Package 'rprimer' reference manual

Title:	Design Degenerate Oligos from a Multiple DNA Sequence Alignment
Description:	Functions, workflow, and a Shiny application for visualizing sequence conservation and designing degenerate primers, probes, and (RT)-(q/d)PCR assays from a multiple DNA sequence alignment. The results can be presented in data frame format and visualized as dashboard-like plots. For more information, please see the package vignette.
Authors:	Sofia Persson [aut, cre]
Maintainer:	Sofia Persson <[email protected]>
License:	GPL-3
Version:	1.11.0
Built:	2024-11-18 04:10:14 UTC
Source:	https://github.com/bioc/rprimer

Check how oligos and assays match to their target sequences

Description

checkMatch() investigates how well oligos or assays match with their intended target sequences within a multiple DNA sequence alignment.

Usage

checkMatch(x, target)

## S4 method for signature 'RprimerOligo'
checkMatch(x, target)

## S4 method for signature 'RprimerAssay'
checkMatch(x, target)
checkMatch(x, target)

## S4 method for signature 'RprimerOligo'
checkMatch(x, target)

## S4 method for signature 'RprimerAssay'
checkMatch(x, target)

Arguments

`x`	An `RprimerOligo` or `RprimerAssay` object.
`target`	A `Biostrings::DNAMultipleAlignment` alignment with intended target sequences. Note that it must be same alignment that was used for generating the oligos/assays in `x`.

Details

The output provides information on the proportion and names of target sequences that match perfectly as well as with one, two, three, or four or more mismatches to the oligo within the intended oligo binding region in the input alignment (on-target match). It also gives the proportion and names of target sequences that match with a maximum of two mismatches to at least one sequence variant of the oligo outside the intended oligo binding region (off-target match). The function is a wrapper to Biostrings::vcountPDict() (Pages et al., 2020).

Value

An RprimerMatchOligo or RprimerMatchAssay object, depending on whether an RprimerOligo or RprimerAssay object was used as input.

RprimerMatchOligo objects contain the following information:

iupacSequence: The oligo sequence in IUPAC format.
perfectMatch: Proportion of target sequences that matches perfectly to the oligo within the intended binding region.
idPerfectMatch: Names of all sequences that matches perfectly.
oneMismatch: Proportion of target sequences with one mismatch to the oligo within the intended binding region.
idOneMismatch: Names of all sequences that matches with one mismatch.
twoMismatches: Proportion of target sequences with two mismatches to the oligo within the intended binding region.
idTwoMismatches: Names of all sequences that matches with two mismatches.
threeMismatches: Proportion of target sequences with three mismatches to the oligo within the intended binding region.
idThreeMismatches: Names of all sequences that matches with three mismatches.
fourOrMoreMismatches: Proportion of target sequences with four or more mismatches to the oligo within the intended binding region.
idFourOrMoreMismatches: Names of all sequences that matches with four or more mismatches.
offTargetMatch: Proportion of target sequences with maximum two mismatches to at least one site outside the intended oligo binding region in the input alignment.
idOffTargetMatch: Names of all off-target matching sequences.

RprimerMatchAssay objects contain the following information:

