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\newcommand{\thymine}{\textsc{m}\oldstylenums{2}\xspace}
\newcommand{\local}{\textsc{m}\oldstylenums{1}\xspace}
\newcommand{\algo}[1]{\textsc{{#1}}}
\newcommand{\andi}{\algo{andi}\xspace}
\newcommand{\word}[1]{\textsf{\small#1}}
\newcommand{\wchar}[1]{\textsf{\small#1}}
\newcommand{\eco}{\textsc{eco}\oldstylenums{29}\xspace}
\newcommand{\pneu}{\textsc{Pneu}\oldstylenums{3085}\xspace}

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\newtheorem{definition}{Definition}


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\title{Documentation of \algo{andi}}
\subtitle{Rapid Estimation of Evolutionary Distances between Genomes\\ {\small\url{https://github.com/EvolBioInf/andi}}}
\author{Fabian Klötzl\\ \href{mailto:kloetzl@evolbio.mpg.de}{kloetzl@evolbio.mpg.de}}
\date{Version \version, \isodate\today \\
\vspace*{2cm}
\centering\includegraphics[width=0.8\textwidth]{andi_labels.pdf}}

\begin{document}

\maketitle

\section*{Abstract}
This is the documentation of the \andi program for estimating the evolutionary distance between closely related genomes. These distances can be used to rapidly infer phylogenies for big sets of genomes. Because \andi does not compute full alignments, it is so efficient that it scales well up to thousands of bacterial genomes.

This is scientific software. Please cite our paper \cite{andi} if you use \andi in your publication. Also refer to the paper for the internals of \andi. Additionally, there is a Master's thesis with even more in depth analysis of \andi \cite{kloetzl}.

\vspace*{1cm}
\section*{License}
This document is release under the Creative Commons Attribution Share-Alike license. This means, you are free to copy and redistribute this document. You may even remix, tweak and build upon this document, as long as you credit me for the work I've done and release your document under the identical terms. The full legal code is available online: {\small\url{https://creativecommons.org/licenses/by-sa/4.0/legalcode}}.

\tableofcontents

\chapter{Installation} %%%%%

\section{Package Manager}

The easiest way to install \andi is via a package manager. This also handles all dependencies for you.


\noindent Debian and Ubuntu:

\begin{lstlisting}
~ %  sudo apt-get install andi
\end{lstlisting}

\noindent macOS with homebrew:

\begin{lstlisting}
~ %  brew tap brewsci/bio
~ %  brew install andi
\end{lstlisting}

\noindent ArchLinux AUR package with aura:

\begin{lstlisting}
~ %  aura -A andi
\end{lstlisting}

\andi is intended to be run in a \algo{Unix} commandline such as \lstinline$bash$ or \lstinline$zsh$. All examples in this document are also intended for that environment. You can verify that \andi was installed correctly by executing \lstinline$andi -h$. This should give you a list of all available options (see Section~\ref{sec:options}).

\section{Source Package} \label{sub:regular}

To build \andi from source, download the latest \href{https://github.com/EvolBioInf/andi/releases}{release} from GitHub. Please note, that \andi requires the \algo{Gnu Scientific Library} and \algo{libdivsufsort}\footnote{\url{https://github.com/y-256/libdivsufsort}} for optimal performance \cite{divsufsort}.

Once you have downloaded the package, unzip it and change into the newly created directory. 

\begin{lstlisting}
~ %  tar -xzvf andi-0.12.tar.gz
~ %  cd andi-0.12
\end{lstlisting}

\noindent Now build and install \andi.

\begin{lstlisting}
~/andi-0.12 %  ./configure
~/andi-0.12 %  make
~/andi-0.12 %  sudo make install
\end{lstlisting}

\noindent This installs \andi for all users on your system. If you do not have root privileges, you will find a working copy of \andi in the \lstinline$src$ subdirectory. For the rest of this documentation, it is assumed, that \andi is in your \textdollar\lstinline!PATH!.

