Type: Package
Title: Develop Hybridization Probes
Version: 1.1.1
Date: 2026-03-18
Description: Hybridization probes for target sequences can be made based on melting temperature value calculated by R package 'TmCalculator' https://CRAN.R-project.org/package=TmCalculator and methods extended from Beliveau, B. J.,(2018) <doi:10.1073/pnas.1714530115>, and those hybridization probes can be used to capture specific target regions in fluorescence in situ hybridization and next generation sequence experiments.
License: GPL-2 | GPL-3 [expanded from: GPL (≥ 2)]
Imports: TmCalculator (≥ 1.0.4),Biostrings(≥ 2.12.0)
Depends: R (≥ 2.10)
RoxygenNote: 7.3.2
NeedsCompilation: no
Packaged: 2026-03-20 16:34:55 UTC; lij11
Repository: CRAN
Date/Publication: 2026-03-23 07:40:02 UTC
Author: Junhui Li ORCID iD [cre, aut]
Maintainer: Junhui Li <ljh.biostat@gmail.com>

Make probes

Description

Probes are made with a FASTA-formatted input file containing the target sequence. User can specify the allowable ranges of probe length, percent GC content, and adjust melting temperature calculated using nearest neighbor thermodynamics or empirical formulas based on GC content. Candidate probe sequences passing all checks output in BED format.

Usage

ProbeMake(
  fafile,
  LN = 90,
  ln = 60,
  TM = 80,
  tm = 60,
  CG = 70,
  cg = 30,
  gap = 0,
  method = c("S2L", "L2S"),
  direction = c("3to5", "5to3"),
  prohibitseq = NULL,
  TmMethod = c("tm_gc", "tm_nn"),
  variant = c("Primer3Plus", "Chester1993", "QuikChange", "Schildkraut1965",
    "Wetmur1991_MELTING", "Wetmur1991_RNA", "Wetmur1991_RNA/DNA", "vonAhsen2001"),
  nn_table = c("DNA_NN_Breslauer_1986", "DNA_NN_Sugimoto_1996", "DNA_NN_Allawi_1998",
    "DNA_NN_SantaLucia_2004", "RNA_NN_Freier_1986", "RNA_NN_Xia_1998",
    "RNA_NN_Chen_2012", "RNA_DNA_NN_Sugimoto_1995"),
  tmm_table = "DNA_TMM_Bommarito_2000",
  imm_table = "DNA_IMM_Peyret_1999",
  de_table = c("DNA_DE_Bommarito_2000", "RNA_DE_Turner_2010"),
  dnac1 = 25,
  dnac2 = 25,
  Na = 0,
  K = 0,
  Tris = 0,
  Mg = 0,
  dNTPs = 0,
  saltcorr = c("Schildkraut2010", "Wetmur1991", "SantaLucia1996", "SantaLucia1998-1",
    "SantaLucia1998-2", "Owczarzy2004", "Owczarzy2008"),
  DMSO = 0,
  fmd = 0,
  DMSOfactor = 0.75,
  fmdfactor = 0.65,
  fmdmethod = c("percent", "molar")
)

Arguments

fafile

Input file with a FASTA format read by function readDNAStringSet in R package 'Biostrings'

LN

The maximum allowed probe length, default is 90

ln

The minimum allowed probe length, default is 60

TM

The maximum allowed melting temperature, default is 80

tm

The minimum allowed melting temperature, default is 60

CG

The maximum allowed percent GC content, default is 70

cg

The minimum allowed percent GC content, default is 30

gap

The minimum gap between adjacent probes, default is 0

method

'S2L' is used to design probe extending from minimal length to the maximum until passing all checks, conversely 'L2S' make probe from maximal length to the minimum. Default is 'S2L'

direction

Design probes from 3 to 5 end or from 5 to 3 end of target sequence, default is '3to5'

prohibitseq

Prohibited sequence list, e.g prohibitseq=c("GGGGG","CCCCC"), default is NULL

TmMethod

The method used to calculate Tm, 'tm_nn' and 'tm_gc' can be seleted

variant

Empirical constants coefficient with 8 variant for 'tm_gc' method: Chester1993, QuikChange, Schildkraut1965, Wetmur1991_MELTING, Wetmur1991_RNA, Wetmur1991_RNA/DNA, Primer3Plus and vonAhsen2001

nn_table

Thermodynamic nearest-neighbor parameters for different nucleic acid hybridizations. Eight parameter sets are available, organized by hybridization type:

