Title: Visually Assessing the Specificity and Informativeness of Primer Pairs
Version: 1.0.6
Description: Identifies potential target sequences for a given set of primers and generates phylogenetic trees annotated with the taxonomies of the predicted amplification products.
License: GPL-2
Depends: R (≥ 3.5.0), directlabels, gridExtra
Imports: ape, foreach, ggplot2, grid, httr, lubridate, plyr, reshape2, scales, stringr, XML
Encoding: UTF-8
LazyData: true
RoxygenNote: 7.1.2
NeedsCompilation: yes
Packaged: 2022-04-04 23:28:09 UTC; MattDellLaptop
Author: Jim Hester [aut], Matt Cannon [aut, cre]
Maintainer: Matt Cannon <matthewvc1@gmail.com>
Repository: CRAN
Date/Publication: 2022-04-05 14:30:02 UTC

Maps a nucleotide database accession to a taxonomy database taxId

Description

Maps a nucleotide database accession to a taxonomy database taxId

Usage

accession2taxid(accessions)

Arguments

accessions

accessions character vector to lookup.

Value

named vector of taxIds.

PrimerTree results for the bryophyte trnL primers

Description

PrimerTree results for the bryophyte trnL primers

Summarize pairwise differences.

Description

Summarize pairwise differences.

Usage

calc_rank_dist_ave(x, ranks = common_ranks)

Arguments

x

a primerTree object

ranks

ranks to show unique counts for, defaults to the common ranks

Details

The purpose of this function is to calculate the average number of nucleotide differences between species within each taxa of given taxonomic level.

For example, at the genus level, the function calculates the average number of nucleotide differences between all species within each genus and reports the mean of those values.

There are several key assumptions and calculations made in this function.

First, the function randomly selects one sequence from each species in the primerTree results. This is to keep any one species (e.g. human, cow, etc.) with many hits from skewing the results.

Second, for each taxonomic level tested, the function divides the sequences by each taxon at that level and calculates the mean number of nucleotide differences within that taxa, then returns the mean of those values.

Third, when calculating the average distance, any taxa for which there is only one species is omitted, as the number of nucleotide differences will always be 0.

Value

returns a data frame of results

Examples

## Not run: 
calc_rank_dist_ave(mammals_16S)

calc_rank_dist_ave(bryophytes_trnL)

# Note that the differences between the results from these two primers
# the mean nucleotide differences is much higher for the mammal primers
# than the byrophyte primers. This suggests that the mammal primers have
# better resolution to distinguish individual species.

## End(Not run)

Multiple sequence alignment with clustal omega

Description

Calls clustal omega to align a set of sequences of class DNAbin. Run without any arguments to see all the options you can pass to the command line clustal omega.

Usage

clustalo(x, exec = "clustalo", quiet = TRUE, original.ordering = TRUE, ...)

Arguments

x

an object of class 'DNAbin'

exec

a character string with the name or path to the program

quiet

whether to supress output to stderr or stdout

original.ordering

use the original ordering of the sequences

...

additional arguments passed to the command line clustalo

Filter out sequences retrieved by search_primer_pair() that are either too short or too long. The alignment and tree will be recalculated after removing unwanted reads.

Description

Filter out sequences retrieved by search_primer_pair() that are either too short or too long. The alignment and tree will be recalculated after removing unwanted reads.

Usage

filter_seqs(x, ...)

## S3 method for class 'primerTree'
filter_seqs(x, min_length = 0, max_length = Inf, ...)

Arguments

x

a primerTree object

...

additional arguments passed to methods.

min_length

the minimum sequence length to keep

max_length

the maximum sequence length to keep

Value

a primerTree object

Methods (by class)

Examples

## Not run: 
# filter out sequences longer or shorter than desired:
mammals_16S_filtered <- filter_seqs(mammals_16S, min_length=131, max_length=156)

## End(Not run)

Retrieves a fasta sequence from NCBI nucleotide database.

Description

Retrieves a fasta sequence from NCBI nucleotide database.

Usage

get_sequence(
  accession,
  start = NULL,
  stop = NULL,
  api_key = Sys.getenv("NCBI_API_KEY")
)

Arguments

accession

nucleotide accession to retrieve.

start

start base to retrieve, numbered beginning at 1. If NULL the beginning of the sequence.

stop

last base to retrieve, numbered beginning at 1. if NULL the end of the sequence.

api_key

NCBI api-key to allow faster sequence retrieval.

Value

an DNAbin object.

See Also

DNAbin

Retrieves fasta sequences from NCBI nucleotide database.

Description

Retrieves fasta sequences from NCBI nucleotide database.

Usage

get_sequences(
  accession,
  start = NULL,
  stop = NULL,
  api_key = Sys.getenv("NCBI_API_KEY"),
  simplify = TRUE,
  .parallel = FALSE,
  .progress = "none"
)

Arguments

accession

the accession number of the sequence to retrieve

start

start bases to retrieve, numbered beginning at 1. If NULL the beginning of the sequence.

stop

stop bases to retrieve, numbered beginning at 1. if NULL the stop of the sequence.

api_key

NCBI api-key to allow faster sequence retrieval.

simplify

simplify the FASTA headers to include only the genbank accession.

