Information about

Melting

-h

Displays a short help and quit.

-L

Prints the legal informations and quit.

-V

Displays the version number and quit.

-p

Return the directory supposed to contain the sets of calorimetric parameters and quit. If the environment variable NN_PATH is set, it is returned. Otherwise, the value defined by default during the compilation is returned.

Mandatory options

-Ssequence

Sequence of one strand of the nucleic acid duplex, entered 5' to 3'. IMPORTANT: Uridine and thymidine are not considered as identical. The bases can be upper or lowercase.

-Csequence

Enters the complementary sequence, from 3' to 5'. This option is mandatory if there are mismatches, inosine(s) or hydroxyadenine(s) between the two strands. If it is not used, the program will compute it as the complement of the sequence entered with the option -S In case of self complementary sequences, the programm can automatically detect the symmetry and deduce the complementary even though there is (are) dangling end(s) and it is not necessary to write the complementary sequence with the option -C IMPORTANT: Uridine and thymidine are not considered as identical. The bases can be upper or lowercase.

-Eion1_name=x.xxe-xx:ion2_name=x.xxe-xx:agent1_name=x.xxe-xx...

Enters the different ion (Na, Mg, Tris, K) or agent (dNTP, DMSO, formamide) concentrations. The effect of ions and denaturing agents on thermodynamic stability of nucleic acid duplexes is complex, and the correcting functions are at best rough approximations. All the concentrations must be positive numeric values and in M. There are some exceptions for the DMSO concentrations (in percent) and the formamide concentrations (in percent or M depending on the used correction method). Be aware, the [Tris+] is about half of the total tris buffer concentration. At least one cation concentration is mandatory, the other agents are optional. See the documentation for the concentration limits. It depends on the used correction.

-Px.xxe-xx

Concentration of the nucleic acid strand in excess. It must be a strict positive numeric value and it is mandatory. The oligomer concentration is in M.

-Hhybridization_type

Specifies the hybridisation type. Moreover this parameter determines the nature of the sequences entered by the user. Possible values are :

dnadna :
DNA sequence (option -S ) and DNA complementary sequence (option -C )

rnarna :
RNA sequence (option -S ) and RNA complementary sequence (option -C )

dnarna :
DNA sequence (option -S ) and RNA complementary sequence (option -C )

rnadna :
RNA sequence (option -S ) and DNA complementary sequence (option -C )

mrnarna :
2-o-methyl RNA sequence (option -S ) and RNA complementary sequence (option -C )

mrnarna :
RNA sequence (option -S ) and 2-o-methyl RNA complementary sequence (option -C )

This option is mandatory to select the default equations and methods to use.

General options

-Txxx

Size threshold before approximative computation. The nearest-neighbour approach will be used by default if the length of the sequence is inferior to this threshold, otherwise it is the approximative approach which will be used by default.

-v

Activates the verbose mode, issuing a lot more information about the current run (try it once to see if you can get something interesting).

-nnpathfolder_pathway

Change the default pathway (Data) where to find the default calorimetric tables (thermodynamic parameters). The program will look for the file in a directory specified during the installation. However, if an environment variable NN_PATH is defined, melting will search in this one first.

-Ooutput_file

The output is directed to this file instead of the standard output. The name of the file must be specified.

-self

To precise that the sequence entered with the option -S
is self complementary. No complementary sequence is mandatory. The program automatically can detect a self complementary sequence for perfect matching sequences or sequences with dangling ends. In these cases, the option -self
is not necessary. Otherwise we need to precise that the sequences are self complementary with this option.
examples:

###beginning###
- The sequence ATCGCGAT is self complementary. The option -self
is not necessary because the programm can automatically detect it.
- The sequence -TCGCGAT is self complementary but with a single dangling end. The option -self
is not necessary because the programm can automatically detect it.
- If the sequence ATCCCGAT is self complementary with a single mismatch (C/C), the option -self
is necessary to precise the self complementarity because the programm can't automatically detect it.

###end###

-Fxxx

This is the correction factor used to modulate the effect of the nucleic acid concentration in the computation of the melting temperature. If the sequences are automatically recognized as self complementary sequences or if the option -self is used, the factor correction is automatically 1. Otherwise F is 4 if the both strands are present in equivalent amount and 1 if one strand is in excess. The default factor value is 4.

Set of thermodynamic parameters and methods (models)

By default, the approximative mode is used for oligonucleotides longer than 60 bases (the default threshold value), otherwise the nearest neighbor model is used.