iupacSequenceFwd: The forward primer sequence in IUPAC format.
perfectMatchFwd: Proportion of target sequences that matches perfectly with the forward primer withing the intended binding region.
idPerfectMatchFwd: Names of all sequences that matches perfectly.
oneMismatchFwd: Proportion of target sequences with one mismatch to the forward primer within the intended binding region.
idOneMismatchFwd: Names of all sequences that matches with one mismatch.
twoMismatchesFwd: Proportion of target sequences with two mismatches to the forward primer within the intended binding region.
idTwoMismatchesFwd: Names of all sequences that matches with two mismatches.
threeMismatchesFwd: Proportion of target sequences with three mismatches to the forward primer within the intended binding region.
idThreeMismatchesFwd: Names of all sequences that matches with three mismatches.
fourOrMoreMismatchesFwd: Proportion of target sequences with four or more mismatches to the forward primer within the intended binding region.
idFourOrMoreMismatchesFwd: Names of all sequences that matches with four or more mismatches.
offTargetMatchFwd: Proportion of target sequences with maximum two mismatches to at least one site outside the intended forward primer binding region in the input alignment.
idOffTargetMatchFwd: Names of all off-target matching sequences.
iupacSequenceRev: The reverse primer sequence in IUPAC format.
perfectMatchRev: Proportion of target sequences that matches perfectly with the reverse primer withing the intended binding region.
idPerfectMatchRev: Names of all sequences that matches perfectly.
oneMismatchRev: Proportion of target sequences with one mismatch to the reverse primer within the intended binding region.
idOneMismatchRev: Names of all sequences that matches with one mismatch.
twoMismatchesRev: Proportion of target sequences with two mismatches to the reverse primer within the intended binding region.
idTwoMismatchesRev: Names of all sequences that matches with two mismatches.
threeMismatchesRev: Proportion of target sequences with three mismatches to the reverse primer within the intended binding region.
idThreeMismatchesRev: Names of all sequences that matches with three mismatches.
fourOrMoreMismatchesRev: Proportion of target sequences with four or more mismatches to the reverse primer within the intended binding region.
idFourOrMoreMismatchesRev: Names of all sequences that matches with four or more mismatches.
offTargetMatchRev: Proportion of target sequences with maximum two mismatches to at least one site outside the intended reverse primer binding region in the input alignment.
idOffTargetMatchRev: Names of all off-target matching sequences.

If the input assay contains probes, the following information is also added:

iupacSequencePr: The probe sequence in IUPAC format.
perfectMatchPr: Proportion of target sequences that matches perfectly with the probe withing the intended binding region.
idPerfectMatchPr: Names of all sequences that matches perfectly.
oneMismatchPr: Proportion of target sequences with one mismatch to the probe within the intended binding region.
idOneMismatchPr: Names of all sequences that matches with one mismatch.
twoMismatchesPr: Proportion of target sequences with two mismatches to the probe within the intended binding region.
idTwoMismatchesPr: Names of all sequences that matches with two mismatches.
threeMismatchesPr: Proportion of target sequences with three mismatches to the probe within the intended binding region.
idThreeMismatchesPr: Names of all sequences that matches with three mismatches.
fourOrMoreMismatchesPr: Proportion of target sequences with four or more mismatches to the probe within the intended binding region.
idFourOrMoreMismatchesPr: Names of all sequences that matches with four or more mismatches.
offTargetMatchPr: Proportion of target sequences with maximum two mismatches to at least one site outside the intended probe binding region in the input alignment.
idOffTargetMatchPr: Names of all off-target matching sequences.

Methods (by class)

RprimerOligo:
RprimerAssay:

Limitations

There are a few limitations with this function, which is important to be aware of:

False negatives or positives may occur due to poorly aligned sequences
The output does not tell which strand (minus or plus) the oligo matches to. This is important to consider when assessing off-target matches to single-stranded targets
Ambiguous bases and gaps in the target sequences are identified as mismatches
The function checks strictly on- and off-target, and may therefore miss off-target matches that partially overlap the intended target

References

Pages, H., Aboyoun, P., Gentleman R., and DebRoy S. (2020). Biostrings: Efficient manipulation of biological strings. R package version 2.57.2.

Examples

#### RprimerOligo objects

data("exampleRprimerOligo")
data("exampleRprimerAlignment")

x <- exampleRprimerOligo[1:2, ]
target <- exampleRprimerAlignment

checkMatch(x, target)

#### RprimerAssay objects

data("exampleRprimerAssay")
data("exampleRprimerAlignment")

x <- exampleRprimerAssay[1:2, ]
target <- exampleRprimerAlignment

checkMatch(x, target)
#### RprimerOligo objects

data("exampleRprimerOligo")
data("exampleRprimerAlignment")

x <- exampleRprimerOligo[1:2, ]
target <- exampleRprimerAlignment

checkMatch(x, target)

#### RprimerAssay objects

data("exampleRprimerAssay")
data("exampleRprimerAlignment")

x <- exampleRprimerAssay[1:2, ]
target <- exampleRprimerAlignment

checkMatch(x, target)

Get sequence information from an alignment

Description

consensusProfile() takes a DNA multiple alignment as input and returns all the data needed for subsequent primer and probe design process. The function is a wrapper to Biostrings::consensusMatrix() (Pages et al., 2020).

