📄PCR primer definition

Usage

If provided, primer definition file are used to:

• Trim primer sequences from input reads to minimize artifacts introduced by primer sequences (e.g. false positive variant called from a mismatch between primer and reference sequences)

• Compute per-amplicon coverage to infer if there is sufficient sample titer for variant calling and consensus sequence generation

Format

If primer coordinates are known (recommended)

A BED-like tab-separated value (TSV) file with no header row, with each row corresponding to a primer, and with 7 columns:

1. accession: reference sequence accession

2. start: primer start position

3. end: primer end position

4. primerName: unique primer name that includes amplicon name and direction tag (e.g. _LEFT)

5. pool: primer pool

6. strand: primer direction (+ or -)

7. sequence: primer sequence

While it is highly recommended to provide all 7 columns, the last three columns (pool, strand, sequence) are optional if both of the following conditions are met:

• All amplicons have exactly one left primer and one right primer (i.e. no alternative primers). This is inferred from theprimerName column.

• At least one entry in accession column appears in custom reference FASTA.

If at least one of the conditions are not met, the pipeline attempts to align the primer sequences against all reference sequences and define primer coordinates on the fly, ignoring the coordinates provided in the BED file. It is therefore highly recommended to provide all 7 columns with all the above conditions met.

Column order must be maintained. If you wish to provide the strand column, for instance, all 5 columns before it must be provided.

Example 1: 5-column format with columns accession, start, end, primerName, pool

seqX    0           15        primer1_LEFT   1
seqX    1745        1760      primer1_RIGHT  1
seqY    0           15        primer2_LEFT   2
seqY    1015        1030      primer2_RIGHT  2

Example 2: 7-column format with columns accession, start, end, primerName, pool, strand, sequence

seqX    0           15        primer1_LEFT   1     +       GGGCAAACCTAAAGG
seqX    1745        1760      primer1_RIGHT  1     -       GTTATGTAAAGGTGC
seqY    0           15        primer2_LEFT   2     +       GGGCGAAACTAAAGG
seqY    1015        1030      primer2_RIGHT  2     -       GTTATGTAAAGGTGC

If primer coordinates are unknown

Options below should be used if primer coordinates are not known but primer sequences are expected to map well to reference sequences with minimal mismatches. Using either option triggers the pipeline to align the primer sequences against all reference sequences in custom reference FASTA and define primer coordinates on the fly.

Option 1. One line per amplicon with 3 columns: ampliconName, forwardSequence,reverseSequence.

amplicon1      GGGCAAACCTAAAGG  GTTATGTAAAGGTGC
amplicon2      GGGCGAAACTAAAGG  GTTATGTAAAGGTGC

Option 2. One line per primer with 3 columns: primerName, sequence, pool.

primer1_LEFT      GGGCAAACCTAAAGG  1
primer1_LEFT_alt  GGGCGAAACTAAAGG  1
primer1_RIGHT     GTTATGTAAAGGTGC  1

Guidelines

• General

  • All text is case sensitive.

  • Any line starting with '#' is ignored. This can be used to add a header line with column names.

  • Every line must have the same number of columns and format (except those starting with '#').

  • 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 start 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 end field (3rd column) minus 1.

  • accession 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 (accession) 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

    • amplicon_4934m_RIGHT_alt

    • amplicon_4934m_RIGHT_alt1

    • amplicon_4934m_R_altprimerB

  • Examples of invalid primer names:

    • LEFT_MY_SEQUENCE_434_A

    • virus1_l

    • amplicon_4934m_RIGHT_L