---
title: "An introduction to Rbowtie"
date: "`r format(Sys.time(), '%d %B, %Y')`"
bibliography: Rbowtie-refs.bib
author:
  - Michael Stadler
  - Dimos Gaidatzis
  - Anita Lerch
package: Rbowtie
output: 
  BiocStyle::html_document:
    toc_float: true
vignette: >
  %\VignetteIndexEntry{An introduction to Rbowtie}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}  
---

# Introduction
The `r Biocpkg("Rbowtie")` package provides an **R** wrapper around the popular
*bowtie* [@bowtie] short read aligner and around *SpliceMap* [@SpliceMap] a *de novo*
splice junction discovery and alignment tool, which makes use of the *bowtie*
software package.

The package is used by the `r Biocpkg("QuasR")` [@QuasR] bioconductor package to
_qu_antify and _a_nnotate _s_hort _r_eads. We recommend to use the `r Biocpkg("QuasR")`
package instead of using `r Biocpkg("Rbowtie")` directly. The `r Biocpkg("QuasR")`
package provides a simpler interface than `r Biocpkg("Rbowtie")` and covers the
whole analysis workflow of typical ultra-high throughput sequencing experiments,
starting from the raw sequence reads, over pre-processing and alignment, up to
quantification.

# Preliminaries
## Citing *Rbowtie*
If you use `r Biocpkg("Rbowtie")` [@Rbowtie] in your work, you can cite it as follows:
```{r cite, eval=TRUE}
citation("Rbowtie")
```

## Installation
`r Biocpkg("Rbowtie")` is a package for the **R** computing environment and it is
assumed that you have already installed **R**. See the **R** project at
(http://www.r-project.org). To install the latest version of `r Biocpkg("Rbowtie")`,
you will need to be using the latest version of **R**. `r Biocpkg("Rbowtie")` is
part of the Bioconductor project at (http://www.bioconductor.org). To get
`r Biocpkg("Rbowtie")` together with its dependencies you can use
```{r install, eval=FALSE}
if (!require("BiocManager"))
    install.packages("BiocManager")
BiocManager::install("Rbowtie")
```


## Loading of *Rbowtie*
In order to run the code examples in this vignette, the `r Biocpkg("Rbowtie")`
library need to be loaded.
```{r loadLibraries, eval=TRUE}
library(Rbowtie)
```

## How to get help
Most questions about `r Biocpkg("Rbowtie")` will hopefully be answered by the
documentation or references. If you've run into a question which isn't addressed
by the documentation, or you've found a conflict between the documentation and
software itself, then there is an active support community which can offer help.

The authors of the package (maintainer: `r maintainer("Rbowtie")`) always appreciate
receiving reports of bugs in the package functions or in the documentation. The
same goes for well-considered suggestions for improvements. 

Any other questions or problems concerning `r Biocpkg("Rbowtie")` should be posted
to the Bioconductor support site (https://support.bioconductor.org). Users posting
to the support site for the first time should read the helpful posting guide at
(https://support.bioconductor.org/info/faq/). Note that each function in `r Biocpkg("Rbowtie")`
has it's own help page, e.g. `help("bowtie")`. Posting etiquette requires that you
read the relevant help page carefully before posting a problem to the site.

# Example usage for individual Rbowtie functions
Please refer to the `r Biocpkg("Rbowtie")` reference manual or the function documentation
(e.g. using `?bowtie`) for a complete description of `r Biocpkg("Rbowtie")` functions.
The descriptions provided below are meant to give and overview over all functions
and summarize the purpose of each one.

## Build the reference index with `bowtie_build`{#bowtieBuild}
To be able to align short reads to a genome, an index has to be build first using
the function `bowtie_build`. Information about arguments can be found with the help
of the `bowtie_build_usage` function or in the manual page `?bowtie_build`.
```{r bowtieBuildUsage, eval=TRUE}
bowtie_build_usage()
```

`refFiles` below is a vector with filenames of the reference sequence in `FASTA`
format, and `indexDir` specifies an output directory for the index files that will
be generated when calling `bowtie_build`:
```{r bowtieBuild, eval=TRUE}
refFiles <- dir(system.file(package="Rbowtie", "samples", "refs"), full=TRUE)
indexDir <- file.path(tempdir(), "refsIndex")

tmp <- bowtie_build(references=refFiles, outdir=indexDir, prefix="index", force=TRUE)
head(tmp)
```

## Create alignment with `bowtie`
Information about the arguments supported by the `bowtie` function can be obtained
with the help of the `bowtie_usage` function or in the manual page `?bowtie`.
```{r bowtieUsage, eval=TRUE}
bowtie_usage()
```

In the example below, `readsFiles` is the name of a file containing short reads to
be aligned with `bowtie`, and `samFiles` specifies the name of the output file with
the generated alignments.
```{r bowtie, eval=TRUE}
readsFiles <- system.file(package="Rbowtie", "samples", "reads", "reads.fastq")
samFiles <- file.path(tempdir(), "alignments.sam")

bowtie(sequences=readsFiles, 
       index=file.path(indexDir, "index"), 
       outfile=samFiles, sam=TRUE,
       best=TRUE, force=TRUE)
strtrim(readLines(samFiles), 65)
```


## Create spliced alignment with `SpliceMap`
While `bowtie` only generates ungapped alignments, the `SpliceMap` function can be
used to generate spliced alignments. `SpliceMap` is itself using `bowtie`. To use
it, it is necessary to create an index of the reference sequence as described in
\@ref(bowtieBuild). `SpliceMap` parameters are specified in the form of a named
list, which follows closely the configure file format of the original `SpliceMap`
program[@SpliceMap]. Be aware that `SpliceMap` can only be used for reads that are
at least 50bp long.
```{r SpliceMap, eval=TRUE}
readsFiles <- system.file(package="Rbowtie", "samples", "reads", "reads.fastq")
refDir <- system.file(package="Rbowtie", "samples", "refs", "chr1.fa")
indexDir <- file.path(tempdir(), "refsIndex")
samFiles <- file.path(tempdir(), "splicedAlignments.sam")

cfg <- list(genome_dir=refDir,
            reads_list1=readsFiles,
            read_format="FASTQ",
            quality_format="phred-33",
            outfile=samFiles,
            temp_path=tempdir(),
            max_intron=400000,
            min_intron=20000,
            max_multi_hit=10,
            seed_mismatch=1,
            read_mismatch=2,
            num_chromosome_together=2,
            bowtie_base_dir=file.path(indexDir, "index"),
            num_threads=4,
            try_hard="yes",
            selectSingleHit=TRUE)
res <- SpliceMap(cfg)
res
strtrim(readLines(samFiles), 65)
```

# Session information
The output in this vignette was produced under:
```{r sessionInfo}
sessionInfo()
```

# References