.parallel

if 'TRUE', perform in parallel, using parallel backend provided by foreach

.progress

name of the progress bar to use, see 'create_progress_bar'

Value

an DNAbin object.

See Also

DNAbin

Retrieve the taxonomy information from NCBI for a set of nucleotide gis.

Description

Retrieve the taxonomy information from NCBI for a set of nucleotide gis.

Usage

get_taxonomy(accessions)

Arguments

accessions

a character vector of the accessions to retrieve

Value

data.frame of the 'accessions, taxIds, and taxonomy

identify the point closest to the mouse click only works on single ranks

Description

identify the point closest to the mouse click only works on single ranks

Usage

## S3 method for class 'primerTree_plot'
identify(x, ...)

Arguments

x

the plot to identify

...

additional arguments passed to annotate

layout a tree using ape, return an object to be plotted by plot_tree

Description

layout a tree using ape, return an object to be plotted by plot_tree

Usage

layout_tree_ape(tree, ...)

Arguments

tree

The phylo tree to be plotted

...

additional arguments to plot.phylo

Value

edge

list of x, y and xend, yend coordinates as well as ids for the edges

tips

list of x, y, label and id for the tips

nodes

list of x, y and id for the nodes

PrimerTree results for the mammalian 16S primers

Description

PrimerTree results for the mammalian 16S primers

Parse the primer hits

Description

Parse the primer hits

Usage

parse_primer_hits(response)

Arguments

response

a httr response object obtained from primer_search

plot function for a primerTree object, calls plot_tree_ranks

Description

plot function for a primerTree object, calls plot_tree_ranks

Usage

## S3 method for class 'primerTree'
plot(x, ranks = NULL, main = NULL, ...)

Arguments

x

primerTree object to plot

ranks

The ranks to include, defaults to all common ranks, if NULL print all ranks. If 'none' just print the layout.

main

an optional title to display, if NULL displays the name as the title

...

additional arguments passed to plot_tree_ranks

See Also

plot_tree_ranks, plot_tree

Examples

library(gridExtra)
library(directlabels)
#plot with all common ranks
plot(mammals_16S)

#plot only the class
plot(mammals_16S, 'class')

#plot the layout only
plot(mammals_16S, 'none')

plots a tree, optionally with colored and labeled points by taxonomic rank

Description

plots a tree, optionally with colored and labeled points by taxonomic rank

Usage

plot_tree(
  tree,
  type = "unrooted",
  main = NULL,
  guide_size = NULL,
  rank = NULL,
  taxonomy = NULL,
  size = 2,
  legend_cutoff = 25,
  ...
)

Arguments

tree

to be plotted, use layout_tree to layout tree.

type

The type of tree to plot, default unrooted.

main

An optional title for the plot

guide_size

The size of the length guide. If NULL auto detects a reasonable size.

rank

The rank to include, if null only the tree is plotted

taxonomy

A data.frame with an accession field corresponding to the tree tip labels.

size

The size of the colored points

legend_cutoff

The number of different taxa names after which the names are no longer printed.

...

additional arguments passed to layout_tree_ape

Value

plot to be printed.

plots a tree along with a series of taxonomic ranks

Description

plots a tree along with a series of taxonomic ranks

Usage

plot_tree_ranks(
  tree,
  taxonomy,
  main = NULL,
  type = "unrooted",
  ranks = common_ranks,
  size = 2,
  guide_size = NULL,
  legend_cutoff = 25,
  ...
)

Arguments

tree

to be plotted, use layout_tree to layout tree.

taxonomy

A data.frame with an accession field corresponding to the tree tip labels.

main

An optional title for the plot

type

The type of tree to plot, default unrooted.

ranks

The ranks to include, defaults to all common ranks, if null print all ranks.

size

The size of the colored points

guide_size

The size of the length guide. If NULL auto detects a reasonable size.

legend_cutoff

The number of different taxa names after which the names are no longer printed.

...

additional arguments passed to layout_tree_ape

See Also

plot_tree to plot only a single rank or the just the tree layout.

Examples

library(gridExtra)
library(directlabels)
#plot all the common ranks
plot_tree_ranks(mammals_16S$tree, mammals_16S$taxonomy)
#plot specific ranks, with a larger dot size
plot_tree_ranks(mammals_16S$tree, mammals_16S$taxonomy,
  ranks=c('kingdom', 'class', 'family'), size=3)

primerTree Visually Assessing the Specificity and Informativeness of Primer Pairs

Description

primerTree has two main commands: search_primer_pair which takes a primer pair and returns an primerTree object of the search results plot.primerTree a S3 method for plotting the primerTree object obtained using search_primer_pair

Description

ambiguity codes, enumerate all possible combinations and combine the results.