DNA/DNA hybridizations: - "DNA_NN_Breslauer_1986": Original DNA/DNA parameters - "DNA_NN_Sugimoto_1996": Improved DNA/DNA parameters - "DNA_NN_Allawi_1998": DNA/DNA parameters with internal mismatch corrections - "DNA_NN_SantaLucia_2004": Updated DNA/DNA parameters

RNA/RNA hybridizations: - "RNA_NN_Freier_1986": Original RNA/RNA parameters - "RNA_NN_Xia_1998": Improved RNA/RNA parameters - "RNA_NN_Chen_2012": Updated RNA/RNA parameters with GU pair corrections

RNA/DNA hybridizations: - "RNA_DNA_NN_Sugimoto_1995": RNA/DNA hybridization parameters

tmm_table

Thermodynamic parameters for terminal mismatches. Default: "DNA_TMM_Bommarito_2000" These parameters account for mismatches at the ends of the duplex.

imm_table

Thermodynamic parameters for internal mismatches. Default: "DNA_IMM_Peyret_1999" These parameters account for mismatches within the duplex, including inosine mismatches.

de_table

Thermodynamic parameters for dangling ends. Default: "DNA_DE_Bommarito_2000" Available options: - "DNA_DE_Bommarito_2000": Parameters for DNA dangling ends - "RNA_DE_Turner_2010": Parameters for RNA dangling ends

dnac1

Concentration of the higher concentrated strand [nM]. Typically this will be the primer (for PCR) or the probe. Default: 25

dnac2

Concentration of the lower concentrated strand [nM]. Default: 25

Na

Millimolar concentration of Na, default is 0

K

Millimolar concentration of K, default is 0

Tris

Millimolar concentration of Tris, default is 0

Mg

Millimolar concentration of Mg, default is 0

dNTPs

Millimolar concentration of dNTPs, default is 0

saltcorr

Salt correction method. Options are "Schildkraut2010", "Wetmur1991","SantaLucia1996","SantaLucia1998-1","Owczarzy2004","Owczarzy2008". Note that "SantaLucia1998-2" is not available for this function.

DMSO

Percent of DMSO

fmd

Formamide concentration in percentage (fmdmethod="percent") or molar (fmdmethod="molar"). Default is 0.

DMSOfactor

Coeffecient of Tm decreases per percent DMSO. Default=0.75 von Ahsen N (2001) <PMID:11673362>. Other published values are 0.5, 0.6 and 0.675.

fmdfactor

Coeffecient of Tm decrease per percent formamide. Default=0.65. Several papers report factors between 0.6 and 0.72.

fmdmethod

"percent" method for formamide concentration in percentage and "molar" for formamide concentration in molar

Value

Returns a bed file in the format TargetID <tab> Chr <tab> Start <tab> End <tab> Sequence <tab> Tm <tab> GC

Author(s)

Junhui Li

References

Beliveau B J, Kishi J Y, Nir G, et al. (2017). OligoMiner: A rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes. bioRxiv.

Breslauer K J , Frank R , Blocker H , et al. Predicting DNA duplex stability from the base sequence.[J]. Proceedings of the National Academy of Sciences, 1986, 83(11):3746-3750.

Sugimoto N , Nakano S , Yoneyama M , et al. Improved Thermodynamic Parameters and Helix Initiation Factor to Predict Stability of DNA Duplexes[J]. Nucleic Acids Research, 1996, 24(22):4501-5.

