- sdfdfadqdqaaaaaaaaaaaaaaaaaaa
+
+These last years the cost of sequencing genomes has been greatly
+reduced, and thus more and more genomes are sequenced. Therefore
+automatic annotation tools are required to deal with this continuously
+increasing amount of genomical data. Moreover, a reliable and accurate
+genome annotation process is needed in order to provide strong
+indicators for the study of life\cite{Eisen2007}.
+
+Various annotation tools (\emph{i.e.}, cost-effective sequencing
+methods\cite{Bakke2009}) producing genomic annotations at many levels
+of detail have been designed by different annotation centers. Among
+the major annotation centers we can notice NCBI\cite{Sayers01012011},
+Dogma \cite{RDogma}, cpBase \cite{de2002comparative},
+CpGAVAS \cite{liu2012cpgavas}, and
+CEGMA\cite{parra2007cegma}. Usually, previous studies used one out of
+three methods for finding genes in annoted genomes using data from
+these centers: \textit{alignment-based}, \textit{composition based},
+or a combination of both~\cite{parra2007cegma}. The alignment-based
+method is used when trying to predict a coding gene (\emph{i.e.}.
+genes that produce proteins) by aligning a genomic DNA sequence with a
+cDNA sequence coding an homologous protein \cite{parra2007cegma}.
+This approach is also used in GeneWise\cite{birney2004genewise}. The
+alternative method, the composition-based one (also known
+as \textit{ab initio}) is based on a probabilistic model of gene
+structure to find genes according to the gene value probability
+(GeneID \cite{parra2000geneid}). Such annotated genomic data will be
+used to overcome the limitation of the first method described in the
+previous section. In fact, the second method we propose finds core
+genes from large amount of chloroplast genomes through genomic
+features extraction.
+
+Figure~\ref{Fig1} presents an overview of the entire method pipeline.
+More precisely, the second method consists of three
+stages: \textit{Genome annotation}, \textit{Core extraction},
+and \textit{Features Visualization} which highlights the
+relationships. To understand the whole core extraction process, we
+describe briefly each stage below. More details will be given in the
+coming subsections. The method uses as starting point some sequence
+database chosen among the many international databases storing
+nucleotide sequences, like the GenBank at NBCI \cite{Sayers01012011},
+the \textit{EMBL-Bank} \cite{apweiler1985swiss} in Europe
+or \textit{DDBJ} \cite{sugawara2008ddbj} in Japan. Different
+biological tools can analyze and annotate genomes by interacting with
+these databases to align and extract sequences to predict genes. The
+database in our method must be taken from any confident data source
+that stores annotated and/or unannotated chloroplast genomes. We have
+considered the GenBank-NCBI \cite{Sayers01012011} database as sequence
+database: 99~genomes of chloroplasts were retrieved. These genomes
+lie in the eleven type of chloroplast families and Table \ref{Tab2}
+summarizes their distribution in our dataset.
+
+\begin{figure}[h]
+ \centering
+ \includegraphics[width=0.75\textwidth]{generalView}
+\caption{A general overview of the annotation-based approach}\label{Fig1}
+\end{figure}
+
+Annotation, which is the first stage, is an important task for
+extracting gene features. Indeed, to extract good gene feature, a good
+annotation tool is obviously required. To obtain relevant annotated
+genomes, two annotation techniques from NCBI and Dogma are used. The
+extraction of gene feature, the next stage, can be anything like gene
+names, gene sequences, protein sequences, and so on. Our method
+considers gene names, gene counts, and gene sequence for extracting
+core genes and producing chloroplast evolutionary tree. The final
+stage allows to visualize genomes and/or gene evolution in
+chloroplast. Therefore we use representations like tables,
+phylogenetic trees, graphs, etc. to organize and show genomes
+relationships, and thus achieve the goal of representing gene
+evolution. In addition, comparing these representations with ones
+issued from another annotation tool dedicated to large population of
+chloroplast genomes give us biological perspectives to the nature of
+chloroplasts evolution. Notice that a local database linked with each
+pipe stage is used to store all the informations produced during the
+process.
+
+\input{population_Table}
+
+\subsection{Genome annotation techniques}
+
+For the first stage, genome annotation, many techniques have been
+developed to annotate chloroplast genomes. These techniques differ
+from each others in the number and type of predicted genes (for
+example: \textit{Transfer RNA (tRNA)} and \textit{Ribosomal RNA
+(rRNA)} genes). Two annotation techniques from NCBI and Dogma are
+considered to analyze chloroplast genomes.
+
+\subsubsection{Genome annotation from NCBI}
+
+The objective is to generate sets of genes from each genome so that
+genes are organized without any duplication. The input is a list of
+chloroplast genomes annotated from NCBI. More precisely, all genomes
+are stored as \textit{.fasta} files which consists in a collection of
+protein coding genes\cite{parra2007cegma,RDogma} (gene that produce
+proteins) organized in coding sequences. To be able build the set of
+core genes, we need to preprocess these genomes
+using \textit{BioPython} package \cite{chapman2000biopython}. This
+step starts by converting each genome from FASTA file format to
+GenVision \cite{geneVision} format from DNASTAR. Each genome is thus
+converted in a list of genes, with gene names and gene counts. Gene
+name duplications can be accumulated during the treatment of a genome.