Now \andi should be ready for use. Try invoking the help.

\begin{lstlisting}
~/andi-0.12 %  Usage: andi [OPTIONS...] FILES...
  FILES... can be any sequence of FASTA files.
  Use '-' as file name to read from stdin.
Options:
  -b, --bootstrap=INT  Print additional bootstrap matrices
      --file-of-filenames=FILE  Read additional filenames from FILE; one per line
  -j, --join           Treat all sequences from one file as a single genome
  -l, --low-memory     Use less memory at the cost of speed
  -m, --model=MODEL    Pick an evolutionary model of 'Raw', 'JC', 'Kimura'; default: JC
  -p FLOAT             Significance of an anchor; default: 0.025
      --progress=WHEN  Print a progress bar 'always', 'never', or 'auto'; default: auto
  -t, --threads=INT    Set the number of threads; by default, all processors are used
      --truncate-names Truncate names to ten characters
  -v, --verbose        Prints additional information
  -h, --help           Display this help and exit
      --version        Output version information and acknowledgments
\end{lstlisting}

\noindent \andi also comes with a man page, which can be accessed via \lstinline$man andi$. % But once you are done with this documentation, you will require it scarcely.

\section{Installing from Git Repository}

To build \andi from the \algo{Git} repo, you will also need the \algo{autotools}. Refer to your OS documentation for installation instructions. Once done, execute the following steps.

\begin{lstlisting}
~ %  git clone git@github.com:EvolBioInf/andi.git
~ %  cd andi
~/andi %  autoreconf -fi -Im4
\end{lstlisting}

\noindent Continue with the \algo{Gnu} trinity as described in Section~\ref{sub:regular}.


\chapter{Usage} %%%%%

The input sequences for \andi should be in \algo{Fasta} format. Any number of files can be passed. Each file may contain more than one sequence.

\begin{lstlisting}
~ %  andi S1.fasta S2.fasta
2
S1        0.0000 0.0979
S2        0.0979 0.0000
\end{lstlisting}

If no file argument is given, \andi reads the input from \algo{stdin}. This makes it convenient to use in \algo{Unix} pipelines.

\begin{lstlisting}
~ %  cat S1.fasta S2.fasta | andi
2
S1        0.0000 0.0979
S2        0.0979 0.0000
\end{lstlisting}

The output of \andi is a matrix in \algo{Phylip} style: On the first line the number of compared sequences is given, \lstinline!2! in our example. Then the matrix is printed, where each line is preceded by the name of the $i$th sequence. Note that the matrix is symmetric and the main diagonal contains only zeros. The numbers themselves are evolutionary distances, estimated from substitution rates.


\section{Input} \label{sec:join}

As mentioned before, \andi reads in \algo{Fasta} files. It recognizes only the four standard bases and is case insensitive (RegEx: \lstinline![acgtACGT]!). All other residue symbols are excluded from the analysis and \andi prints a warning, when this happens.

If instead of distinct sequences, a \algo{Fasta} file contains contigs belonging to a single taxon, \andi will treat them as a unit when switched into \algo{join} mode. This can be achieved by using the \lstinline!-j! or \lstinline!--join! command line switch.

\begin{lstlisting}
~ %  andi --join E_coli.fasta Shigella.fasta
[Output]
\end{lstlisting}

When the \algo{join} mode is active, the file names are used to label the individual sequences. Thus, in \algo{join} mode, each genome has to be in its own file, and furthermore, at least one filename has to be given via the command line.

If not enough file names are provided, \andi will try to read sequences from the standard input stream. This behaviour can be explicitly triggered by passing a single dash (\lstinline$-$) as a file name, which is useful in pipelines.

If \andi seems to take unusually long, or requires huge amounts of memory, then you might have forgotten the \algo{join} switch. This makes \andi compare each contig instead of each genome, resulting in many more comparisons! Since version 0.12 \andi produces a progressmeter on the standard error stream. \andi tries to be smart about when to show or hide the progress bar. You can manually change this behaviour using the \lstinline!--progress! option.

Starting with version 0.11 \andi supports an extra way of input. Instead of passing file names directly to \andi via the commandline arguments, the file names may also be read from a file itself. Using this new \lstinline$--file-of-filenames$ argument can work around limitations imposed be the shell.

The following three snippets have the same functionality.

\begin{lstlisting}
~ %  andi --join *.fasta
[Output]
\end{lstlisting}

\begin{lstlisting}
~ %  ls *.fasta > filenames.txt
~ %  andi --join --file-of-filenames filenames.txt
[Output]
\end{lstlisting}

\begin{lstlisting}
~ %  ls *.fasta | andi --join --file-of-filenames -
[Output]
\end{lstlisting}

\section{Output}

The output of \andi is written to \lstinline$stdout$. This makes it easy to use on the command line and within shell scripts. As seen before, the matrix, computed by \algo{andi}, is given in \algo{Phylip} format \cite{phylip}.

\begin{lstlisting}
~ %  cat S1.fasta S2.fasta | andi
2
S1        0.0000 0.0979
S2        0.0979 0.0000
\end{lstlisting}

If the computation completed successfully, \andi exits with the status code 0. Otherwise, the value of \lstinline$errno$ is used as the exit code. \andi can also produce warnings and error messages for the user's convenience. These messages are printed to \lstinline$stderr$ and thus do not interfere with the normal output.