Allawi, H. Thermodynamics of internal C.T mismatches in DNA[J]. Nucleic Acids Research, 1998, 26(11):2694-2701.

Hicks L D , Santalucia J . The thermodynamics of DNA structural motifs.[J]. Annual Review of Biophysics & Biomolecular Structure, 2004, 33(1):415-440.

Freier S M , Kierzek R , Jaeger J A , et al. Improved free-energy parameters for predictions of RNA duplex stability.[J]. Proceedings of the National Academy of Sciences, 1986, 83(24):9373-9377.

Xia T , Santalucia , J , Burkard M E , et al. Thermodynamic Parameters for an Expanded Nearest-Neighbor Model for Formation of RNA Duplexes with Watson-Crick Base Pairs,[J]. Biochemistry, 1998, 37(42):14719-14735.

Chen J L , Dishler A L , Kennedy S D , et al. Testing the Nearest Neighbor Model for Canonical RNA Base Pairs: Revision of GU Parameters[J]. Biochemistry, 2012, 51(16):3508-3522.

Bommarito S, Peyret N, Jr S L. Thermodynamic parameters for DNA sequences with dangling ends[J]. Nucleic Acids Research, 2000, 28(9):1929-1934.

Turner D H , Mathews D H . NNDB: the nearest neighbor parameter database for predicting stability of nucleic acid secondary structure[J]. Nucleic Acids Research, 2010, 38(Database issue):D280-D282.

Sugimoto N , Nakano S I , Katoh M , et al. Thermodynamic Parameters To Predict Stability of RNA/DNA Hybrid Duplexes[J]. Biochemistry, 1995, 34(35):11211-11216.

Allawi H, SantaLucia J: Thermodynamics and NMR of internal G-T mismatches in DNA. Biochemistry 1997, 36:10581-10594.

Santalucia N E W J . Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes[J]. Nucleic Acids Research, 2005, 33(19):6258-67.

Peyret N , Seneviratne P A , Allawi H T , et al. Nearest-Neighbor Thermodynamics and NMR of DNA Sequences with Internal A-A, C-C, G-G, and T-T Mismatches, [J]. Biochemistry, 1999, 38(12):3468-3477.

Marmur J , Doty P . Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature.[J]. Journal of Molecular Biology, 1962, 5(1):109-118.

Schildkraut C . Dependence of the melting temperature of DNA on salt concentration[J]. Biopolymers, 2010, 3(2):195-208.

Wetmur J G . DNA Probes: Applications of the Principles of Nucleic Acid Hybridization[J]. CRC Critical Reviews in Biochemistry, 1991, 26(3-4):33.

Untergasser A , Cutcutache I , Koressaar T , et al. Primer3–new capabilities and interfaces[J]. Nucleic Acids Research, 2012, 40(15):e115-e115.

von Ahsen N, Wittwer CT, Schutz E , et al. Oligonucleotide melting temperatures under PCR conditions: deoxynucleotide Triphosphate and Dimethyl sulfoxide concentrations with comparison to alternative empirical formulas. Clin Chem 2001, 47:1956-1961.

Examples


## Not run: 
data(samplefa)
ProbeMake(samplefa,LN=90,ln=60,TM=80,tm=70,CG=80,cg=20,TmMethod="tm_nn",Na=50)

## End(Not run)

sample data for target sequence region with class 'DNAStringSet'

Description

sample data read by function readDNAStringSet in R package 'Biostrings' from fasta format file, there are two target sequence resion in this data

Usage

data("samplefa")

Format

Formal class 'DNAStringSet' [package "Biostrings"] with 5 slots

sample data read by function readDNAStringSet in R package 'Biostrings' from fasta format file, which is from ncbiRefSeq database for Homo Sapiens with referece genome version hg19

Examples

data(samplefa)