PCR amplicon prediction
Search for predicted amplicons. The database contains two or more primers (oligonucleotide
sequences). Each query sequence is searched for matches to a pair of primers
that would generate a PCR amplicon. To search for matches to single primers or
probes, use search_oligodb.
letters indicating degenerate positions in the primer are supported. See
IUPAC codes for details.
An amplicon is predicted when two primer sequences
(which can be the same) align to a query sequence within a given distance range.
The primers must match opposite strands of the query. The distance from the
first base in the first hit to the last base in the second hit is the length of
the predicted amplicon. The range of amplicon lengths is given by -minamp
(minimum length, default 50) and -maxamp (default 1000).
Pairs of primers satisfying the criteria for amplicons
are reported in the -pcrout tabbed text output file. See
pcrout file for details.
The -ampout option
specifies a FASTA output file for predicted amplicon sequences. The predicted amplicon
extends from the first base matching the first primer to the last base matching
the second primer.
The algorithm uses a fast and exact method; there are
no heuristics, so all matches meeting the accept criteria are guaranteed to be
found. Alignments are global; all letters of the database sequence must be
aligned to a letter in the query sequence. Gaps are not permitted, except for
terminal gaps in the query sequence.
are supported. By default, termination is disabled, equivalent to -maxaccepts 0
-maxrejects 0. In other words, by default the entire database (all primers) is searched.
Accept options are
supported. By default, -maxdiffs 2 is assumed and other accept criteria are not
used. Note that accept criteria are applied to the alignments of one primer to a
query sequence, not to the pair of alignments that constitute a hit. So with the
default criteria, there could be 2 diffs to the first primer and 2 diffs to the
second primer also, for a total of 4 diffs.
If the -pcr_strip_primers
option is specified, the query sequence segments matching the primers are
removed from the predicted amplicon before writing to the -prcrout and -ampout
files. This option does not change the amplicon length range set by the -minamp
and -maxamp options, which always include the primers.
The query file may be in
FASTA or FASTQ format.
file must be specified using the ‑db option and must be in
option is required.
usearch -search_pcr greengenes.fa -db 16s_primers.fa
-strand both \
-maxdiffs 3 -minamp 30 -maxamp 2000 -pcrout hits.txt -ampout