\section{Options} \label{sec:options}

\andi takes a small number of commandline options, of which even fewer are of interest on a day-to-day basis. If \lstinline$andi -h$ displays a \lstinline$-t$ option, then \andi was compiled with multi-threading support (implemented using \algo{OpenMP}). By default, \andi uses all available processors. However, to restrict the number of threads, use \lstinline$-t$.

\begin{lstlisting}
~ %  time andi ../test/1M.1.fasta -t 1
2
S1        0.0000 0.0995
S2        0.0995 0.0000
./andi ../test/1M.1.fasta  0,60s user 0,01s system 99% cpu 0,613 total
~ %  time andi ../test/1M.1.fasta -t 2
2
S1        0.0000 0.0995
S2        0.0995 0.0000
./andi ../test/1M.1.fasta -t 2  0,67s user 0,03s system 195% cpu 0,362 total
\end{lstlisting}

In the above examples the runtime dropped from \SI{0.613}{\second}, to \SI{0.362}{\second} using two threads. Giving \andi more threads than input genomes leads to no further speed improvement. \, The other important option is \lstinline$--join$ (see Section~\ref{sec:join}).

By default, the distances computed by \andi are \emph{Jukes-Cantor} corrected \cite{jukescantor}. Other evolutionary models are also implemented (Kimura, raw). The \lstinline$--model$ parameter can be used to switch between them.

Since version 0.9.4 \andi includes a bootstrapping method. It can be activated via the \lstinline$--bootstrap$ or \lstinline$-b$ switch. This option takes a numeric argument representing the number of matrices to create. The output can then be piped into \algo{phylip}. For more information on computing support values from distance matrices see \cite{afra}.

\begin{lstlisting}
~ %  andi -b 2 ../test/1M.1.fasta
2
S1        0.0000 0.1067
S2        0.1067 0.0000
2
S1        0.0000 0.1071
S2        0.1071 0.0000
\end{lstlisting}

The original \algo{phylip} only supports distance matrices with names no longer than ten characters. However, this sometimes leads to problems with long accession numbers. Starting with version 0.11 \andi prints the full name of a sequence, even if it is longer than ten characters. If your downstream tools have trouble with this, use \lstinline$--truncate-names$ to reimpose the limit.

Also new in version 0.11 is the \lstinline$--file-of-filenames$ option. See Section~\ref{sec:join} for details.

\section{Example: \algo{eco29}}

Here follows a real-world example of how to use \algo{andi}. It makes heavy use of the commandline and tools like \algo{Phylip}. If you prefer \algo{R}, check out this excellent blog post by Kathryn Holt.\footnote{\url{http://holtlab.net/2015/05/08/r-code-to-infer-tree-from-andi-output/}}

As a data set we use \algo{eco29}; 29 genomes of \textit{E. Coli} and \textit{Shigella}. You can download the data from here: {\small{\url{http://guanine.evolbio.mpg.de/andi/eco29.fasta.gz}}}. The genomes have an average length of 4.9~million nucleotides amounting to a total \SI{138}{\mega\byte}.

\algo{eco29} comes a single \algo{fasta} file, where each sequence is a genome. To calculate their pairwise distances, enter

\begin{lstlisting}
~ % andi eco29.fasta > eco29.mat
andi: The input sequences contained characters other than acgtACGT. These were automatically stripped to ensure correct results.
\end{lstlisting}

\noindent The \algo{eco29} data set includes non-canonical nucleotides, such as \word{Y}, \word{N}, and \word{P}, which get stripped from the input sequences. The resulting matrix is stored in \lstinline$eco29.mat$; Here is a small excerpt:

\begin{lstlisting}
~ % head -n 5 eco29.mat | cut -d ' ' -f 1-5
29
gi|563845 0.0000e+00 1.8388e-02 1.8439e-02 2.6398e-02
gi|342360 1.8388e-02 0.0000e+00 4.4029e-04 2.6166e-02
gi|300439 1.8439e-02 4.4029e-04 0.0000e+00 2.6123e-02
gi|261117 2.6398e-02 2.6166e-02 2.6123e-02 0.0000e+00
\end{lstlisting}

