---
title: "Computation of melting temperature of nucleic acid duplexes with `rmelting`"
author: "Aravind, J.^1^ and Krishna, G. K.^2^"
date: "`r Sys.Date()`"
output:
  pdf_document:
    dev: cairo_pdf
    fig_caption: no
    number_sections: yes
    toc: no
  html_document:
    number_sections: yes
    toc: yes
documentclass: article
classoption: table, twoside
header-includes:
  - \usepackage{fancyhdr}
  - \usepackage{wrapfig}
  - \usepackage{float}
  - \pagestyle{fancyplain}
  - \fancyhf{}
  - \fancyhead[LE]{ \fancyplain{}{Computation of melting temperature of nucleic acid duplexes with \texttt{rmelting}} }
  - \fancyhead[RO]{ \fancyplain{}{\slshape \nouppercase{\leftmark}} }
  - \fancyfoot[LE]{ \fancyplain{}{\thepage} }
  - \fancyfoot[RO]{ \fancyplain{}{\thepage} }
  - \usepackage{hyperref}
  - \hypersetup{colorlinks=true}
  - \hypersetup{linktoc=all}
  - \hypersetup{linkcolor=blue}
  - \usepackage{pdflscape}
  - \usepackage{booktabs}
  - \newcommand{\blandscape}{\begin{landscape}}
  - \newcommand{\elandscape}{\end{landscape}}
  - \renewcommand\thesection{\arabic{section}}
csl: frontiers.csl
bibliography: REFERENCES.bib
vignette: |
  %\VignetteIndexEntry{Tutorial}
  %\usepackage[utf8]{inputenc}
  %\VignetteEngine{knitr::rmarkdown_notangle}
---


```{r, echo=FALSE}
out_type <- knitr::opts_knit$get("rmarkdown.pandoc.to")

r = getOption("repos")
r["CRAN"] = "https://cran.rstudio.com/"
#r["CRAN"] = "https://cloud.r-project.org/"
#r["CRAN"] = "https://ftp.iitm.ac.in/cran/"
options(repos = r)
```


```{r, results='asis', echo=FALSE}
switch(out_type,
    html = {cat("<p>1. Division of Germplasm Conservation, ICAR-National Bureau of Plant Genetic Resources, New Delhi.</p>

<p>2. Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi.</p>")},
    latex = cat("\\begin{center}
1. Division of Germplasm Conservation, ICAR-National Bureau of Plant Genetic Resources, New Delhi.

2. Division of Crop Physiology, ICAR-Indian Agricultural Research Institute, New Delhi.

\\end{center}" )
)
```

\begin{center}
\vspace{6pt}
\hrule
\end{center}

```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = TRUE)

library(readxl)

#  x <- read_excel("vignettes/Tables.xlsx", sheet = "Approx.methods", col_names = TRUE)
rmeltingtables <- function(file, sheet, def="default",
                           code="Formula", ref = "Reference") {
  
  if (requireNamespace("readxl", quietly = TRUE)) {
    
    x <- read_excel(path = file, sheet = sheet, col_names = TRUE)
    
    x[,code] <- paste0("`", unlist(x[,code]), "`", sep = "")
  
  if (!is.null(def)) {
    ind <- which(is.na(x[,def]) %in% FALSE)
    x[ind,code] <- paste0(unlist(x[ind,code]), unlist(x[ind,def]), sep = "")
    rm(ind)
  }

  x[, def] <- NULL
  
  x[, ref] <- gsub("\\[|\\]", "", unlist(x[, ref]))
  x[, ref] <- gsub(";", ",", unlist(x[, ref]))
  x[, "Limits/Remarks"] <- gsub("(\\\\code\\{)(.+)(\\})", "`\\2`", unlist(x[, "Limits/Remarks"]))
  
  # x <- data.frame(x)
  
  pander::pander(x, style = 'multiline',
               split.cell = c(13, 10, 20, 20),
               justify = c('left'), split.table = Inf, keep.line.breaks = FALSE,
               use.hyphening = FALSE, missing = "")
} else {
  print("Package 'readxl' is required to generate this table.")
}
}
```

\tableofcontents

\begin{wrapfigure}{r}{0.35\textwidth}
  \vspace{-10pt}
  \begin{center}
    \includegraphics[width=0.33\textwidth]{`r system.file("extdata", "rmelting.png", package = "rmelting")`}
  \end{center}
    \vspace{-10pt}
\end{wrapfigure}

<img src="https://raw.githubusercontent.com/aravind-j/rmelting/master/inst/extdata/rmelting.png" align="right" alt="logo" width="173" height = "200" style = "padding: 10px; border: none; float: right;">

# Introduction

The `R` package `rmelting` is an interface to the [MELTING 5](https://www.ebi.ac.uk/biomodels/tools/melting/) program [@le_novere_melting_2001; @dumousseau_melting_2012]  to compute melting temperatures of nucleic acid duplexes (DNA/DNA, DNA/RNA, RNA/RNA or 2'-O-MeRNA/RNA) along with other thermodynamic parameters such as hybridisation enthalpy and entropy.

Melting temperatures are computed by Nearest-neighbour methods for short sequences or approximative estimation formulae for long sequences. Apart from these, multiple corrections are available to take into account the presence of Cations (Na, Tris, K and Mg) or denaturing agents (DMSO and formamide).

# Installation
The package can be installed from Bioconductor as follows.

```{r, eval=FALSE}
if (!"BiocManager" %in% rownames(installed.packages())) 
  install.packages("BiocManager")
BiocManager::install("rmelting")
```

The development version can be installed from github as follows.

```{r, eval=FALSE}
if (!require('devtools')) install.packages('devtools')
devtools::install_github("aravind-j/rmelting")
```

Then the package can be loaded as follows.
```{r, message=FALSE}
library(rmelting)
```

# Basic usage

Melting temperatures are computed in `rmelting` through the core function `melting` which takes a number of arguments (see `?melting`). The following are the essential arguments which are mandatory for computation.

