| ProbeMake {ProbeDeveloper} | R Documentation |
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_NN4", "DNA_NN1", "DNA_NN2", "DNA_NN3", "RNA_NN1", "RNA_NN2",
"RNA_NN3", "R_DNA_NN1"),
tmm_table = "DNA_TMM1",
imm_table = "DNA_IMM1",
de_table = c("DNA_DE1", "RNA_DE1"),
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("concentration", "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 NN values, eight tables are implemented. For DNA/DNA hybridizations: DNA_NN1,DNA_NN2,DNA_NN3,DNA_NN4 For RNA/RNA hybridizations: RNA_NN1,RNA_NN2,RNA_NN3 For RNA/DNA hybridizations: R_DNA_NN1 |
tmm_table |
Thermodynamic values for terminal mismatches. Default: DNA_TMM1 |
imm_table |
Thermodynamic values for internal mismatches, may include insosine mismatches. Default: DNA_IMM1 |
de_table |
Thermodynamic values for dangling ends: DNA_DE1(default),RNA_DE1 |
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="concentration") or molar (fmdmethod="molar") |
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 |
"concentration" 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
data(samplefa)
ProbeMake(samplefa,LN=90,ln=60,TM=80,tm=70,CG=80,cg=20,TmMethod="Tm_NN",Na=50)