-ammethod_name

Forces to use a specific approximative formula, based on G+C content. You can use one of the following :

DNA duplexes
ahs01 (from von Ahsen et al. 2001)
che93 (from Marmur 1962, Chester et al. 1993)
che93corr (from von Ahsen et al. 2001, Marmur 1962, Chester et al. 1993)
schdot (Marmur-Schildkraut-Doty formula)
owe69 (from Owen et al. 1969)
san98 (from Santalucia et al. 1998)
wetdna91 (from Wetmur 1991) (by default)

RNA duplexes
wetrna91 (from Wetmur 1991) (by default)

DNA/RNA duplexes
wetdnarna91 (from Wetmur 1991) (by default)

If there is no formula name after the option -am , we will compute the melting temperature with the default approximative formula. This option has to be used with caution. Note that such a calcul is increasingly incorrect when the length of the duplex decreases. Moreover, it does not take into account nucleic acid concentration, which is a strong mistake. examples :

###beginning###
- "-am" if you want to force the approximative approach with the default formula.
- "-am ahs01" if you want to use the approximative formula from Ahsen et al. 2001.

###end###

-nnmethod_name

Forces to use a specific nearest neighbor model. You can use one of the following :

DNA duplexes
all97 (from Allawi and Santalucia 1997) (by default)
bre86 (from Breslauer et al. 1986)
san04 (from Hicks and Santalucia 2004)
san96 (from Santalucia et al. 1996)
sug96 (from Sugimoto et al 1996)
tan04 (from Tanaka et al. 2004)

RNA duplexes
fre86 (from Freier al. 1986)
xia98 (from Xia et al. 1998) (by default)

DNA/RNA duplexes
sug95 (from Sugimoto et al. 1995) (by default)

mRNA/RNA duplexes
tur06 (from Kierzeck et al. 2006) (by default)

If there is no formula name after the option -nn , we will compute the melting temperature with the default nearest neighbor model. Each nearest neighbor model uses a specific xml file containing the thermodynamic values. If you want to use another file, write the file name or the file pathway preceded by ':' (-nn [optionalname:optionalfile]). examples:

###beginning###
- "-nn" if you want to force the nearest neighbor computation with the default model.
- "-nn tan04" if you want to use the nearest neighbor model from Tanaka et al. 2004 with the thermodynamic parameters in the default xml file.
- "-nn tan04:fileName" if you want to use the nearest neighbor model from Tanaka et al. 2004 with the thermodynamic parameters in the file fileName.
- "-nn :fileName" if you want to use the default nearest neighbor model with the thermodynamic parameters in the file fileName.

###end###

-sinMMmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of single mismatch to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
allsanpey (from Allawi, Santalucia and Peyret 1997, 1998 and 1999) (by default)

RNA duplexes
tur06 (from Lu et al. 2006)
zno07 (from Davis et al. 2007) (by default)
zno08 (from Davis et al. 2008)

DNA/RNA duplexes
wat10 (from Watkins et al. 2011) (by default)

To change the file containing the thermodynamic parameters for single mismatch computation, the same syntax as the one for the -nn
option is used. Single mismatches are not taken into account by the approximative mode.

-GUMmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of GU base pairs to the thermodynamic of helix-coil transition. You can use one of the following :

RNA duplexes
tur99 (from Mathews et al. 1999)
ser12 (from Serra et al. 2012) (by default)

To change the file containing the thermodynamic parameters for GU base pair computation, the same syntax as the one for the -nn
option is used. GU base pairs are not taken into account by the approximative mode.

-tanMMmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of tandem mismatches to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
allsanpey (from Allawi, Santalucia and Peyret 1997, 1998 and 1999) (by default)

RNA duplexes
tur99 (from Mathews et al. 1999) (by default)

To change the file containing the thermodynamic parameters for tandem mismatch computation, the same syntax as the one for the -nn
option is used. Tandem mismatches are not taken into account by the approximative mode. Note that not all the mismatched Crick's pairs have been investigated.

-intLPmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of internal loop to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes}]
san04 (from Hicks and Santalucia 2004) (by default)

RNA duplexes
tur06 (from Lu et al. 2006) (by default)
zno07 (from Badhwarr et al. 2007, only for 1x2 loop)

To change the file containing the thermodynamic parameters for internal loop computation, the same syntax as the one for the -nn
option is used. Internal loops are not taken into account by the approximative mode.

-sinDEmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of single dangling end to the thermodynamic of helix-coil transition.
You can use one of the following :

DNA duplexes
bom00 (from Bommarito et al. 2000) (by default)
sugdna02 (from Ohmichi et al. 2002, only for polyA dangling ends)

RNA duplexes
sugrna02 (from Ohmichi et al. 2002, only for polyA dangling ends)
ser08 (from Miller et al. 2008) (by default)

To change the file containing the thermodynamic parameters for single dangling end computation, the same syntax as the one for the -nn
option is used. Single dangling ends are not taken into account by the approximative mode.

-secDEmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of double dangling end to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
sugdna02 (from Ohmichi et al. 2002, only for polyA dangling ends) (by default)

RNA duplexes
sugrna02 (from Ohmichi et al. 2002, only for polyA dangling ends)
ser05 (from O'toole et al. 2005)
ser06 (from O'toole et al. 2006) (by default)

To change the file containing the thermodynamic parameters for double dangling end computation, the same syntax as the one for the -nn
option is used. Double dangling ends are not taken into account by the approximative mode.

-lonDEmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of long dangling end to the thermodynamic of helix-coil transition. You can use one of the following : DNA duplexes
sugdna02 (from Ohmichi et al. 2002, only for polyA dangling ends) (by default)

RNA duplexes
sugrna02 (from Ohmichi et al. 2002, only for polyA dangling ends)

To change the file containing the thermodynamic parameters for long dangling end computation, the same syntax as the one for the -nn
option is used. Long dangling ends are not taken into account by the approximative mode.

-sinBUmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of single bulge loop to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
san04 (from Hicks and Santalucia 2004)
tan04 (from Tanaka et al. 2004) (by default)

RNA duplexes
ser07 (from Blose et al. 2007)
tur06 (from Lu et al. 1999 and 2006) (by default)

To change the file containing the thermodynamic parameters for single bulge loop computation, the same syntax as the one for the -nn
option is used. Single bulge loops are not taken into account by the approximative mode.

-lonBUmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of long bulge loop to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
san04 (from Hicks and Santalucia 2004) (by default)

RNA duplexes
tur06 (from Lu et al. 1999 and 2006) (by default)

To change the file containing the thermodynamic parameters for long bulge loop computation, the same syntax as the one for the -nn
option is used. Long bulge loops are not taken into account by the approximative mode.

-CNGmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of CNG repeats to the thermodynamic of helix-coil transition. N represents a single mismatch of type N/N. You can use one of the following : RNA duplexes
bro05 (from Magdalena et al. 2005) (by default)

To change the file containing the thermodynamic parameters for CNG repeats computation, the same syntax as the one for the -nn
option is used. CNG repeats are not taken into account by the approximative mode. Be aware : Melting can compute the contribution of CNG repeats to the thermodynamic of helix-coil transition for only 2 to 7 CNG repeats.

-inomethod_name

Forces to use a specific nearest neighbor model to compute the contribution of inosine bases (I) to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
san05 (from Watkins and Santalucia et al. 2005) (by default)

RNA duplexes
zno07 (from Wright et al. 2007) (by default)

To change the file containing the thermodynamic parameters for inosine bases computation, the same syntax as the one for the -nn
option is used. Inosine bases (I) are not taken into account by the approximative mode.

-hamethod_name

Forces to use a specific nearest neighbor model to compute the contribution of hydroxyadenine bases (A*) to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
sug01 (from Kawakami et al. 2001)

To change the file containing the thermodynamic parameters for hydroxyadenine bases computation, the same syntax as the one for the -nn
option is used. Hydroxyadenine bases (A*) are not taken into account by the approximative mode.

-azomethod_name

Forces to use a specific nearest neighbor model to compute the contribution of azobenzenes (X_T for trans azobenzenes and X_C for cis azobenzenes) to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
asa05 (from Asanuma et al. 2005)(by default)

To change the file containing the thermodynamic parameters for azobenzene computation, the same syntax as the one for the -nn
option is used. Azobenzenes (X_T for trans azobenzenes and X_C for cis azobenzenes) are not taken into account by the approximative mode.

-lckmethod_name

Forces to use a specific nearest neighbor model to compute the contribution of single locked nucleic acids (AL, GL, TL and CL) to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
mct04 (from McTigue et al. 2004)
owc11 (from Owczarzy et al.) (by default)

To change the file containing the thermodynamic parameters for single locked nucleic acids computation, the same syntax as the one for the -nn
option is used. Locked nucleic acids (AL, GL, TL and CL) are not taken into account by the approximative mode. -tanLckmethod_name Forces to use a specific nearest neighbor model to compute the contribution of consecutive locked nucleic acids (AL, GL, TL and CL) to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
owc11 (from Owczarzy et al. 2011) (by default)

To change the file containing the thermodynamic parameters for consecutive locked nucleic acids computation, the same syntax as the one for the -nn
option is used. Locked nucleic acids (AL, GL, TL and CL) are not taken into account by the approximative mode. -sinMMLckmethod_name Forces to use a specific nearest neighbor model to compute the contribution of consecutive locked nucleic acids with a single mismatch (AL, GL, TL and CL) to the thermodynamic of helix-coil transition. You can use one of the following :

DNA duplexes
owc11 (from Owczarzy et al. 2011) (by default)

To change the file containing the thermodynamic parameters for consecutive locked nucleic acids computation with single mismatch, the same syntax as the one for the -nn
option is used. Locked nucleic acids (AL, GL, TL and CL) are not taken into account by the approximative mode.