* `sequence`
    + 5' to 3' sequence of one strand of the nucleic acid duplex as a character string.  Recognises A, C, G, T, U, I, X_C, X_T, A*, AL, TL, GL and CL (**Table 1**). U and T are not considered identical.

**Table 1**: Recognized sequences
```{r, echo=FALSE}
if (requireNamespace("readxl", quietly = TRUE)) {
  ntides <- read_excel("Tables.xlsx", sheet = "MELTING", col_names = TRUE)
  knitr::kable(ntides)
} else {
  print("Package 'readxl' is required to generate this table.")
}
```
    
* `Comp.sequence`
    + Mandatory if there are mismatches, inosine(s) or hydroxyadenine(s) between the two strands. If not specified, it is computed as the complement of `sequence`. Self-complementarity in `sequence` is detected even though there may be (are) dangling end(s) and `comp.sequence` is computed.

* `nucleic.acid.conc`
    + In molar concentration (M or mol L^-1^).
    
* `Na.conc, Mg.conc, Tris.conc, K.conc`
    + At least one cation (Na, Mg, Tris, K) concentration is mandatory, the other agents(dNTP, DMSO, formamide) are optional.
    
* `hybridisation.type`
    + The possible options for hybridisation type are as follows (**Table 2**).

**Table 2**: Hybridisation type options
```{r, echo=FALSE}
if (requireNamespace("readxl", quietly = TRUE)) {
  hybtype <- read_excel("Tables.xlsx", sheet = "HybType", col_names = TRUE)
  hybtype$Option <- paste("`", hybtype$Option, "`", "")
  knitr::kable(hybtype)
} else {
  print("Package 'readxl' is required to generate this table.")
}
```

With these arguments, the melting temperature can be computed as follows.

```{r}
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1)
```

Only the melting temperature is given as a console output. However, the output can be assigned to an object which contains the details of the environment, options and the thermodynamics results as a list.

```{r}
# Get output as list
out <- melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
               hybridisation.type = "dnadna", Na.conc = 1)
# Environment output
out$Environment
# Options used
out$Options
# Thermodynamics results
out$Results

```

The command for the MELTING 5 java version is saved as an attribute in the list `out` and can be retrieved as follows.

```{r}
# Command for MELTING 5
attributes(out)$command
```

# Melting temperature computation

Melting temperature is computed by either approximative or nearest neighbour methods according to the length of the oligonucleotide sequences. For longer sequences (longer than the threshold value, the threshold value set by `size.threshold` with the default value 60) approximative method is used, while for others, nearest neighbour method is used. 

## Approximative methods

The approximative method for computation can be specified by the argument `method.approx`. The available methods are given in **Table 3**.

**Table 3**: Details of approximative methods
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "Approx.methods",
               def = "default", code = "Formula", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:TCTAATGTGCTGTTAGATGTATCCAGAGATAGCCGAGCATAAACTTCAACACACGAGACGTTGATTGGATTTAACCATAG
        ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
    DNA:AGATTACACGACAATCTACATAGGTCTCTATCGGCTCGTATTTGAAGTTGTGTGCTCTGCAACTAACCTAAATTGGTATC

    RNA:UUAAUCUCCGUCAUCUUUAAGCCGUGGAGAGACUGUAGACUUGAACAGGGGUAAGCGGAGGCACGUAGGAUUCACAUCAU
        ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
    RNA:AAUUAGAGGCAGUAGAAAUUCGGCACCUCUCUGACAUCUGAACUUGUCCCCAUUCGCCUCCGUGCAUCCUAAGUGUAGUA
    
    DNA:TCTAATGTGCTGTTAGATGTATCCAGAGATAGCCGAGCATAAACTTCAACACACGAGACGTTGATTGGATTTAACCATAG
        ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
    RNA:AGAUUACACGACAAUCUACAUAGGUCUCUAUCGGCUCGUAUUUGAAGUUGUGUGCUCUGCAACUAACCUAAAUUGGUAUC
    
```

```{r}
# Long Nucleotide sequence
DNAseq <- c("TCTAATGTGCTGTTAGATGTATCCAGAGATAGCCGAGCATAAACTTCAACACACGAGACGTTGATTGGATTTAACCATAG")
RNAseq <- c("UUAAUCUCCGUCAUCUUUAAGCCGUGGAGAGACUGUAGACUUGAACAGGGGUAAGCGGAGGCACGUAGGAUUCACAUCAU")

# Approximative method - default (DNA/DNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1)

# Approximative method - wetdna91 (DNA/DNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1,
        method.approx = "wetdna91")

# Approximative method - ahs01 (DNA/DNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1,
        method.approx = "ahs01")

# Approximative method - che93 (DNA/DNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1,
        method.approx = "che93")

# Approximative method - che93corr (DNA/DNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1,
        method.approx = "che93corr")

# Approximative method - schdot (DNA/DNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1,
        method.approx = "schdot")

# Approximative method - owe69 (DNA/DNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1,
        method.approx = "owe69")

# Approximative method - san98 (DNA/DNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1,
        method.approx = "san98")

# Approximative method - default (RNA/RNA)
melting(sequence = RNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "rnarna", Na.conc = 1)

# Approximative method - wetrna91 (RNA/RNA)
melting(sequence = RNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "rnarna", Na.conc = 1,
        method.approx = "wetrna91")

# Approximative method - wetdnarna91 (DNA/RNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnarna", Na.conc = 1)

# Approximative method - wetdnarna91 (DNA/RNA)
melting(sequence = DNAseq, nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnarna", Na.conc = 1,
        method.approx = "wetdnarna91")
```

## Nearest neighbour methods

### Perfectly matching sequences

The nearest neighbour model for computation in case of perfectly matching sequences can be specified by the argument `method.nn`. The available methods are given in **Table 4**.

**Table 4**: Details of nearest neighbour methods for perfectly matching sequences

```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "NN.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:CAGTGAGACAGCAATGGTCG
        ||||||||||||||||||||
    DNA:GTCACTCTGTCGTTACCAGC