Usage

consensusProfile(x, ambiguityThreshold = 0)
consensusProfile(x, ambiguityThreshold = 0)

Arguments

`x`	A `Biostrings::DNAMultipleAlignment` object.
`ambiguityThreshold`	"Detection level" for ambiguous bases. All DNA bases that occur with a relative frequency higher than the specified value will be included when the IUPAC consensus character is determined. Can range from 0 to 0.2, defaults to `0`.

Value

An RprimerProfile object, which contains the following information:

position: Position in the alignment.
a: Proportion of A.
c: Proportion of C.
g: Proportion of G.
t: Proportion of T.
other: Proportion of bases other than A, C, G, T.
gaps: Proportion of gaps (recognized as "-" in the alignment).
majority: Majority consensus sequence. Denotes the most frequently occurring nucleotide. If two or more bases occur with the same frequency, the consensus nucleotide will be randomly selected among these.
identity: Proportion of sequences, among all sequences with a DNA base (i.e., A, C, G or T), that has the majority consensus base.
iupac: The consensus sequence expressed in IUPAC format. The IUPAC consensus sequence only takes 'A', 'C', 'G', 'T' and '-' as input. Degenerate bases will be skipped. If a position only contains degenerate bases, the IUPAC consensus will be NA at that position.
coverage: Proportion of sequences in the target alignment, among all sequences with a DNA base, that are covered the IUPAC consensus character. The value will be 1 if there are no "remaining" DNA bases (and/or if ambiguityThreshold = 0).

References

Pages, H., Aboyoun, P., Gentleman R., and DebRoy S. (2020). Biostrings: Efficient manipulation of biological strings. R package version 2.57.2.

Examples

data("exampleRprimerAlignment")
consensusProfile(exampleRprimerAlignment)

consensusProfile(exampleRprimerAlignment, ambiguityThreshold = 0.05)
data("exampleRprimerAlignment")
consensusProfile(exampleRprimerAlignment)

consensusProfile(exampleRprimerAlignment, ambiguityThreshold = 0.05)

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)
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))
data("exampleRprimerOligo")

## Design assays using default settings
designAssays(exampleRprimerOligo)

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

Design primers and probes

Description

designOligos() designs oligos (primers and probes) from a consensus profile.

Usage

designOligos(
  x,
  maxGapFrequency = 0.01,
  lengthPrimer = c(18, 22),
  maxDegeneracyPrimer = 4,
  gcClampPrimer = TRUE,
  avoidThreeEndRunsPrimer = TRUE,
  gcPrimer = c(0.4, 0.65),
  tmPrimer = c(50, 65),
  concPrimer = 500,
  designStrategyPrimer = "ambiguous",
  probe = TRUE,
  lengthProbe = c(18, 22),
  maxDegeneracyProbe = 4,
  avoidFiveEndGProbe = TRUE,
  gcProbe = c(0.4, 0.65),
  tmProbe = c(50, 70),
  concProbe = 250,
  concNa = 0.05
)
designOligos(
  x,
  maxGapFrequency = 0.01,
  lengthPrimer = c(18, 22),
  maxDegeneracyPrimer = 4,
  gcClampPrimer = TRUE,
  avoidThreeEndRunsPrimer = TRUE,
  gcPrimer = c(0.4, 0.65),
  tmPrimer = c(50, 65),
  concPrimer = 500,
  designStrategyPrimer = "ambiguous",
  probe = TRUE,
  lengthProbe = c(18, 22),
  maxDegeneracyProbe = 4,
  avoidFiveEndGProbe = TRUE,
  gcProbe = c(0.4, 0.65),
  tmProbe = c(50, 70),
  concProbe = 250,
  concNa = 0.05
)

