primersearch 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 Search DNA sequences for matches with primer pairs Description primersearch reads in primer pairs from an input file and searches them against DNA sequence(s) specified by the user. Each of the primers in a pair is searched against the sequence and potential amplimers are reported. The output file is in the format of Whitehead primer3_core program. The user can specify a maximum percent mismatch level; for example, 10% mismatch on a primer of length 20bp means that the program will classify a primer as matching a sequence if 18 of the 20 base pairs matches. It will only report matches if both primers in the pair have a match in opposite orientations. Algorithm Each primer pair is specified in the input file by a name, followed by two primer sequences, primerA and primerB. The program first compares primerA to the forward strand and if it matches, primerB is compared to the reverse strand. The approach is then reversed, with the primerB being compared to the forward strand and primerA to the reverse. In this way all possible amplimers are reported. Usage Here is a sample session with primersearch % primersearch tembl:z52466 Search DNA sequences for matches with primer pairs Primer pairs file: primers Allowed percent mismatch [0]: Whitehead primer3_core program output file [z52466.primersearch]: Go to the input files for this example Go to the output files for this example Example 2 Here we run the same example but allowing 20% mismatch between the primers and the sequence: % primersearch tembl:z52466 Search DNA sequences for matches with primer pairs Primer pairs file: primers Allowed percent mismatch [0]: 20 Whitehead primer3_core program output file [z52466.primersearch]: Go to the output files for this example Command line arguments Search DNA sequences for matches with primer pairs Version: EMBOSS:6.4.0.0 Standard (Mandatory) qualifiers: [-seqall] seqall Nucleotide sequence(s) filename and optional format, or reference (input USA) [-infile] infile Primer pairs file [-mismatchpercent] integer [0] Allowed percent mismatch (Any integer value) [-outfile] outfile [*.primersearch] Whitehead primer3_core program output file Additional (Optional) qualifiers: (none) Advanced (Unprompted) qualifiers: (none) Associated qualifiers: "-seqall" 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 -odirectory4 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 primersearch reads in 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:z52466' is a sequence entry in the example nucleic acid database 'tembl' File: primers # This is my primer file D1S243 cacacaggctcacatgcc gctccagcgtcatggact D1S468 aattaaccgttttggtcct gcgacacacacttccc D1S2845 ccaaagggtgcttctc gtggcattccaacctc D1S1608 gatggcttttggggactatt cactgagccaagtgacacag D1S2893 aaaacatcaactctcccctg ctcaaaccccaataagcctt D1S2660 cacacatgcacatgcac agtgacaccagcaggg Database entry: tembl:z52466 ID Z52466; SV 1; linear; genomic DNA; STS; HUM; 389 BP. XX AC Z52466; XX DT 18-MAR-1996 (Rel. 47, Created) DT 09-SEP-2004 (Rel. 81, Last updated, Version 4) XX DE H.sapiens (D1S2660) DNA segment containing (CA) repeat; clone AFMa203yc1; DE single read. XX KW CA repeat; dinucleotide repeat; GT repeat; microsatellite DNA; KW microsatellite marker; repeat polymorphism; STS. 