    RNA:CAGUGAGACAGCAAUGGUCG
        ||||||||||||||||||||
    RNA:GUCACUCUGUCGUUACCAGC
    
    DNA:CAGTGAGACAGCAATGGTCG
        ||||||||||||||||||||
    RNA:GUCACUCUGUCGUUACCAGC
    
```

```{r}
# Nearest neighbour method - default (DNA/DNA: No Self-Complimentarity)
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1)

# Nearest neighbour method - all97 (DNA/DNA: No Self-Complimentarity)
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "all97")

# Nearest neighbour method - bre86 (DNA/DNA: No Self-Complimentarity)
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "bre86")

# Nearest neighbour method - san04 (DNA/DNA: No Self-Complimentarity)
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "san04")

# Nearest neighbour method - san96 (DNA/DNA: No Self-Complimentarity)
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "san96")

# Nearest neighbour method - sug96 (DNA/DNA: No Self-Complimentarity)
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "sug96")

# Nearest neighbour method - tan04 (DNA/DNA: No Self-Complimentarity)
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "tan04")

# Nearest neighbour method - default (RNA/RNA: No Self-Complimentarity)
melting(sequence = "CAGUGAGACAGCAAUGGUCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "rnarna", Na.conc = 1)

# Nearest neighbour method - xia98 (RNA/RNA: No Self-Complimentarity)
melting(sequence = "CAGUGAGACAGCAAUGGUCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "rnarna", Na.conc = 1, method.nn = "xia98")

# Nearest neighbour method - fre86 (RNA/RNA: No Self-Complimentarity)
melting(sequence = "CAGUGAGACAGCAAUGGUCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "rnarna", Na.conc = 1, method.nn = "fre86")

# Nearest neighbour method - default (mRNA/RNA: No Self-Complimentarity)
melting(sequence = "CAGUGAGACAGCAAUGGUCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "mrnarna", Na.conc = 1)

# Nearest neighbour method - tur06 (mRNA/RNA: No Self-Complimentarity)
melting(sequence = "CAGUGAGACAGCAAUGGUCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "mrnarna", Na.conc = 1, method.nn = "tur06")

# Nearest neighbour method - default (DNA/RNA: No Self-Complimentarity)
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnarna", Na.conc = 1)

# Nearest neighbour method - sug95 (DNA/RNA: No Self-Complimentarity)
melting(sequence = "CAGTGAGACAGCAATGGTCG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnarna", Na.conc = 1, method.nn = "sug95")
```

Self complementarity for perfect matching sequences or sequences with dangling ends is detected automatically. However it can be enforced by the argument `force.self = TRUE`.

_**Examples**_

```
    DNA:CATATGGCCATATG
        ||||||||||||||
    DNA:GTATACCGGTATAC

    RNA:AUGUACAU
        ||||||||
    RNA:UACAUGUA
```

```{r}
# Nearest neighbour method - default (DNA/DNA: Self-Complimentarity)
melting(sequence = "CATATGGCCATATG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1)

# Nearest neighbour method - all97 (DNA/DNA: Self-Complimentarity)
melting(sequence = "CATATGGCCATATG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "all97")

# Nearest neighbour method - bre86 (DNA/DNA: Self-Complimentarity)
melting(sequence = "CATATGGCCATATG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "bre86")

# Nearest neighbour method - san04 (DNA/DNA: Self-Complimentarity)
melting(sequence = "CATATGGCCATATG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "san04")

# Nearest neighbour method - san96 (DNA/DNA: Self-Complimentarity)
melting(sequence = "CATATGGCCATATG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "san96")

# Nearest neighbour method - sug96 (DNA/DNA: Self-Complimentarity)
melting(sequence = "CATATGGCCATATG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "sug96")

# Nearest neighbour method - tan04 (DNA/DNA: Self-Complimentarity)
melting(sequence = "CATATGGCCATATG", nucleic.acid.conc = 2e-06,
        hybridisation.type = "dnadna", Na.conc = 1, method.nn = "tan04")

# Nearest neighbour method - default (RNA/RNA: Self-Complimentarity)
melting(sequence = "AUGUACAU", nucleic.acid.conc = 2e-06,
        hybridisation.type = "rnarna", Na.conc = 1)

# Nearest neighbour method - xia98 (RNA/RNA: Self-Complimentarity)
melting(sequence = "AUGUACAU", nucleic.acid.conc = 2e-06,
        hybridisation.type = "rnarna", Na.conc = 1, method.nn = "xia98")

# Nearest neighbour method - fre86 (RNA/RNA: Self-Complimentarity)
melting(sequence = "AUGUACAU", nucleic.acid.conc = 2e-06,
        hybridisation.type = "rnarna", Na.conc = 1, method.nn = "fre86")

```


### GU wobble base pairs effect

The nearest neighbour model for computation in case of sequences with GU wobble base pairs can be specified by the argument `method.GU`. The available methods are given in **Table 5**.

**Table 5**: Details of methods for sequences with GU wobble base pairs
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "GU.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    RNA:CCAGCGUCCU
        ||||||||||
    RNA:GGTCGCAGGA
```

```{r}
# GU wobble base pairs effect - default (RNA/RNA)
melting(sequence = "CCAGCGUCCU", nucleic.acid.conc = 0.0001,
        hybridisation.type = "rnarna", Na.conc = 1)

# GU wobble base pairs effect - ser12 (RNA/RNA)
melting(sequence = "CCAGCGUCCU", nucleic.acid.conc = 0.0001,
        hybridisation.type = "rnarna", Na.conc = 1, method.GU = "ser12")

# GU wobble base pairs effect - tur99 (RNA/RNA)
melting(sequence = "CCAGCGUCCU", nucleic.acid.conc = 0.0001,
        hybridisation.type = "rnarna", Na.conc = 1, method.GU = "tur99")
```

### Single mismatch effect

The nearest neighbour model for computation in case of sequences with a single mismatch can be specified by the argument `method.singleMM`. The available methods are given in **Table 6**.