Arguments

`x`	An `RprimerProfile` object.
`maxGapFrequency`	Maximum allowed gap frequency at the primer and probe binding sites in the target alignment. A number [0, 1], defaults to `0.01`.
`lengthPrimer`	Primer length range. A numeric vector [15, 30], defaults to `c(18, 22)`.
`maxDegeneracyPrimer`	Maximum number of variants of each primer. A number [1, 64], defaults to `4`.
`gcClampPrimer`	If primers must have a GC clamp. A GC clamp is identified as two to three G or C:s within the last five bases (3' end) of the primer. `TRUE` or `FALSE`, defaults to `TRUE`.
`avoidThreeEndRunsPrimer`	If primers with more than two runs of the same nucleotide at the terminal 3' end should be avoided. `TRUE` or `FALSE`, defaults to `TRUE`.
`gcPrimer`	GC-content range for primers. A numeric vector [0, 1], defaults to `c(0.40, 0.65)`.
`tmPrimer`	Tm range for primers (in Celcius degrees). A numeric vector [30, 90], defaults to `c(55, 65)`.
`concPrimer`	Primer concentration in nM, for tm calculation. A number [20, 2000], defaults to `500`.
`designStrategyPrimer`	`"ambiguous"` or `"mixed"`. Defaults to `"ambiguous"` (see details below).
`probe`	If probes should be designed. `TRUE` or `FALSE`, defaults to `TRUE`.
`lengthProbe`	Probe length range. A numeric vector [15, 40], defaults to `c(18, 22)`.
`maxDegeneracyProbe`	Maximum number of variants of each probe. A number [1, 64], defaults to `4`.
`avoidFiveEndGProbe`	If probes with G at the 5' end should be avoided. `TRUE` or `FALSE`, defaults to `TRUE`.
`gcProbe`	GC-content range for probes. A numeric vector [0, 1], defaults to `c(0.40, 0.65)`.
`tmProbe`	Tm range for probes (in Celcius degrees). A numeric vector [30, 90], defaults to `c(55, 70)`.
`concProbe`	Primer concentration in nM, for tm calculation. A numeric vector [20, 2000], defaults to `250`.
`concNa`	Sodium ion (equivalent) concentration in the PCR reaction (in M). For calculation of tm and delta G. A numeric vector [0.01, 1], defaults to `0.05` (50 mM).

Details

Valid oligos

For an oligo to be considered as valid, all sequence variants must fulfill all the specified design constraints.

Furthermore, oligos with at least one sequence variant containing more than four consecutive runs of the same nucleotide (e.g. "AAAAA") and/or more than three consecutive runs of the same di-nucleotide (e.g. "TATATATA") will be excluded from consideration.

Calculation of tm and delta G

Melting temperatures are calculated for perfectly matching DNA duplexes using the nearest-neighbor method (SantaLucia and Hicks, 2004), by using the following equation:

\[Tm = (\Delta H ^o \cdot 1000) / (\Delta S ^o + R \cdot \log [\mathrm{oligo}]) - 273.15\]

where $\Delta H ^o$ is the change in enthalpy (in cal/mol) and $\Delta S ^o$ is the change in entropy (in cal/K/mol) when an oligo and a perfectly matching target sequence goes from random coil to duplex formation. $K$ is the gas constant (1.9872 cal/mol K).

Delta G is calculated at 37 Celcius degrees, for when an oligo and a perfectly matching target sequence goes from random coil to duplex state, by using the following equation:

\[ \Delta G ^o _T = ( \Delta H ^o \cdot 1000 - T \cdot \Delta S ^o ) / 1000\]

ASCII representation For both tm and delta G, the following salt correction method is used for $ \Delta S^o $, as described in SantaLucia and Hicks (2004):

\[ \Delta S^o [\mathrm{Na^+}] = \Delta S^o [\mathrm{1 M NaCl}] + 0.368 \cdot N / 2 \cdot \log [\mathrm{Na^+}] \]

where $N$ is the total number of phosphates in the duplex, and [Na+] is the total concentration of monovalent cations.

