 # unacceptable product size 3770 # high end compl 29 # ok 336 # Start Len Tm GC% Sequence 1 PRODUCT SIZE: 199 FORWARD PRIMER 201 20 59.90 65.00 ACCGGTTCTCCATCCCCGCA REVERSE PRIMER 380 20 59.84 65.00 CCGCGCGGTTCCCTGCTATT 2 PRODUCT SIZE: 207 FORWARD PRIMER 826 20 60.04 70.00 GACCGGCCAGGAAACGGTCG REVERSE PRIMER 1013 20 59.96 70.00 TGGCCTCATCCTCCAGGGGC 3 PRODUCT SIZE: 202 FORWARD PRIMER 831 20 60.32 70.00 GCCAGGAAACGGTCGCCCAG REVERSE PRIMER 1013 20 59.96 70.00 TGGCCTCATCCTCCAGGGGC 4 PRODUCT SIZE: 205 FORWARD PRIMER 600 20 60.18 70.00 GTGGCCCTGCTCCAGTCCCT REVERSE PRIMER 785 20 59.97 65.00 TGGGCGCGGACAAAGAGCTG 5 PRODUCT SIZE: 197 FORWARD PRIMER 203 20 59.84 70.00 CGGTTCTCCATCCCCGCAGC REVERSE PRIMER 380 20 59.84 65.00 CCGCGCGGTTCCCTGCTATT If the '-explain' flag has been used, as in the example, then statistics are output describing the number of primers that were considered and rejected for various reasons. Headers describing the input file name and the names of the output columns are displayed. The best 5 primer pairs are displayed with the product size. The reverse sequence is displayed as the reverse complement to the input forward sense sequence. The 'Start' positions are given counting from the start of the sequence for both the forward and reverse primer (and for the internal oligos). Note that if you compare the results to the output from the public Primer3 web site you will see a difference in the reverse primer positions - this is because the original Primer3 program reports the reverse primer positions as counted from the 3' end. The convention in EMBOSS is to report both forward and reverse features as counted from the 5' end, so the reverse primer positions are given counted from the 5' start of the sequence. Data files None. Notes The Whitehead Institute's primer3 program is not part of this program, but it must be set up on your system and on your path. The Whitehead Institute program that is run by this program is available from: http://primer3.sourceforge.net/ (Then see the link Download and then 'Release 1.1.4') Earlier versions are also supported. We expect later versions to also work - please contact the EMBOSS team if you find any problems. The Whitehead Institute's primer3 program states: We request but do not require that use of this software be cited in publications as: Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386 Source code available at http://fokker.wi.mit.edu/primer3/. The paper above is available at http://jura.wi.mit.edu/rozen/papers/rozen-and-skaletsky-2000-primer3.pd f References None. Warnings None. Diagnostic Error Messages The message: "EMBOSS An error in eprimer32.c at line 346: The program 'primer32_core' must be on the path. It is part of the 'primer3' package, available from the Whitehead Institute. See: http://primer3.sourceforge.net/" is output if you do not have the Whitehead Institute's primer3 program set up and on your path. Note that compiling the primer3 package will produce an executable called primer3_core. This must be renamed to primer32_core (or a link of that name added) for eprimer32 to work. This is to avoid conflict with the primer3 version 1.x series primer3_core executables required by the EMBOOS application eprimer3. Exit status It always exits with status 0. Known bugs None. See also Program name Description eprimer3 Picks PCR primers and hybridization oligos primersearch Search DNA sequences for matches with primer pairs stssearch Search a DNA database for matches with a set of STS primers History Author(s) Gary Williams formerly at: MRC Rosalind Franklin Centre for Genomics Research Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SB, UK Please report all bugs to the EMBOSS bug team (emboss-bug (c) emboss.open-bio.org) not to the original author. History Target users This program is intended to be used by everyone and everything, from naive users to embedded scripts. Comments None