**Table 6**: Details of methods for sequences with single mismatch
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "singleMM.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}


_**Examples**_

```
    DNA:CAACTTGATATTAATA
        |||||||| |||||||
    DNA:GTTGAACTCTAATTAT

    RNA:GACAGGCUG
        |||| ||||
    RNA:CUGUGCGAC
        
    DNA:CCATAACTACC
        |||| ||||||
    RNA:GGUAAUGAUGG
```

```{r}
# Single mismatch effect - default (DNA/DNA)
melting(sequence = "CAACTTGATATTAATA", comp.sequence = "GTTGAACTCTAATTAT",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna", Na.conc = 1)

# Single mismatch effect - allsanpey (DNA/DNA)
melting(sequence = "CAACTTGATATTAATA", comp.sequence = "GTTGAACTCTAATTAT",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna",
        Na.conc = 1, method.singleMM = "allsanpey")

# Single mismatch effect - default (RNA/RNA)
melting(sequence = "GACAGGCUG", comp.sequence = "CUGUGCGAC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna", Na.conc = 1)

# Single mismatch effect - zno07 (RNA/RNA)
melting(sequence = "GACAGGCUG", comp.sequence = "CUGUGCGAC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.singleMM = "zno07")

# Single mismatch effect - zno08 (RNA/RNA)
melting(sequence = "CAGUACGUC", comp.sequence = "GUCGGGCAG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.singleMM = "zno08")

# Single mismatch effect - tur06 (RNA/RNA)
melting(sequence = "GACAGGCUG", comp.sequence = "CUGUGCGAC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.singleMM = "tur06")

# Single mismatch effect - default (DNA/RNA)
melting(sequence = "CCATAACTACC", comp.sequence = "GGUAAUGAUGG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnarna", Na.conc = 1)

# Single mismatch effect - wat11 (DNA/RNA)
melting(sequence = "CCATAACTACC", comp.sequence = "GGUAAUGAUGG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnarna",
        Na.conc = 1, method.singleMM = "wat11")
```

### Tandem mismatches effect

The nearest neighbour model for computation in case of sequences with tandem mismatches can be specified by the argument `method.tandemMM`. The available methods are given in **Table 7**.

**Table 7**: Details of methods for sequences with tandem mismatches
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "tandemMM.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:GACGTTGGAC
        ||||  ||||
    DNA:CTGCGGCCTG

    RNA:GAGCGGAG
        |||  |||
    RNA:CUCCACUC
```

```{r}
# Tandem mismatches effect - default (DNA/DNA)
melting(sequence = "GACGTTGGAC", comp.sequence = "CTGCGGCCTG",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna", Na.conc = 1)

# Tandem mismatches effect - allsanpey (DNA/DNA)
melting(sequence = "GACGTTGGAC", comp.sequence = "CTGCGGCCTG",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna",
        Na.conc = 1, method.tandemMM = "allsanpey")

# Tandem mismatches effect - default (RNA/RNA)
melting(sequence = "GAGCGGAG", comp.sequence = "CUCCACUC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna", Na.conc = 1)

# Tandem mismatches effect -  tur06 (RNA/RNA)
melting(sequence = "GAGCGGAG", comp.sequence = "CUCCACUC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.tandemMM = "tur99")
```

### Single dangling end effect

The nearest neighbour model for computation in case of sequences with a single dangling end can be specified by the argument `method.single.dangle`. The available methods are given in **Table 8**.

**Table 8**: Details of methods for sequences with single dangling end
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "single.dangle.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:-GTAGCTACA
         |||||||| 
    DNA:ACATCGATG-

    RNA:-GGCGCUG
         |||||||
    RNA: CCGCGAC
        
    DNA:-GGCGCUG
         |||||||
    RNA: CCGCGAC
```

```{r}
# Single dangling end effect - default (DNA/DNA)
melting(sequence = "-GTAGCTACA",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna",
        Na.conc = 1)

# Single dangling end effect - bom00 (DNA/DNA)
melting(sequence = "-GTAGCTACA",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna",
        Na.conc = 1, method.single.dangle = "bom00")

# Single dangling end effect - sugdna02 (DNA/DNA)
melting(sequence = "-GTAGCTACA",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna",
        Na.conc = 1, method.single.dangle = "sugdna02")

# Single dangling end effect - default (RNA/RNA)
melting(sequence = "-GGCGCUG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1)

# Single dangling end effect -  ser08 (RNA/RNA)
melting(sequence = "-GGCGCUG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.single.dangle = "ser08")

# Single dangling end effect -  sugrna02 (RNA/RNA)
melting(sequence = "-GGCGCUG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.single.dangle = "sugrna02")
```

### Double dangling end effect

The nearest neighbour model for computation in case of sequences with a double or secondary dangling ends can be specified by the argument `method.double.dangle`. The available methods are given in **Table 9**.

**Table 9**: Details of methods for sequences with double dangling ends
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "double.dangle.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:--ATGCATAA
          ||||||
    DNA:AATACGTA--

    RNA:--AUGCAUAA
          ||||||
    RNA:AAUACGUA--
```

```{r}
# Double dangling end effect - default (DNA/DNA)
melting(sequence = "--ATGCATAA",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna",
        Na.conc = 1)

# Double dangling end effect - sugdna02 (DNA/DNA)
melting(sequence = "--ATGCATAA",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna",
        Na.conc = 1, method.double.dangle = "sugdna02")

# Double dangling end effect - default (RNA/RNA)
melting(sequence = "--AUGCAUAA",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1)

# Double dangling end effect -  ser06 (RNA/RNA)
melting(sequence = "--AUGCAUAA",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.double.dangle = "ser06")

# Double dangling end effect -  sugrna02 (RNA/RNA)
melting(sequence = "--AUGCAUAA",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.double.dangle = "sugrna02")