Nearest neighbor table values for $\Delta S^o$ and $\Delta H^o$ are from SantaLucia and Hicks, 2004, and can be retrieved calling rprimer:::lookup$nn.

Primer design strategies

Primers can be generated by using one of the two following strategies:

The ambiguous strategy (default) generates primers from the IUPAC consensus sequence alone, which means that ambiguous bases can occur at any position in the primer.
The mixed strategy generates primers from both the majority and the IUPAC consensus sequence. These primers consist of a shorter degenerate part at the 3' end (approx. 1/3 of the primer, targeting a conserved region) and a longer consensus part at the 5' end (approx. 2/3 of the primer), which instead of having ambiguous bases contains the most frequently occuring nucleotide at each position. This strategy resembles the widely-adopted Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) principle (Rose et al., 1998), and aims to to allow amplification of highly variable targets using primers with low degeneracy. The idea is that the degenerate 3' end part will bind specifically to the target sequence in the initial PCR cycles, and promote amplification in spite of eventual mismatches at the 5' consensus part (since 5' end mismatches are generally less detrimental than 3' end mismatches). In this way, the generated products will match the 5' ends of all primers perfectly, which allows them to be efficiently amplified in later PCR cycles. To provide a sufficiently high tm in spite of mismatches, it is recommended to design relatively long primers (at least 25 bases) when using this strategy.

Probes are always designed using the ambiguous strategy.

Scoring system for oligos

All valid oligos are scored based on their identity, coverage and average GC content. The scoring system is presented below.

Identity and coverage

Value range	Score
$(0.99, 1]$	0
$(0.95, 0.99]$	1
$(0.90, 0.95]$	2
$\leq 0.90$	3

Average GC-content

This score is based on how much the average GC-content deviates from 0.5 (in absolute value).

Value range	Score
$[0, 0.05)$	0
$[0.05, 0.1)$	1
$[0.1, 0.2)$	2
$\geq 0.2$	3

These scores are summarized. The weight of each individual score is 1, and thus, the lowest and best possible score for an oligo is 0, and the worst possible score is 9.

Value

An RprimerOligo object, containing the following information:

type: Whether the oligo is a primer or probe.
fwd: TRUE if the oligo is valid in forward direction, FALSE otherwise.
rev: TRUE if the oligo is valid in reverse direction, FALSE otherwise.
start: Start position of the oligo.
end: End positon of the oligo.
length: Oligo length.
iupacSequence: Oligo sequence in IUPAC format (i.e. with ambiguous bases).
iupaSequenceRc: The reverse complement of the iupacSequence.
identity: For ambiguous oligos: average identity of the oligo. For mixed oligos: average identity of the 5' (consensus) part of the oligo. The value can range from 0 to 1.
coverage: For ambiguous oligos: average coverage of the oligo. For mixed oligos: average coverage of the 3' (degenerate) part of the oligo. The value can range from 0 to 1.
degeneracy: Number of sequence variants of the oligo.
gcContentMean: Mean GC-content of all sequence variants of the oligo.
gcContent: Range in GC-content of all sequence variants of the oligo.
tmMean: Mean tm of all sequence variants of the oligo (in Celcius degrees).
tm: Range in tm of all sequence variants of the oligo (in Celcius degrees).
deltaGMean: Mean delta G of all sequence variants of the oligo (in kcal/mol).
deltaG: Range in delta G of all sequence variants of the oligo (in kcal/mol).
sequence: All sequence variants of the oligo.
sequenceRc: Reverse complements of all sequence variants.
gcContent: GC-content of all sequence variants.
tm: Tm of all sequence variants (in Celcius degrees).
deltaG: Delta G of all sequence variants (in kcal/mol).
method: Design method used to generate the oligo: "ambiguous", "mixedFwd" or "mixedRev".
score: Oligo score, the lower the better.
roiStart: First position of the input RprimerProfile object (roi = region of interest).
roiEnd: Last position of the input RprimerProfile object.