\noindent From this we compute a tree via neighbor-joining using a \algo{Phylip} wrapper called \algo{Embassy}.\footnote{\url{http://emboss.sourceforge.net/embassy/\#PHYLIP}}

\begin{lstlisting}
~ % fneighbor -datafile eco29.mat -outfile eco29.phylipdump
\end{lstlisting}
\noindent To make this tree easier to read, we can midpoint-root it.
\begin{lstlisting}
~ % fretree -spp 29 -intreefile eco29.treefile -outtreefile eco29.tree <<EOF
M
X
Y
R
EOF
\end{lstlisting}

\noindent The file \lstinline$eco29.tree$ now contains the tree in Newick format. This can be plotted using \cite{figtree}

\begin{lstlisting}
~ % figtree eco29.tree &
\end{lstlisting}

\noindent to yield

\begin{figure}[h]
  \centering\includegraphics[width=0.8\textwidth]{andi_labels.pdf}
\end{figure}


\chapter{Warnings and Errors}

Here be an explanation of all possible errors. Other errors may occur and are due to the failure of underlying functions (e.\,g.~\lstinline$read(3)$). All warning messages are printed to \lstinline$stderr$. Most errors are non-recoverable and will result in \andi exiting with a non-zero state.

\section{Sequence Related Messages}

\subsection*{Unexpected Character}

\andi is pretty pedantic about the formatting of \algo{FASTA} files. If you violate the syntax, \andi will print the file name, the line and the problematic character. These errors are non-recovering, meaning no further sequences are read from the invalid file. The checks are implemented by the \href{https://github.com/kloetzl/pfasta}{\algo{pfasta}} library.


\subsection*{Non acgtACGT Nucleotides Stripped}

Our models of genome evolution (JC, Kimura) only work on the four canonical types of nucleotides. All others are stripped from the sequences. This can be ignored in most cases.

\subsection*{Too Short Sequence}

\andi was designed for big data sets of whole genomes. On short sequences the distance estimates are inaccurate. Use an multiple sequence alignment instead.

\subsection*{Too Long Sequence}

\algo{libdivsufsort} limits the length of a sequence to 31 bits. That count includes the reverse complement. So the technical limit for a sequence analysis is $2^{30} = 1.073.741.824$. Unfortunately, that excludes (full) human and mice genomes. Per-chromosome analysis works just fine.

\subsection*{Empty Sequence}

One of the given sequences contained either no nucleotides at all, or only non-canonical ones.

\subsection*{Less than two sequences given}

As \andi tries to compare sequences, at least two need to be supplied. Note that \andi may have regarded some of your given sequences as unusable.

\subsection*{Maximum Number of Sequences}

The maximum number of sequences \andi can possible compare is huge (roughly $457.845.052$). I doubt anyone will ever reach that limit. Please send me a mail, if you do.

\section{Technical Messages}

\subsection*{Out of Memory}

If \andi runs out of memory, it gives up. Either free memory, run \andi on a bigger machine, try the \lstinline$--low-memory$ mode or reduce the number of threads.

\subsection*{RNG allocation}

Some technical thing failed. If it keeps failing repeatedly, file a bug.

\subsection*{Bootstrapping failed}

This should not happen.

\subsection*{Failed index creation}

This should not happen, either.

\subsection*{Skipped and ignored Arguments}

Some command line parameters of \andi require arguments. If these are not of the expected type, a warning is given. See Section~\ref{sec:options} for their correct usage.


\section{Output-related Warnings}

As the input sequences get more evolutionary divergent, \andi finds less homologous anchors. With less anchors, less nucleotides are considered homologous between two sequences. If no anchors are found, comparison fails and \lstinline!nan! is printed instead. See our paper and especially Figure~2 for details.

\subsection*{NaN}

No homologous sections were found. Your sequences are very divergent ($d>0.5$) or sprout a lot of indels that make comparison difficult.

\subsection*{Little Homology}

Very few anchors were found and thus only a tiny part of the sequences is considered homologous. Expect that the given distance is erroneous.

\subsection*{Too long name}

If you added the \lstinline$--truncate-names$ switch and an input name is longer than ten characters, you will receive this warning.

\chapter{DevOps} %%%%%

\andi is written in C/C++; mostly C99 with some parts in C++11. The sources are released on \algo{GitHub} as \emph{free software} under the \textsc{Gnu General Public License version~3} \cite{GPL}. Prebundled packages using \algo{autoconf} are also available, with the latest release being {\version} at the time of writing.

