                                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:6.5.6.0

   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
   -scircular1         boolean    Sequence is circular
   -sformat1           string     Input sequence format
   -iquery1            string     Input query fields or ID list
   -ioffset1           integer    Input start position offset
   -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 INSDC.
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     Pick PCR primers and hybridization oligos
                    eprimer32    Pick 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