An error message will return if no oligos are found. If so, a good idea could be to re-run the design process with relaxed constraints.

References

Rose, TM., Schultz ER., Henikoff JG., Pietrokovski S., McCallum CM., and Henikoff S. 1998. Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Distantly Related Sequences. Nucleic Acids Research 26 (7): 1628-35.

SantaLucia Jr, J., & Hicks, D. (2004). The thermodynamics of DNA structural motifs. Annu. Rev. Biophys. Biomol. Struct., 33, 415-440.

Examples

data("exampleRprimerProfile")
x <- exampleRprimerProfile

## Design primers and probes with default values
designOligos(x)
data("exampleRprimerProfile")
x <- exampleRprimerProfile

## Design primers and probes with default values
designOligos(x)

Example datasets

Description

The purpose of these datasets is to illustrate the functionality of rprimer. The following datasets are provided:

exampleRprimerAlignment - a Biostrings::DNAMultipleAlignment object (Pages et al., 2020) containing an alignment of 50 hepatitis E virus sequences collected from NCBI GenBank. See "documentation_example_alignment.txt" within the inst/script folder of this package for more details.
exampleRprimerProfile - an RprimerProfile object, generated from the alignment above.
exampleRprimerOligo - an RprimerOligo object, generated from the consensus profile above.
exampleRprimerAssay - an RprimerAssay object, generated from the oligos above.
exampleRprimerMatchOligo - an RprimerMatchOligo object, describing how well some oligos match with the sequences in exampleRprimerAlignment.
exampleRprimerMatchAssay - an RprimerMatchAssay object, describing how well some assays match with the seuqences in exampleRprimerAlignment.

Usage

data("exampleRprimerAlignment")

data("exampleRprimerProfile")

data("exampleRprimerOligo")

data("exampleRprimerAssay")

data("exampleRprimerMatchOligo")

data("exampleRprimerMatchAssay")
data("exampleRprimerAlignment")

data("exampleRprimerProfile")

data("exampleRprimerOligo")

data("exampleRprimerAssay")

data("exampleRprimerMatchOligo")

data("exampleRprimerMatchAssay")

Format

An object of class DNAMultipleAlignment with 50 rows and 7597 columns.

An object of class RprimerProfile with 7597 rows and 12 columns.

An object of class RprimerOligo with 322 rows and 26 columns.

An object of class RprimerAssay with 4883 rows and 65 columns.

An object of class RprimerMatchOligo with 10 rows and 13 columns.

An object of class RprimerMatchAssay with 5 rows and 39 columns.

References

H. Pages, P. Aboyoun, R. Gentleman and S. DebRoy (2020). Biostrings: Efficient manipulation of biological strings. R package version 2.57.2.

Plot an Rprimer object

Description

plotData visualizes objects from all different Rprimer classes.

Usage

plotData(x, ...)