Usage

primer_search(
  forward,
  reverse,
  num_aligns = 500,
  num_permutations = 25,
  ...,
  .parallel = FALSE,
  .progress = "none"
)

Arguments

forward

forward primer to search by 5'-3' on plus strand

reverse

reverse primer to search by 5'-3' on minus strand

num_aligns

number of alignment results to keep

num_permutations

the number of primer permutations to search, if the degenerate bases cause more than this number of permutations to exist, this number will be sampled from all possible permutations.

...

additional arguments passed to Primer-Blast

.parallel

if 'TRUE', perform in parallel, using parallel backend provided by foreach

.progress

name of the progress bar to use, see 'create_progress_bar'

Value

httr response object of the query, pass to parse_primer_hits to parse the results.

Automatic primer searching Search a given primer pair, retrieving the alignment results, their product sequences, the taxonomic information for the sequences, a multiple alignment of the products

Description

Automatic primer searching Search a given primer pair, retrieving the alignment results, their product sequences, the taxonomic information for the sequences, a multiple alignment of the products

Usage

search_primer_pair(
  forward,
  reverse,
  name = NULL,
  num_aligns = 500,
  num_permutations = 25,
  simplify = TRUE,
  clustal_options = list(),
  distance_options = list(model = "N", pairwise.deletion = T),
  api_key = Sys.getenv("NCBI_API_KEY"),
  ...,
  .parallel = FALSE,
  .progress = "none"
)

Arguments

forward

forward primer to search by 5'-3' on plus strand

reverse

reverse primer to search by 5'-3' on minus strand

name

name to give to the primer pair

num_aligns

number of alignment results to keep

num_permutations

the number of primer permutations to search, if the degenerate bases cause more than this number of permutations to exist, this number will be sampled from all possible permutations.

simplify

use simple names for primer hit results or complex

clustal_options

a list of options to pass to clustal omega, see link{clustalo} for a list of options

distance_options

a list of options to pass to dist.dna, see link{dist.dna} for a list of options

api_key

NCBI api-key to allow faster sequence retrieval

...

additional arguments passed to Primer-Blast

.parallel

if 'TRUE', perform in parallel, using parallel backend provided by foreach

.progress

name of the progress bar to use, see create_progress_bar

Value

A list with the following elements,

name

name of the primer pair

BLAST_result

html blast results from Primer-BLAST as 'a response

object.

taxonomy

taxonomy for the primer products from NCBI

sequence

sequence of the primer products

alignment

multiple alignment of the primer products

tree

phylogenetic tree of the reconstructed from the 'multiple alignment

See Also

primer_search, clustalo

Examples

## Not run: 
#simple search
mammals_16S = search_primer_pair(name='Mammals 16S',
 'CGGTTGGGGTGACCTCGGA', 'GCTGTTATCCCTAGGGTAACT')
#returning 1000 alignments, allow up to 3 mismatches in primer
mammals_16S = search_primer_pair(name='Mammals 16S',
 'CGGTTGGGGTGACCTCGGA', 'GCTGTTATCCCTAGGGTAACT',
 num_aligns=1000, total_primer_specificity_mismatch=3)

## End(Not run)

Get a summary of sequence lengths from a primerTree object

Description

Get a summary of sequence lengths from a primerTree object

Usage

seq_lengths(x, summarize = TRUE)

Arguments

x

a primerTree object.

summarize

a logical indicating if a summary should be displayed

Value

a table of sequence length frequencies

Examples


# Show the counts for each length
seq_lengths(mammals_16S)

# Plot the distribution of lengths
seqLengths <- seq_lengths(mammals_16S)
barplot(seqLengths, 
 main = "Frequency of sequence lengths for 16S mammal primers", 
 xlab="Amplicon length (in bp)", 
 ylab=("Frequency"))

Method for primerTree objects

Description

Method for primerTree objects

Usage

## S3 method for class 'primerTree'
seq_lengths(x, summarize = TRUE)

Arguments

x

a primerTree object.

summarize

a logical indicating if a summary should be displayed

Summarize a primerTree result, printing quantiles of sequence length and pairwise differences.

Description

Summarize a primerTree result, printing quantiles of sequence length and pairwise differences.

Usage

## S3 method for class 'primerTree'
summary(object, ..., probs = c(0, 0.05, 0.5, 0.95, 1), ranks = common_ranks)

Arguments

object

the primerTree object to summarise

...

Ignored options

probs

quantile probabilities to compute, defaults to 0, 5, 50, 95, and 100 probabilities.

ranks

ranks to show unique counts for, defaults to the common ranks

Value

invisibly returns a list containing the printed results

Construct a neighbor joining tree from a dna alignment

Description

Construct a neighbor joining tree from a dna alignment

Usage

tree_from_alignment(dna, pairwise.deletion = TRUE, ...)

Arguments

dna

fasta dna object the tree is to be constructed from

pairwise.deletion

a logical indicating if the distance matrix should be constructed using pairwise deletion

...

furthur arguments to dist.dna

See Also

dist.dna, nj