-ionmethod_name

Forces to use a specific ion correction. You can use one of the following corrections :

Sodium corrections

DNA duplexes
ahs01 (from von Ahsen et al. 2001)
kam71 (from Frank-Kamenetskii et al 2001)
owc1904 (equation 19 from Owczarzy et al. 2004)
owc2004 (equation 20 from Owczarzy et al. 2004)
owc2104 (equation 21 from Owczarzy et al. 2004)
owc2204 (equation 21 from Owczarzy et al. 2004) (by default)
san96 (from Santalucia et al. 1996)
san04 (from Santalucia et al. 1998, 2004)
schlif (from Schildkraut and Lifson 1965)
tanna06 (from Tan et al. 2006)
wetdna91 (from Wetmur 1991)

RNA duplexes or mRNA/RNA duplexes
tanna07 (from Tan et al. 2007) (by default)
wetrna91 (from Wetmur 1991)

DNA/RNA duplexes
wetdnarna91 (from Wetmur 1991)

Magnesium corrections

DNA duplexes
owcmg08 (from Owczarzy et al. 2008) (by default)
tanmg06 (from Tan et al. 2006)

RNA duplexes or mRNA/RNA duplexes
tanmg07 (from Tan et al. 2007) (by default)

Mixed Na Mg corrections

DNA duplexes
owcmix08 (from Owczarzy et al. 2008) (by default)
tanmix07 (from Tan et al. 2007)

RNA duplexes or mRNA/RNA duplexes}]
tanmix07 (from Tan et al. 2007) (by default)

The effect of ions on thermodynamic stability of nucleic acid duplexes is complex, and the correcting functions are at best rough approximations. By default, the program use the algorithm from Owczarzy et al 2008 : ratio = Mg^0.5 and monovalent = Na + Tris + K if monovalent = 0, a magnesium correction is used. if ratio < 0.22, a sodium correction is used. if 0.22 <= ratio < 6, a mixed Na Mg correction is used. if ratio >= 6, a magnesium correction is used. examples :

###beginning###
- "-ion owcmg08" if you want to force the use of the magnesium correction from Owczarzy et al 2008. This correction will be used independently of the cations present in the solution.

###end###

-naeqmethod_name

Forces to use a specific ion correction which gives a sodium equivalent concentration if other cations are present. You can use one of the following :

DNA duplexes
ahs01 (from von Ahsen et al 2001) (by default)
mit96 (from Mitsuhashi et al. 1996)
pey00 (from Peyret 2000)

For the other types of hybridization, the DNA default correction is used but there is no guaranty of accuracy. If there are other cations when an approximative approach is used, a sodium equivalence is automatically computed. The correcting functions are at best rough approximations. examples :

###beginning###
- "-naeq ahs01" if you want to force the use of the magnesium correction from Ahsen et al 2001. This sodium equivalence will be used in case of approximative approach. In case of nearest neighbor approach, the sodium equivalence will be used only if a sodium correction is selected by the user. - "-naeq ahs01 -ion san04" means that the sodium equivalence computed by the method ahs01 (from Ahsen et al 2001) will be combined with the sodium correction san04 (from Santalucia 2004)

###end###

-DMSOmethod_name

Forces to use a specific DMSO correction (DMSO is always in percent). You can use one of the following :

DNA duplexes}]
ahs01 (from von Ahsen et al 2001) (by default)
mus81 (from Musielski et al. 1981)
cul76 (from Cullen et al. 1976)
esc80 (from Escara et al. 1980)

For the other types of hybridization, the DNA default correction is used but there is no guaranty of accuracy. If there are DMSO when an approximative approach is used, a DMSO correction is automatically computed. The correcting functions are at best rough approximations. example :

###beginning###
- "-DMSO ahs01" if you want to force the use of the DMSO correction from Ahsen et al 2001. This DMSO correction will be used if there is DMSO present in the solutions in case of nearest neighbor approach and approximative approach.

###end###

-formethod_name

Forces to use a specific formamide correction. You can use one of the following : DNA duplexes}]
bla96 (from Blake et al 1996) with formamide concentration in M (by default)
lincorr (linear correction) with a percent of formamide volume

For the other types of hybridization, the DNA default correction is used but there is no guaranty of accuracy. If there are formamide when an approximative approach is used, a formamide correction is automatically computed. The correcting functions are at best rough approximations. example :

###beginning###
- "-for lincorr" if you want to force the use of the linear formamide correction. This formamide correction will be used if there is formamide present in the solutions in case of nearest neighbor approach and approximative approach.

###end###