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 1-389 RA Weissenbach J.; RT ; RL Submitted (01-SEP-1995) to the EMBL/GenBank/DDBJ databases. RL Genethon, B.P. 60, 91002 Evry Cedex France. E-mail: RL Jean.Weissenbach@genethon.fr XX RN [2] RP 1-389 RX DOI; 10.1038/380152a0. RX PUBMED; 8600387. RA Dib C., Faure S., Fizames C., Samson D., Drouot N., Vignal A., RA Millasseau P., Marc S., Hazan J., Seboun E., Lathrop M., Gyapay G., RA Morissette J., Weissenbach J.; RT "A comprehensive genetic map of the human genome based on 5,264 RT microsatellites"; RL Nature 380(6570):152-154(1996). XX DR GDB; 606855. XX CC full automatic; XX FH Key Location/Qualifiers FH FT source 1..389 FT /organism="Homo sapiens" FT /chromosome="1" FT /mol_type="genomic DNA" FT /clone_lib="genomic DNA" FT /cell_line="CEPH 134702" FT /note="cloning vector is M13mp18" FT /db_xref="taxon:9606" XX SQ Sequence 389 BP; 118 A; 124 C; 86 G; 57 T; 4 other; agctgtgtgc acacaacatg anggggcaca catgcacatg cacacatgcc cacatgcata 60 tgcacacaca cacacacaca cacacacaca ttcatgccca agcacgccca ccctcatgtc 120 tcaccatgtg cacataacac acagtcacat ataccctggc acacatgccc acatgcagac 180 acgaaacaca ggcccacgnt tncatgcaca caggtatggg cacacatacc atgcacacat 240 aangacaaat accaggccag acatgatttg cccctgctgg tgtcactgtt aagtgtgaca 300 gacaagcaga ggacacacac ccacctggga cgcggggctt caggagagag gcagacctaa 360 tagggcccgg attcggggct ggggaggct 389 // The input primer file has the following format: Comment lines start with a '#' Lines with primer information have three fields separated by spaces or TAB characters. The columns contain: 1. The name of the primer pair - this is reported in the output. 2. The sequence of the first primer. 3. The sequence of the second primer. Empty files will cause primersearch to note that no primers have been found, and to exit. Output file format Output files for usage example File: z52466.primersearch Primer name D1S243 Primer name D1S468 Primer name D1S2845 Primer name D1S1608 Primer name D1S2893 Primer name D1S2660 Amplimer 1 Sequence: Z52466 Z52466 H.sapiens (D1S2660) DNA segment containing (CA) repeat; clone AFMa203yc1 ; single read. CACACATGCACATGCAC hits forward strand at 27 with 0 mismatches AGTGACACCAGCAGGG hits reverse strand at [103] with 0 mismatches Amplimer length: 261 bp Output files for usage example 2 File: z52466.primersearch Primer name D1S243 Primer name D1S468 Primer name D1S2845 Primer name D1S1608 Primer name D1S2893 Primer name D1S2660 Amplimer 1 Sequence: Z52466 Z52466 H.sapiens (D1S2660) DNA segment containing (CA) repeat; clone AFMa203yc1 ; single read. CACACATGCACATGCAC hits forward strand at 49 with 2 mismatches AGTGACACCAGCAGGG hits reverse strand at [103] with 0 mismatches Amplimer length: 239 bp Amplimer 2 Sequence: Z52466 Z52466 H.sapiens (D1S2660) DNA segment containing (CA) repeat; clone AFMa203yc1 ; single read. CACACATGCACATGCAC hits forward strand at 27 with 0 mismatches AGTGACACCAGCAGGG hits reverse strand at [103] with 0 mismatches Amplimer length: 261 bp Data files None. Notes Every potential amplimer will be reported; if one primer matches the forward strand twice and the other matches the reverse strand only once, two potential amplimers are reported. If the reverse primer matches twice, four potential amplimers are reported. References None. Warnings This program is processor-intensive. You should probably not use it, for example, to search the whole of EMBL or even the human section of EMBL. It will take longer with more primer pairs and if mismatches are allowed. Diagnostic Error Messages "No suitable primers found - exiting" means that either the primers file was empty or there were no compilable primer pairs contained in it. Exit status It always exits with status 0 Known bugs None. See also Program name Description eprimer3 Picks PCR primers and hybridization oligos eprimer32 Picks PCR primers and hybridization oligos stssearch Search a DNA database for matches with a set of STS primers stssearch does something similar, but doesn't allow you to find mismatches and will report any match in any orientation and doesn't require you to have both primers matching. Author(s) Val Curwen formerly at: Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. Please report all bugs to the EMBOSS bug team (emboss-bug (c) emboss.open-bio.org) not to the original author. History Written Aug 2000 - Val Curwen Target users This program is intended to be used by everyone and everything, from naive users to embedded scripts. Comments None