If you are interested in the internals of \algo{andi}, consult the paper \cite{andi} or my Master's thesis \cite{kloetzl}. Both explain the used approach in detail. The latter emphasizes the used algorithms, data structures and their efficient implementation.

\section{Dependencies}

Here is a complete list of dependencies required for developing \algo{andi}.

\begin{itemize}
  \item A C and a C++11 compiler,
  \item the \algo{autotools},
  \item the \algo{Gnu Scientific Library},
  \item \algo{Pdflatex} with various packages for the manual,
  \item \algo{Git},
  \item \algo{glib2} for the unit tests,
  \item \algo{doxygen},
  \item and \algo{libdivsufsort}.
\end{itemize}


\section{Code Documentation}

\emph{Every} function in \andi is documented using \algo{doxygen} style comments. To create the documentation run \lstinline$make code-docs$ in the main directory. You will then find the documentation under \lstinline$./docs$.


\section{Unit Tests}

The unit tests are located in the \andi repository under the \lstinline$./test$ directory. Because they require \algo{glib2}, and a C++11 compiler, they are deactivated by default. To enable them, execute

\begin{lstlisting}
~/andi %   ./configure --enable-unit-tests
\end{lstlisting}

\noindent during the installation process. You can then verify the build via 

\begin{lstlisting}
~/andi %   make check
\end{lstlisting}

\noindent The unit tests are also checked each time a commit is sent to the repository. This is done via \algo{TravisCI}.\footnote{\url{https://travis-ci.org/EvolBioInf/andi}} Thus, a warning is produced, when the builds fail, or the unit tests did not run successfully. Currently, the unit tests cover more than 75\% of the code. This is computed via the \algo{Travis} builds and a service called \algo{Coveralls}.\footnote{\url{https://coveralls.io/r/EvolBioInf/andi}}

\section{Known Issues}

These minor issues are known. I intend to fix them, when I have time.

\begin{enumerate}
  \item This code will not work under Windows. At two places Unix-only code is used: filepath-separators are assumed to be \lstinline$/$ and file-descriptors are used for I/O.
  \item Unit tests for the bootstrapped matrices are missing.
  \item Cached intervals are sometimes not “as deep as they could be”. If that got fixed \lstinline$get_match_cache$ could bail out on \lstinline$ij.lcp < CACHE_LENGTH$. However the \lstinline$esa_init_cache$ code is the most fragile part and should be handled with care.
\end{enumerate}


\section{Creating a Release}

A release should be a stable version of \andi with significant improvements over the last version. dotdot releases should be avoided.

%\subsection{Preparing a new Release}

Once \andi is matured, the new features implemented, and all tests were run, a new release can be created. First, increase the version number in \lstinline$configure.ac$. Commit that change in git, and tag this commit with \lstinline$vX.y$. Tags should be annotated and signed, if possible. This manual then needs manual rebuilding.

Ensure that \andi is ready for packaging with \algo{autoconf}.

\begin{lstlisting}
~ % make distcheck
make  dist-gzip am__post_remove_distdir='@:'
make[1]: Entering directory `/home/kloetzl/Projects/andi'
if test -d "andi-0.9.1-beta"; then find "andi-0.9.1-beta" -type d ! -perm -200 -exec chmod u+w {} ';' && rm -rf "andi-0.9.1-beta" || { sleep 5 && rm -rf "andi-0.9.1-beta"; }; else :; fi
test -d "andi-0.9.1-beta" || mkdir "andi-0.9.1-beta"
 (cd src && make  top_distdir=../andi-0.9.1-beta distdir=../andi-0.9.1-beta/src \
     am__remove_distdir=: am__skip_length_check=: am__skip_mode_fix=: distdir)

... Loads of output ...

=================================================
andi-0.9.1-beta archives ready for distribution: 
andi-0.9.1-beta.tar.gz
=================================================
\end{lstlisting}

If the command does not build successfully, no tarballs will be created. This may necessitate further study of \algo{autoconf} and \algo{automake}.

Also verify that the recent changes did not create a performance regression. This includes testing both ends of the scale: \eco and \pneu. Both should be reasonable close to previous releases.

Create another commit, where you set the version number to the next release (e.\,g., \lstinline$vX.z-beta$). This assures that there is only one commit and build with that specific version.

\backmatter
%\addcontentsline{toc}{chapter}{Bibliography}
\bibliography{references}

\end{document}