Electronic PCR (ePCR)
Electronic PCR (ePCR) is used to identify sequence landmarks within a nucleotide sequence by looking at the alignment identity, coverage, and orientation of primer pairs to determine the ePCR product size compared to the expected product size.
The input data for this tool is a four (4) columns tab-demimited text file with the below specifications. Each row in the text file will correspond to one primer-pair. A sample of the input file can be obtained by clicking here.
- Column 1 - Forward Primer Name (Required): A string that assigns an unique identifier for the forward primer.
- Column 2 - Forward Primer Sequence (Required): Nucleotide sequence of forward primer.
- Column 3 - Reverse Primer Name (Required): A string that assigns an unique identifier for the reverse primer.
- Column 4 - Reverse Primer Sequence (Required): Nucleotide sequence of reverse primer.
- Column 5 - Expected Product Size (Optional): This column is optional and if specified, this tool will use this value to determine the base pairs difference between "actual" and ePCR product size.
USAGE: perl checkAmplicons.pl --length <length file> --blast <tabular blast output file> --pairs <primer pairs file> [OPTIONS] WHERE: <length file> : Two column text file separated by tab of which the first column contains the primer name and the second column contains the primer sequence length in base pairs. <tabular blast output file> : Blast output file of primer sequences blasted (BLASTN) to the reference sequence with -m 8 output option <primer pairs file> : 2 or 3 column text file separated by tab. The first two columns are the names of the primers of a given pair. The third column is optional and if included it should contain the expected product size in base pairs [OPTIONS] --ident or -i : Minimum identity of each primer hit from 0 to 1 (DEFAULT: 1) --coverage or -c : Minimum coverage of each primer hit from 0 to 1 (DEFAULT: 1) --maxpcr or -e : Integer value to filter all the amplicons that are less than --maxpcr