Primer definition file formats

Any number of spaces can separate the columns. A value within a single column should not have any space.

BED format

• Per standard BED conventions, sequence coordinates are given as 0-based, half-open intervals, such that the chromStart field (2nd column) contains the first nucleotide in the primer binding site and the last nucleotide in the primer binding site is the value in the chromEnd field (3rd column) minus 1.

• chrom field must contain a sequence identifier that matches the header of the FASTA file containing the sequence that the coordinates are relative to.

• Multiple sequence identifiers (chrom) are permitted within one file.

Primer name

• primerName must be unique and encode the name of the amplicon for which the primer is designed, the direction tag indicating which side of the amplicon, left or right, the primer belongs to, and an optional indicator that the primer is an alternative primer for that amplicon.

• In addition to _LEFT and _RIGHT, we permit _L and _R as direction tags in primerName. Any text after the direction tag should be separated by an underscore.

• Text in primerName before the direction tag is considered to be an amplicon identifier. Ensure that the text of the amplicon identifier is unique for that amplicon and that the direction tag occurs only once in primerName.

• Each amplicon must have at least one left and right primer (including alternative primers) associated with it.

• Alternative primers are used to bind to locations that avoid sequence variation in the default primer binding site that may disrupt hybridization. An amplicon may have an arbitrary number of alternative primers (as long as the primer name is unique), but most amplicons will have none. Alternative primers are indicated by the presence of the _alt after the direction tag in primerName, followed by optional text to distinguish between different alternative primers, such as a number.

• Examples of valid primer names:

  • MY_SEQUENCE_434_A_LEFT

  • virus1_L

• Examples of invalid primer names:

  • LEFT_MY_SEQUENCE_434_A

  • virus1_l

amplicon_4934m_RIGHT_alt1

amplicon_4934m_R_altprimerB