TsIO-class: TsIO class

In TAPseq: Targeted scRNA-seq primer design for TAP-seq

Description

TsIO objects store TAP-seq Primer3 input and output.

Usage

TsIO(
  sequence_id,
  target_sequence,
  beads_oligo,
  reverse_primer,
  product_size_range,
  target_annot = NULL,
  primer_num_return = 5,
  min_primer_region = 100,
  primer_opt_tm = NA,
  primer_min_tm = NA,
  primer_max_tm = NA
)

## S4 method for signature 'TsIO'
sequence_id(x)

## S4 replacement method for signature 'TsIO'
sequence_id(x) <- value

## S4 method for signature 'TsIO'
target_sequence(x)

## S4 replacement method for signature 'TsIO'
target_sequence(x) <- value

## S4 method for signature 'TsIO'
beads_oligo(x)

## S4 replacement method for signature 'TsIO'
beads_oligo(x) <- value

## S4 method for signature 'TsIO'
reverse_primer(x)

## S4 replacement method for signature 'TsIO'
reverse_primer(x) <- value

## S4 method for signature 'TsIO'
target_annot(x)

## S4 replacement method for signature 'TsIO'
target_annot(x) <- value

## S4 method for signature 'TsIO'
product_size_range(x)

## S4 replacement method for signature 'TsIO'
product_size_range(x) <- value

## S4 method for signature 'TsIO'
primer_num_return(x)

## S4 replacement method for signature 'TsIO'
primer_num_return(x) <- value

## S4 method for signature 'TsIO'
min_primer_region(x)

## S4 replacement method for signature 'TsIO'
min_primer_region(x) <- value

## S4 method for signature 'TsIO'
primer_opt_tm(x)

## S4 replacement method for signature 'TsIO'
primer_opt_tm(x) <- value

## S4 method for signature 'TsIO'
primer_min_tm(x)

## S4 replacement method for signature 'TsIO'
primer_min_tm(x) <- value

## S4 method for signature 'TsIO'
primer_max_tm(x)

## S4 replacement method for signature 'TsIO'
primer_max_tm(x) <- value

## S4 method for signature 'TsIO'
sequence_template(x)

## S4 method for signature 'TsIO'
tapseq_primers(x)

## S4 method for signature 'TsIO'
pcr_products(x)

Arguments

sequence_id	Name (character) of the target sequence, e.g. the gene name. It's adviced to use meaningful sequence ids to savely assign designed primers to their targets.
target_sequence	A DNAString or character object containing the target sequence for which primers should be designed. Usually a transcript sequence.
beads_oligo	Beads-oligo-dT sequence for the used droplet sequencing protocol (10x, Drop-seq).
reverse_primer	Reverse primer sequence used for all PCR reactions.
product_size_range	Numerical vector of length 2 specifying the desired length of the resulting amplicons.
target_annot	(optional) A GRanges object with transcript annotation in case the target is a transcript of a gene locus. If provided, it should contain all exons of the targeted transcript.
primer_num_return	How many forward primers should be designed? (default: 5)
min_primer_region	Minimum sequence length required for primer design. Mostly relevant in case the sequence template is too short to allow the specified product_size_range.
primer_opt_tm, primer_min_tm, primer_max_tm	Optimal, minumum and maximum primer melting temperature.
x	A TsIO object.
value	A valid value to assign to the chosen slot.
tapseq_primers	Slot where designed TAP-seq primers are stored. Not set by user.
pcr_products	Slot where PCR products of primers are stored. Not set by user.

Details

The TsIO class is based on the Boulder IO records used by Primer3 (Primer3 manual). These objects are used to store the sequence templates and parameters needed for TAP-seq primer design. Primers designed with Primer3 are also stored in the same TsIO objects.

Use TsIO() to construct a new TsIO object from scratch.

Value

A TsIO object.

Methods (by generic)

• sequence_id: Get sequence_id

• sequence_id<-: Set sequence_id

• target_sequence: Get target_sequence

• target_sequence<-: Set target_sequence

• beads_oligo: Get beads_oligo

• beads_oligo<-: Set beads_oligo

• reverse_primer: Get reverse_primer

• reverse_primer<-: Set reverse_primer

• target_annot: Get target_annot

• target_annot<-: Set target_annot

• product_size_range: Get product_size_range

• product_size_range<-: Set product_size_range

• primer_num_return: Get primer_num_return

• primer_num_return<-: Set primer_num_return

• min_primer_region: Get min_primer_region

• min_primer_region<-: Set min_primer_region

• primer_opt_tm: Get primer_opt_tm

• primer_opt_tm<-: Set primer_opt_tm

• primer_min_tm: Get primer_min_tm

• primer_min_tm<-: Set primer_min_tm

• primer_max_tm: Get primer_max_tm

• primer_max_tm<-: Set primer_max_tm

• sequence_template: Create sequence_template

• tapseq_primers: Get tapseq_primers

• pcr_products: Get pcr_products

See Also

http://primer3.org/manual.html for Primer3 manual.

Examples

# get example transcript sequence
data("chr11_truncated_txs_seq")
tx_seq <- chr11_truncated_txs_seq[[1]]
tx_id <- names(chr11_truncated_txs_seq)[1]

# 10x beads-oligo-dt sequence
beads_oligo <- "CTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT"

# reverse primer used in all PCR reactions
reverse_primer <- "CTACACGACGCTCTTCCGATCT"

# create TsIO object
obj <- TsIO(sequence_id = tx_id, target_sequence = tx_seq, beads_oligo = beads_oligo,
            reverse_primer = reverse_primer, product_size_range = c(350, 500))

# slot values can be accessed using accessor functions
sequence_id(obj)
sequence_id(obj) <- "Gene1"
sequence_id(obj)

# the sequence template (target sequence + reverse complement of beads-oligo-dt) for primer
# design can be viewed as well
sequence_template(obj)

TAPseq documentation built on Nov. 8, 2020, 7:51 p.m.