# Double dangling end effect -  ser05 (RNA/RNA)
melting(sequence = "--AUGCAUAA",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.double.dangle = "ser05")
```

### Long dangling end effect

The nearest neighbour model for computation in case of sequences with a double or secondary dangling ends can be specified by the argument `method.long.dangle`. The available methods are given in **Table 10**.

**Table 10**: Details of methods for sequences with long dangling ends
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "long.dangle.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:----GCATATGCAAAA
            |||||||| 
    DNA:AAAACGTATACG----

    RNA:AAAAGCAUAUGC----
            ||||||||
    RNA:----CGUAUACGAAAA
```

```{r}
# Long dangling end effect - default (DNA/DNA)
melting(sequence = "----GCATATGCAAAA",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna",
        Na.conc = 1)

# Long dangling end effect - sugdna02 (DNA/DNA)
melting(sequence = "----GCATATGCAAAA",
        nucleic.acid.conc = 0.0004, hybridisation.type = "dnadna",
        Na.conc = 1, method.long.dangle = "sugdna02")

# Long dangling end effect - default (RNA/RNA)
melting(sequence = "AAAAGCAUAUGC----",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1)

# Long dangling end effect -  sugrna02 (RNA/RNA)
melting(sequence = "AAAAGCAUAUGC----",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.long.dangle = "sugrna02")
```

### Internal loop effect

The nearest neighbour model for computation in case of sequences with an internal loop (more than two adjacent mismatches) can be specified by the argument `method.internal.loop`. The available methods are given in **Table 11**.

**Table 11**: Details of methods for sequences with internal loops
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "internal.loop.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:GCGATTGGCACTTTGGTGAAC
        |||||    ||||||||||||
    DNA:CGCTACATATGAAACCACTTG

    RNA:GACAC-GCUG
        ||||  ||||
    RNA:CUGUAUCGAC
```

```{r}
# Internal loop effect - default (DNA/DNA)
melting(sequence = "GCGATTGGCACTTTGGTGAAC", comp.sequence = "CGCTACATATGAAACCACTTG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1)

# Internal loop effect - san04 (DNA/DNA)
melting(sequence = "GCGATTGGCACTTTGGTGAAC", comp.sequence = "CGCTACATATGAAACCACTTG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.internal.loop = "san04")

# Internal loop effect - default (RNA/RNA)
melting(sequence = "GACAC-GCUG", comp.sequence = "CUGUAUCGAC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1)

# Internal loop effect -  zno07 (RNA/RNA)
melting(sequence = "GACAC-GCUG", comp.sequence = "CUGUAUCGAC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.internal.loop = "zno07")

# Internal loop effect -  tur06 (RNA/RNA)
melting(sequence = "GACAC-GCUG", comp.sequence = "CUGUAUCGAC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.internal.loop = "tur06")
```

### Single bulge loop effect

The nearest neighbour model for computation in case of sequences with a single bulge loop can be specified by the argument `method.single.bulge.loop`. The available methods are given in **Table 12**.

**Table 12**: Details of methods for sequences with single bulge loop
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "single.bulge.loop.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:TCGATTAGCGACACAGG
        |||||||| ||||||||
    DNA:AGCTAATC-CTGTGTCC

    RNA:GACUCUGUC
        |||| ||||
    RNA:CUGA-ACAG
```

```{r}
# Single bulge loop effect - default (DNA/DNA)
melting(sequence = "TCGATTAGCGACACAGG", comp.sequence = "AGCTAATC-CTGTGTCC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1)

# Single bulge loop effect - tan04 (DNA/DNA)
melting(sequence = "TCGATTAGCGACACAGG", comp.sequence = "AGCTAATC-CTGTGTCC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.single.bulge.loop = "tan04")

# Single bulge loop effect - san04 (DNA/DNA)
melting(sequence = "TCGATTAGCGACACAGG", comp.sequence = "AGCTAATC-CTGTGTCC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.single.bulge.loop = "san04")

# Single bulge loop effect - default (RNA/RNA)
melting(sequence = "GACUCUGUC", comp.sequence = "CUGA-ACAG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1)

# Single bulge loop effect -  tur06 (RNA/RNA)
melting(sequence = "GACUCUGUC", comp.sequence = "CUGA-ACAG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.single.bulge.loop = "tur06")

# Single bulge loop effect -  ser07 (RNA/RNA)
melting(sequence = "GACUCUGUC", comp.sequence = "CUGA-ACAG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.single.bulge.loop = "ser07")
```

### Long bulge loop effect

The nearest neighbour model for computation in case of sequences with long bulge loop can be specified by the argument `method.long.bulge.loop`. The available methods are given in **Table 13**.

**Table 13**: Details of methods for sequences with long bulge loop
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "long.bulge.loop.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:ATATGACGCCACAGCG
        |||||   ||||||||
    DNA:TATAC---GGTGTCGC

    RNA:AUAUGACGCCACAGCG
        |||||   ||||||||
    RNA:UAUAC---GGUGUCGC
```

```{r}
# Long bulge loop effect - default (DNA/DNA)
melting(sequence = "ATATGACGCCACAGCG", comp.sequence = "TATAC---GGTGTCGC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1)

# Long bulge loop effect - san04 (DNA/DNA)
melting(sequence = "ATATGACGCCACAGCG", comp.sequence = "TATAC---GGTGTCGC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.long.bulge.loop = "san04")

# Long bulge loop effect - default (RNA/RNA)
melting(sequence = "AUAUGACGCCACAGCG", comp.sequence = "UAUAC---GGUGUCGC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1)

# Long bulge loop effect -  tur06 (RNA/RNA)
melting(sequence = "AUAUGACGCCACAGCG", comp.sequence = "UAUAC---GGUGUCGC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.long.bulge.loop = "tur06")
```

### CNG repeats effect

The nearest neighbour model for computation in case of sequences with CNG repeats can be specified by the argument `method.CNG`. The available methods are given in **Table 14**.

**Table 14**: Details of methods for sequences with CNG repeats
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "CNG.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    RNA:GCGGCGGCGGC
        |||||||||||
    RNA:CGCCGCCGCCG
```

```{r}
# CNG repeats effect - default (RNA/RNA)
melting(sequence = "GCGGCGGCGGC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1)

# CNG repeats effect -  bro05 (RNA/RNA)
melting(sequence = "GCGGCGGCGGC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.CNG = "bro05")
```

### Inosine bases effect

The nearest neighbour model for computation in case of sequences with inosine bases (I) can be specified by the argument `method.inosine`. The available methods are given in **Table 15**.

