designAssays: Design (RT)-PCR assays

In sofpn/rprimer: Design Degenerate Oligos from a Multiple DNA Sequence Alignment

Design (RT)-PCR assays

Description

designAssays() combines primers to (RT)-PCR assays from an RprimerOligo object. If probes are present in the input dataset, only assays with a probe present between the primer pair will be kept.

Usage

designAssays(x, length = c(65, 120), tmDifferencePrimers = NULL)

Arguments

x	An RprimerOligo object, which can be with or without probes.
length	Amplicon length range, a numeric vector [40, 5000], defaults to c(65, 120).
tmDifferencePrimers	Maximum allowed difference between the mean tm of the forward and reverse primer (in Celcius degrees, as an absolute value). Defaults to NULL, which means that primers will be paired regardless of their tm.

Value

An RprimerAssay object, containing the following information:

start

Position where the assay starts.

end

Position where the assay ends.

length

Length of the amplicon.

totalDegeneracy

Total number of oligos in the assay.

score

Average oligo score. The best possible score is 0 and the worst possible score is 9. See ?oligos for more information about the scoring system.

startFwd

Start position of the forward primer.

endFwd

End positon of the forward primer.

lengthFwd

Length of the forward primer.

iupacSequenceFwd

Forward primer sequence in IUPAC format (i.e. with ambiguous bases).

identityFwd

For ambiguous primers: average identity of the forward primer. For mixed primers: average identity of the 5' (consensus) part of the forward primer. The value can range from 0 to 1.

coverageFwd

For ambiguous primers: average coverage of the forward primer. For mixed primers: average coverage of the 3' (degenerate) part of the forward primer. The value can range from 0 to 1.

degeneracyFwd

Number of sequence variants of the forward primer.

gcContentMeanFwd

Mean GC-content of all sequence variants of the forward primer.

gcContentRangeFwd

Range in GC-content of all sequence variants of the forward primer.

tmMeanFwd

Mean tm of all sequence variants of the forward primer (in Celcius degrees).

tmRangeFwd

Range in tm of all sequence variants of the forward primer (in Celcius degrees).

deltaGMeanFwd

Mean delta G of all sequence variants of the forward primer (in kcal/mol).

deltaGRangeFwd

Range in delta G of all sequence variants of the forward primer (in kcal/mol).

sequenceFwd

All sequence variants of the forward primer.

gcContentFwd

GC-content of all sequence variants of the forward primer.

tmFwd

Tm of all sequence variants of the forward primer (in Celcius degrees).

deltaGFwd

Delta G of all sequence variants of the forward primer (in kcal/mol).

methodFwd

Design method used to generate the forward primer: "ambiguous" or "mixedFwd".

startRev

Start position of the reverse primer.

endRev

End positon of the reverse primer.

lengthRev

Length of the reverse primer.

iupacSequenceRev

Reverse primer sequence in IUPAC format (i.e. with ambiguous bases).

identityRev

For ambiguous primers: average identity of the reverse primer. For mixed primers: average identity of the 5' (consensus) part of the reverse primer. The value can range from 0 to 1.

coverageRev

For ambiguous primers: average coverage of the reverse primer. For mixed primers: average coverage of the 3' (degenerate) part of the reverse primer. The value can range from 0 to 1.

degeneracyRev

Number of sequence variants of the reverse primer.

gcContentMeanRev

Mean GC-content of all sequence variants of the reverse primer.

gcContentRangeRev

Range in GC-content of all sequence variants of the reverse primer.

tmMeanRev

Mean tm of all sequence variants of the reverse primer (in Celcius degrees).

tmRangeRev

Range in tm of all sequence variants of the reverse primer (in Celcius degrees).

deltaGMeanRev

Mean delta G of all sequence variants of the reverse primer (in kcal/mol).

deltaGRangeRev

Range in delta G of all sequence variants of the reverse primer (in kcal/mol).

sequenceRev

All sequence variants of the reverse primer.

gcContentRev

GC-content of all sequence variants of the reverse primer.

tmRev

Tm of all sequence variants of the reverse primer (in Celcius degrees).

deltaGRev

Delta G of all sequence variants of the reverse primer (in kcal/mol).

methodRev

Design method used to generate the forward primer: "ambiguous" or "mixedRev.

roiStart

Start position of the input consensus profile used for oligo design.

roiEnd

End position of the input consensus profile used for oligo design.

If a probe is included in the input RprimerOligo object, the following columns are also included:

startPr

Start position of the probe.

endPr

End positon of the probe.

lengthPr

Length of the probe.

iupacSequencePr

Probe sequence in plus sense, in IUPAC format.

iupacSequenceRcPr

Probe sequence in minus sense, in IUPAC format.

identityPr

For ambiguous primers: average identity of the probe. For mixed primers: average identity of the 5' (consensus) part of the probe. The value can range from 0 to 1.

coveragePr

For ambiguous primers: average coverage of the probe. For mixed primers: average coverage of the 3' (degenerate) part of the probe. The value can range from 0 to 1.

degeneracyPr

Number of sequence variants of the probe.

gcContentMeanPr

Mean GC-content of all sequence variants of the probe.

gcContentRangePr

Range in GC-content of all sequence variants of the probe.

tmMeanPr

Mean tm of all sequence variants of the probe (in Celcius degrees).

tmRangePr

Range in tm of all sequence variants of the forward primer (in Celcius degrees).

deltaGMeanPr

Mean delta G of all sequence variants of the probe (in kcal/mol).

deltaGRangePr

Range in delta G of all sequence variants of the probe (in kcal/mol).

sequencePr

All sequence variants of the probe, in plus sense.

sequenceRcPr

All sequence variants of the probe, in minus sense.

gcContentPr

GC-content of all sequence variants of the probe.

tmPr

Tm of all sequence variants of the probe (in Celcius degrees).

deltaGPr

Delta G of all sequence variants of the probe (in kcal/mol).

methodPr

Design method used to generate the probe.

plusPr

If the probe is valid in plus sense.

minusPr

If the probe is valid in minus sense.

An error message will return if no assays are found.

Examples

data("exampleRprimerOligo")

## Design assays using default settings
designAssays(exampleRprimerOligo)

## Modify the length range
designAssays(exampleRprimerOligo, length = c(1000, 2000))

sofpn/rprimer documentation built on Aug. 19, 2024, 4:19 p.m.