## S4 method for signature 'RprimerProfile'
plotData(x, type = "overview", highlight = NULL, rc = FALSE)

## S4 method for signature 'RprimerOligo'
plotData(x)

## S4 method for signature 'RprimerAssay'
plotData(x)

## S4 method for signature 'RprimerMatchOligo'
plotData(x)

## S4 method for signature 'RprimerMatchAssay'
plotData(x)
plotData(x, ...)

## S4 method for signature 'RprimerProfile'
plotData(x, type = "overview", highlight = NULL, rc = FALSE)

## S4 method for signature 'RprimerOligo'
plotData(x)

## S4 method for signature 'RprimerAssay'
plotData(x)

## S4 method for signature 'RprimerMatchOligo'
plotData(x)

## S4 method for signature 'RprimerMatchAssay'
plotData(x)

Arguments

`x`	An `RprimerProfile`, `RprimerOligo` `RprimerAssay`, `rprimerMatchOligo` or `RprimerMatchAssay` object.
`...`	Optional arguments for `RprimerProfile` objects.
`type`	For `Rprimeroligo` objects: Type of plot: `"overview"`, or `"nucleotide"`, defaults to `"overview"`.
`highlight`	For `Rprimeroligo` objects: If a specific region within an overview plot should be highlighted. A numeric vector indicating the start and end position, e.g. `c(100, 1000)`, defaults to `NULL` (i.e., no highlight).
`rc`	For `Rprimeroligo` objects, and `type = "nucleotide"`: If the plotted sequence should be displayed as reverse complement or not. `TRUE` or FALSE, defaults to `FALSE`. See examples below.

Value

A plot.

Methods (by class)

RprimerProfile:
RprimerOligo:
RprimerAssay:
RprimerMatchOligo:
RprimerMatchAssay:

Examples

#### Plot an RprimerProfile object

data("exampleRprimerProfile")
prof <- exampleRprimerProfile

## Plot an overview
plotData(prof, highlight = c(500, 1000))

## Select a region of interest
roi <- prof[prof$position >= 500 & prof$position <= 550, ]

## Plot an overview of the roi
plotData(roi)

## Plot the nucleotide distribution of the roi
plotData(roi, type = "nucleotide")

#### Plot an RprimerOligo object

data("exampleRprimerOligo")
plotData(exampleRprimerOligo)

#### Plot an RprimerAssay object

data("exampleRprimerAssay")
plotData(exampleRprimerAssay)

#### Plot an RprimerMatchOligo object

data("exampleRprimerMatchOligo")
plotData(exampleRprimerMatchOligo)

#### Plot an RprimerMatchAssay object

data("exampleRprimerMatchAssay")
plotData(exampleRprimerMatchAssay)
#### Plot an RprimerProfile object

data("exampleRprimerProfile")
prof <- exampleRprimerProfile

## Plot an overview
plotData(prof, highlight = c(500, 1000))

## Select a region of interest
roi <- prof[prof$position >= 500 & prof$position <= 550, ]

## Plot an overview of the roi
plotData(roi)

## Plot the nucleotide distribution of the roi
plotData(roi, type = "nucleotide")

#### Plot an RprimerOligo object

data("exampleRprimerOligo")
plotData(exampleRprimerOligo)

#### Plot an RprimerAssay object

data("exampleRprimerAssay")
plotData(exampleRprimerAssay)

#### Plot an RprimerMatchOligo object

data("exampleRprimerMatchOligo")
plotData(exampleRprimerMatchOligo)

#### Plot an RprimerMatchAssay object

data("exampleRprimerMatchAssay")
plotData(exampleRprimerMatchAssay)

S4 classes for representation of different Rprimer objects

Description

The rprimer package contains five different S4 classes. Each class is used as input or output for the different functions within the oligo and assay design workflow:

RprimerProfile: output from consensusProfile(), input for oligos().
RprimerOligo: output from oligos(), input for assays() and checkMatch().
RprimerAssay: output from assays(), input for checkMatch().
RprimerMatchOligo: output from checkMatch().
RprimerMatchAssay: output from checkMatch().

These classes extends the DFrame class from S4vectors (Pages et al., 2020), without any additional slots, but with some additional checks for validity.

Usage

RprimerProfile(...)

RprimerOligo(...)

RprimerAssay(...)

RprimerMatchOligo(...)

RprimerMatchAssay(...)
RprimerProfile(...)

RprimerOligo(...)

RprimerAssay(...)

RprimerMatchOligo(...)

RprimerMatchAssay(...)

Arguments

...

A data frame or list to be converted into an Rprimer-object.

Value

An Rprimer-object if validation succeeds, an error message otherwise.

Coercion

Each class can be converted to a traditional data frame, by using either as() or as.data.frame().