**Table 15**: Details of methods for sequences with inosine bases
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "inosine.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
    DNA:CCGICTGTIGCG
        ||| |||| |||
    DNA:GGCCGACACCGC

    RNA:GCAICGC
        ||| |||
    RNA:CGUUGCG
```

```{r}
# Inosine bases effect - default (DNA/DNA)
melting(sequence = "CCGICTGTIGCG", comp.sequence = "GGCCGACACCGC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1)

# Inosine bases effect - san05 (DNA/DNA)
melting(sequence = "CCGICTGTIGCG", comp.sequence = "GGCCGACACCGC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.inosine = "san05")

# Inosine bases effect - default (RNA/RNA)
melting(sequence = "GCAICGC", comp.sequence = "CGUUGCG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1)

# Inosine bases effect -  zno07 (RNA/RNA)
melting(sequence = "GCAICGC", comp.sequence = "CGUUGCG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "rnarna",
        Na.conc = 1, method.inosine = "zno07")
```

### Hydroxyadenine bases effect

The nearest neighbour model for computation in case of sequences with hydroxyadenine bases can be specified by the argument `method.hydroxyadenine`. The available methods are given in **Table 16**.

**Table 16**: Details of methods for sequences with hydroxyadenine bases
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "hydroxyadenine.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
               *
    DNA:AGAAATGACACGGTG
        |||||||||||||||
    DNA:TCTTTACCGTGCCAC
```

```{r}
# Hydroxyadenine bases effect - default (DNA/DNA)
melting(sequence = "AGAAATGA*CACGGTG", comp.sequence = "TCTTTACCGTGCCAC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1)

# Hydroxyadenine bases effect - sug01 (DNA/DNA)
melting(sequence = "AGAAATGA*CACGGTG", comp.sequence = "TCTTTACCGTGCCAC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.hydroxyadenine = "sug01")
```

### Azobenzenes effect

The nearest neighbour model for computation in case of sequences with azobenzenes (X_T for trans azobenzenes and X_C for cis azobenzenes) can be specified by the argument `method.azobenzenes`. The available methods are given in **Table 17**.

**Table 17**: Details of methods for sequences with azobenzenes
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "azobenzenes.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
          C    C    C    C    C
    DNA:CTXTTAAXGAAGXGAGAXTATAXCC
        || |||| |||| |||| |||| ||
    DNA:GA AATT CTTC CTCT ATAT GG
```

```{r}
# Azobenzenes effect - default (DNA/DNA)
melting(sequence = "CTX_CTTAAX_CGAAGX_CGAGAX_CTATAX_CCC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1)

# Azobenzenes effect - asa05 (DNA/DNA)
melting(sequence = "CTX_CTTAAX_CGAAGX_CGAGAX_CTATAX_CCC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.azobenzenes = "asa05")
```

### Single Locked nucleic acid effect

The nearest neighbour model for computation in case of sequences with single locked nucleic acids can be specified by the argument `method.locked`. The available methods are given in **Table 18**.

**Table 18**: Details of methods for sequences with single locked nucleic acids
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "single.locked.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
             L  
    DNA:CCATTGCTACC
        |||||||||||
    DNA:GGTAACGATGG
```

```{r}
# Single locked nucleic acids effect - default (DNA/DNA)
melting(sequence = "CCATTLGCTACC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1)

# Single locked nucleic acids effect - mct04 (DNA/DNA)
melting(sequence = "CCATTLGCTACC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.locked = "mct04")

# Single locked nucleic acids effect - owc11 (DNA/DNA)
melting(sequence = "CCATTLGCTACC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.locked = "owc11")
```
## Consecutive Locked nucleic acids effect

The nearest neighbour model for computation in case of sequences with consecutive locked nucleic acids can be specified by the argument `method.consecutive.locked`. The available methods are given in **Table 19**.

**Table 19**: Details of methods for sequences with single locked nucleic acids
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "consq.locked.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
          LL  
    DNA:GACC
        ||||
    DNA:CTGG
```

```{r}
# Consecutive locked nucleic acids effect - default (DNA/DNA)
melting(sequence = "GALCLC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1)

# Consecutive locked nucleic acids effect - owc11 (DNA/DNA)
melting(sequence = "GALCLC",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.consecutive.locked = "owc11")
```

## Consecutive Locked nucleic acids with a single mismatch effect


The nearest neighbour model for computation in case of sequences with consecutive locked nucleic acids with single mismatch can be specified by the argument `method.consecutive.locked.singleMM`. The available methods are given in **Table 20**.

**Table 20**: Details of methods for sequences with single locked nucleic acids
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "consq.locked.SMM.methods",
               def = "default", code = "Model", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

_**Examples**_

```
          LLL  
    DNA:GACGC
        || ||
    DNA:CTTCG
```

```{r}
# Consecutive locked nucleic acids effect - default (DNA/DNA)
melting(sequence = "GALCLGLC", comp.sequence = "CTTCG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1)

# Consecutive locked nucleic acids effect - owc11 (DNA/DNA)
melting(sequence = "GALCLGLC", comp.sequence = "CTTCG",
        nucleic.acid.conc = 0.0001, hybridisation.type = "dnadna",
        Na.conc = 1, method.consecutive.locked.singleMM = "owc11")
```

# Corrections

Once the melting temperature is computed, a correction is applied to it according to the concentration of nucleic acids, cations and/or denaturing agents.

## Nucleic acid concentration

For self complementary sequences (auto detected or specified by `force.self`) it is 1. Otherwise it is 4 if the both strands are present in equivalent amount and 1 if one strand is in excess. 

## Ion corrections

Melting temperature is computed initially for $[\textrm{Na}^{+}]$ = 1 M, after which a correction for the presence of cations ($[\textrm{Na}^{+}]$, $[\textrm{K}^{+}]$, $[\textrm{Tris}^{+}]$ and $[\textrm{Mg}^{+}]$) is applied either directly on the computed melting temperature or on the computed entropy.

Th correction methods for cation concentration can be specified by the argument `correction.ion`.

### Sodium corrections

The available correction methods for sodium concentration are given in **Table 21**.

**Table 21**: Details of the corrections for sodium concentration
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "na.correction",
               def = "default", code = "Correction", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

```{r}
# Na correction - default (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069)

# Na correction - owc2204 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "owc2204")

# Na correction - ahs01 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "ahs01")

# Na correction - kam71 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "kam71")

# Na correction - marschdot (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "marschdot")

# Na correction - owc1904 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "owc1904")

# Na correction - owc2004 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "owc2004")

# Na correction - owc2104 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "owc2104")

# Na correction - san96 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "san96")

# Na correction - san04 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "san04")

# Na correction - schlif (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "schlif")

# Na correction - tanna06 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "tanna06")

# Na correction - wet91 (DNA/DNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, correction.ion = "wet91")

# Na correction - default (RNA/RNA)
melting(sequence = "CCAGCCAGUCUCUCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "rnarna",
        Na.conc = 0.069)

# Na correction -  tanna07 (RNA/RNA)
melting(sequence = "CCAGCCAGUCUCUCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "rnarna",
        Na.conc = 0.069, correction.ion = "tanna07")

# Na correction -  wet91 (RNA/RNA)
melting(sequence = "CCAGCCAGUCUCUCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "rnarna",
        Na.conc = 0.069, correction.ion = "wet91")

# Na correction - default (mRNA/RNA)
melting(sequence = "UACGCGUCAAUAACGCUA",
        nucleic.acid.conc = 0.000002, hybridisation.type = "mrnarna",
        Na.conc = 0.069)

# Na correction -  tanna07 (mRNA/RNA)
melting(sequence = "UACGCGUCAAUAACGCUA",
        nucleic.acid.conc = 0.000002, hybridisation.type = "mrnarna",
        Na.conc = 0.069, correction.ion = "tanna07")

# Na correction - default (DNA/RNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnarna",
        Na.conc = 0.069)

# Na correction - wet91 (DNA/RNA)
melting(sequence = "CCAGCCAGTCTCTCC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnarna",
        Na.conc = 0.069, correction.ion = "wet91")
```

### Magnesium corrections

The available correction methods for magnesium concentration are given in **Table 22**.

**Table 22**: Details of the corrections for magnesium concentration
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "mg.correction",
               def = "default", code = "Correction", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

```{r}
# Mg correction - default (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Mg.conc = 0.0015)

# Mg correction - owcmg08 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Mg.conc = 0.0015, correction.ion = "owcmg08")

# Mg correction - tanmg06 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Mg.conc = 0.0015, correction.ion = "tanmg06")

# Mg correction - default (RNA/RNA)
melting(sequence = "CAGCCUCGUCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "rnarna",
        Mg.conc = 0.0015)

# Mg correction -  tanmg07 (RNA/RNA)
melting(sequence = "CAGCCUCGUCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "rnarna",
        Mg.conc = 0.0015, correction.ion = "tanmg07")

# Mg correction - default (mRNA/RNA)
melting(sequence = "UACGCGUCAAUAACGCUA",
        nucleic.acid.conc = 0.000002, hybridisation.type = "mrnarna",
        Mg.conc = 0.0015)

# Mg correction -  tanmg07 (mRNA/RNA)
melting(sequence = "UACGCGUCAAUAACGCUA",
        nucleic.acid.conc = 0.000002, hybridisation.type = "mrnarna",
        Mg.conc = 0.0015, correction.ion = "tanmg07")
```

### Mixed Sodium and Magnesium corrections

The available correction methods for mixed sodium magnesium concentration are given in **Table 23**.

**Table 23**: Details of the corrections for mixed sodium and magnesium concentration
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "NaMg.correction",
               def = "default", code = "Correction", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

```{r}
# Mixed Na & Mg correction - default (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, Mg.conc = 0.0015)

# Mixed Na & Mg correction - owcmix08 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, Mg.conc = 0.0015, correction.ion = "owcmix08")

# Mixed Na & Mg correction - tanmix07 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, Mg.conc = 0.0015, correction.ion = "tanmix07")

# Mixed Na & Mg correction - default (RNA/RNA)
melting(sequence = "CAGCCUCGUCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "rnarna",
        Na.conc = 0.069, Mg.conc = 0.0015)

# Mixed Na & Mg correction - tanmix07 (RNA/RNA)
melting(sequence = "CAGCCUCGUCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "rnarna",
        Na.conc = 0.069, Mg.conc = 0.0015, correction.ion = "tanmix07")

# Mixed Na & Mg correction - default (mRNA/RNA)
melting(sequence = "UACGCGUCAAUAACGCUA",
        nucleic.acid.conc = 0.000002, hybridisation.type = "mrnarna",
        Na.conc = 0.069, Mg.conc = 0.0015)

# Mixed Na & Mg correction - tanmix07 (mRNA/RNA)
melting(sequence = "UACGCGUCAAUAACGCUA",
        nucleic.acid.conc = 0.000002, hybridisation.type = "mrnarna",
        Na.conc = 0.069, Mg.conc = 0.0015, correction.ion = "tanmix07")
```

The ion correction by @owczarzy_predicting_2008 is used by default according to the $\frac{[\textrm{Mg}^{2+}]^{0.5}}{[\textrm{Mon}^{+}]}$ ratio, where $[\textrm{Mon}^{+}] = \textrm{Na}^{+}]+[\textrm{Tris}^{+}]+[\textrm{K}^{+}]$.

If,

* $[\textrm{K}^{+}] = 0$, default sodium correction is used;
* Ratio $<$ 0.22, default sodium correction is used;
* 0.22 $\leq$ Ratio $<$ 6, default mixed Na and Mg correction is used and
* Ratio $\geq$ 6,default magnesium correction is used.

Note that $[\textrm{Tris}^{+}]$ is about half of the total tris buffer concentration. 

### Sodium equivalent concentration methods

The available correction methods for mixed sodium magnesium concentration are given in **Table 24**.

**Table 24**: Details of the methods for computation of sodium equivalent concentration in the presence of other ions
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "Naeq.methods",
               def = "default", code = "Correction", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