Moreover, as() can also be used for converting oligo sequences within an RprimerOligo or RprimerAssay object into a Biostrings::DNAStringSet object (Pages et al., 2020). Note that all oligo sequences will be written in the same direction as the input alignment that was used to generate the oligos.

References

Pages, H., Lawrence, M., and Aboyoun, R. (2020). S4Vectors: Foundation of vector-like and list-like containers in Bioconductor. R package version 0.28.0.

Pages, H., Aboyoun, P., Gentleman R., and DebRoy S. (2020). Biostrings: Efficient manipulation of biological strings. R package version 2.57.2.

Examples

## Constructors

data("exampleRprimerProfile")
x <- as.data.frame(exampleRprimerProfile)
RprimerProfile(x)

data("exampleRprimerOligo")
x <- as.data.frame(exampleRprimerOligo)
RprimerOligo(x)

data("exampleRprimerAssay")
x <- as.data.frame(exampleRprimerAssay)
RprimerAssay(x)

data("exampleRprimerMatchOligo")
x <- as.data.frame(exampleRprimerMatchOligo)
RprimerMatchOligo(x)

data("exampleRprimerMatchAssay")
x <- as.data.frame(exampleRprimerMatchAssay)
RprimerMatchAssay(x)

## Coercion methods for RprimerOligo and RprimerAssay objects

## Convert an RprimerOligo object to a DNAStringSet
data("exampleRprimerOligo")

## Pick rows to convert
x <- exampleRprimerOligo[1:2, ]
as(x, "DNAStringSet")

## Convert an RprimerAssay object to a DNAStringSet
data("exampleRprimerAssay")

## Pick rows to convert
x <- exampleRprimerAssay[1:2, ]
as(x, "DNAStringSet")
## Constructors

data("exampleRprimerProfile")
x <- as.data.frame(exampleRprimerProfile)
RprimerProfile(x)

data("exampleRprimerOligo")
x <- as.data.frame(exampleRprimerOligo)
RprimerOligo(x)

data("exampleRprimerAssay")
x <- as.data.frame(exampleRprimerAssay)
RprimerAssay(x)

data("exampleRprimerMatchOligo")
x <- as.data.frame(exampleRprimerMatchOligo)
RprimerMatchOligo(x)

data("exampleRprimerMatchAssay")
x <- as.data.frame(exampleRprimerMatchAssay)
RprimerMatchAssay(x)

## Coercion methods for RprimerOligo and RprimerAssay objects

## Convert an RprimerOligo object to a DNAStringSet
data("exampleRprimerOligo")

## Pick rows to convert
x <- exampleRprimerOligo[1:2, ]
as(x, "DNAStringSet")

## Convert an RprimerAssay object to a DNAStringSet
data("exampleRprimerAssay")

## Pick rows to convert
x <- exampleRprimerAssay[1:2, ]
as(x, "DNAStringSet")

rprimer Shiny application

Description

runRprimerApp() starts a Shiny application where the workflow of the rprimer package can be run through a graphical user interface.

Usage

runRprimerApp()
runRprimerApp()

Value

Opens the Shiny application.

Examples

## Only run this in interactive R sessions:
if (interactive()) {
    runRprimerApp()
}
## Only run this in interactive R sessions:
if (interactive()) {
    runRprimerApp()
}

Package 'rprimer'

Help Index

Check how oligos and assays match to their target sequences

Description

Usage

Arguments

Details

Value

Methods (by class)

Limitations

References

Examples

Get sequence information from an alignment

Description

Usage

Arguments

Value

References

Examples

Design (RT)-PCR assays

Description

Usage

Arguments

Value

Examples

Design primers and probes

Description

Usage

Arguments

Details

Value

References

Examples

Example datasets

Description

Usage

Format

References

Plot an Rprimer object

Description

Usage

Arguments

Value

Methods (by class)

Examples

S4 classes for representation of different Rprimer objects

Description

Usage

Arguments

Value

Coercion

References

See Also

Examples

rprimer Shiny application

Description

Usage

Value

Examples