```{r}
# Na equivalent concentration method - default (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, Mg.conc = 0.0015)

# Na equivalent concentration method - ahs01 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, Mg.conc = 0.0015, method.Naeq = "ahs01")

# Na equivalent concentration method - mit96 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, Mg.conc = 0.0015, method.Naeq = "mit96")

# Na equivalent concentration method - pey00 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 0.069, Mg.conc = 0.0015, method.Naeq = "pey00")
```

## Denaturing agent corrections

These include melting temperature corrections for concentration of formamide and DMSO.

### DMSO corrections

The available correction methods for DMSO concentration are given in **Table 25**.

**Table 25**: Details of the corrections for DMSO concentration
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "DMSO.methods",
               def = "default", code = "Correction", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

```{r}
# DMSO correction - default (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 1, DMSO.conc = 10)

# DMSO correction - ahs01 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 1, DMSO.conc = 10, correction.DMSO = "ahs01")

# DMSO correction - cul76 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 1, DMSO.conc = 10, correction.DMSO = "cul76")

# DMSO correction - esc80 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 1, DMSO.conc = 10, correction.DMSO = "esc80")

# DMSO correction - mus80 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 1, DMSO.conc = 10, correction.DMSO = "mus81")
```

### Formamide corrections

The available correction methods for formamide concentration are given in **Table 26**.

**Table 26**: Details of the corrections for formamide concentration
```{r, echo=FALSE}
rmeltingtables(file = "Tables.xlsx", sheet = "formamide.methods",
               def = "default", code = "Correction", ref = "Reference")
```

\* Default method for computation.
\begin{center}
\vspace{2pt}
\end{center}

```{r}
# Formamide correction - default (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 1, formamide.conc = 0.06)

# Formamide correction - bla96 (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 1, formamide.conc = 0.06, correction.formamide = "bla96")

# Formamide correction - lincorr (DNA/DNA)
melting(sequence = "CAGCCTCGTCGCAGC",
        nucleic.acid.conc = 0.000002, hybridisation.type = "dnadna",
        Na.conc = 1, formamide.conc = 10, correction.formamide = "lincorr")
```

# Equivalent options in MELTING 5

The options in MELTING 5 command line equivalent to the arguments in `rmelting` are given in **Table 27**.

**Table 27**: Arguments in `rmelting` and their equivalent options in MELTING 5 command line.
```{r, echo=FALSE}
options <- read_excel("Tables.xlsx", sheet = "commands", col_names = TRUE)[1:34, c(2,1)]
options$`MELTING 5` <- paste("-", options$`MELTING 5`, sep = "")
options$rmelting <- paste("`", options$rmelting, "`", sep = "")
colnames(options) <- c("`rmelting`", "MELTING 5 (command line)")
knitr::kable(options)
```

# Batch computation

Melting temperature for multiple nucleic acid duplexes can be computed using the `meltingBatch` function.

```{r}
sequence <- c("CAAAAAG", "CAAAAAAG", "TTTTATAATAAA", "CCATCGCTACC",
              "CAAACAAAG", "CCATTGCTACC", "CAAAAAAAG", "GTGAAC", "AAAAAAAA",
              "CAACTTGATATTATTA", "CAAATAAAG", "GCGAGC", "GGGACC",
              "CAAAGAAAG", "CTGACAAGTGTC", "GCGAAAAGCG")

meltingBatch(sequence, nucleic.acid.conc = 0.0004,
             hybridisation.type = "dnadna", Na.conc = 1)
```

Complementary sequences are computed by default, but need to be specified in case of mismatches, inosine(s) or hydroxyadenine(s) between the two strands.

```{r}
seq <- c("GCAUACG", "CAGUAGGUC", "CGCUCGC", "GAGUGGAG", "GACAGGCUG",
         "CAGUACGUC", "GACAUCCUG", "GACCACCUG", "CAGAAUGUC", "GCGUCGC",
         "CGUCCGG", "GACUCUCUG", "CAGCUGGUC", "GACUAGCUG", "CUCUGCUC",
         "GCGUCCG", "GUCCGCG", "CGAUCAC", "GACUACCUG", "GACGAUCUG")

comp.seq <- c("CGUUUGC", "GUCGGCCAG", "GCGUGCG", "CUCUUCUC", "CUGUGCGAC",
              "GUCGGGCAG", "CUGUUGGAC", "CUGGGGGAC", "GUCUGGCAG", "CGCUGCG",
              "GCUGGCC", "CUGAUAGAC", "GUCGUUCAG", "CUGAGCGAC", "GAGUUGAG",
              "CGCUGGC", "CUGGCGC", "GCUUGUG", "CUGAGGGAC", "CUGCCAGAC")

meltingBatch(sequence = seq, comp.seq = comp.seq, nucleic.acid.conc = 0.0004,
             hybridisation.type = "rnarna", Na.conc = 1,
             method.singleMM = "tur06")
```

# Further reading

Further details about algorithm, formulae and methods are available in the [MELTING 5 documentation](https://www.ebi.ac.uk/biomodels/tools/melting/melting5-doc/melting.html).

# Citing `rmelting`

```{r, echo = FALSE, collapse = TRUE}
detach("package:rmelting", unload = TRUE)
suppressPackageStartupMessages(library(rmelting))
cit <- citation("rmelting")
# yr <- format(Sys.Date(), "%Y")
# cit[1]$year <- yr
# oc <- class(cit)
# 
# cit <- unclass(cit)
# attr(cit[[1]],"textVersion") <- gsub("\\(\\)",
#                                      paste("\\(", yr, "\\)", sep = ""),
#                                      attr(cit[[1]],"textVersion"))
# class(cit) <- oc
cit
```

# Session Info

```{r}
sessionInfo()
```

# References