+These duplications come from gene fragments (\emph{e.g.} gene
+fragments treated with NCBI) and from chloroplast DNA sequences. To
+ensure that all the duplications are removed, each list of gene is
+translated into a set of genes. Note that NCBI genome annotation
+produces genes except \textit{Ribosomal (rRNA)} genes.
+
+\subsubsection{Genome annotation from Dogma}
+
+Dogma stands for \textit{Dual Organellar GenoMe Annotator}. It is an
+annotation tool developed at University of Texas in 2004 for plant
+chloroplast and animal mitochondrial genomes. This tool has its own
+database for translating a genome in all six reading frames and
+queries the amino acid sequence database using
+BLAST \cite{altschul1990basic} (\emph{i.e.} Blastx) with various
+parameters. Protein coding genes are identified in an input genome
+using sequence similarity of genes in Dogma database. In addition in
+comparison with NCBI annotation tool, Dogma can produce
+both \textit{Transfer RNAs (tRNA)} and \textit{Ribosomal RNAs (rRNA)},
+verify their start and end positions. Another difference is also that
+there is no gene duplication with Dogma after solving gene
+fragmentation. In fact, genome annotation with Dogma can be the key
+difference when extracting core genes.
+
+The Dogma annotation process is divided into two tasks. First, we
+manually annotate chloroplast genomes using Dogma web tool. The output
+of this step is supposed to be a collection of coding genes files for
+each genome, organized in GeneVision file. The second task is to solve
+the gene duplication problem and therefore we have use two
+methods. The first method, based on gene name, translates each genome
+into a set of genes without duplicates. The second method avoid gene
+duplication through a defragment process. In each iteration, this
+process starts by taking a gene from gene list, searches for gene
+duplication, if a duplication is found, it looks on the orientation of
+the fragment sequence. If it is positive it appends directly the
+sequence to gene files. Otherwise reverse complement operations are
+applied on the sequence, which is then also append to gene files.
+Finally, a check for missing start and stop codons is performed. At
+the end of the annotation process, all the genomes are fully
+annotated, their genes are defragmented, and gene counts are
+available.
+
+\subsection{Core genes extraction}
+
+The goal of this stage is to extract maximum core genes from sets of
+genes. To find core genes, the following methodology is applied.
+
+\subsubsection{Preprocessing}
+
+In order to extract core genomes in a suitable manner, the genomic
+data are preprocessed with two methods: on the one hand a method based
+on gene name and count, and on the other hand a method based on a
+sequence quality control test.
+
+In the first method, we extract a list of genes from each chloroplast
+genome. Then we store this list of genes in the database under genome
+nam and genes counts can be extracted by a specific length command.
+The \textit{Intersection Core Matrix}, described in next subsection,
+is then computed to extract the core genes. The problem with this
+method can be stated as follows: how can we ensure that the gene which
+is predicted in core genes is the same gene in leaf genomes? The
+answer to this problem is that if the sequences of any gene in a
+genome annotated from Dogma and NCBI are similar with respect to a
+given threshold, then we do not have any problem with this
+method. When the sequences are not similar we have a problem, because
+we cannot decide which sequence belongs to a gene in core genes.
+
+The second method is based on the underlying idea: we can predict the
+the best annotated genome by merging the annotated genomes from NCBI
+and Dogma according to a quality test on genes names and sequences. To
+obtain all quality genes of each genome, we consider the following
+hypothesis: any gene will appear in the predicted genome if and only
+if the annotated genes in NCBI and Dogma pass a specific threshold
+of \textit{quality control test}. In fact, the Needle-man Wunch
+algorithm is applied to compare both sequences with respect to a
+threshold. If the alignment score is above the threshold, then the
+gene will be retained in the predicted genome, otherwise the gene is
+ignored. Once the prediction of all genomes is done,
+the \textit{Intersection Core Matrix} is computed on these new genomes
+to extract core genes, as explained in Algorithm \ref{Alg3:thirdM}.
+
+\begin{algorithm}[H]
+\caption{Extract new genome based on gene quality test}
+\label{Alg3:thirdM}
+\begin{algorithmic}
+\REQUIRE $Gname \leftarrow \text{Genome Name}, Threshold \leftarrow 65$
+\ENSURE $geneList \leftarrow \text{Quality genes}$
+\STATE $dir(NCBI\_Genes) \leftarrow \text{NCBI genes of Gname}$
+\STATE $dir(Dogma\_Genes) \leftarrow \text{Dogma genes of Gname}$
+\STATE $geneList=\text{empty list}$
+\STATE $common=set(dir(NCBI\_Genes)) \cap set(dir(Dogma\_Genes))$
+\FOR{$\text{gene in common}$}
+ \STATE $g1 \leftarrow open(NCBI\_Genes(gene)).read()$
+ \STATE $g2 \leftarrow open(Dogma\_Genes(gene)).read()$
+ \STATE $score \leftarrow geneChk(g1,g2)$
+ \IF {$score > Threshold$}
+ \STATE $geneList \leftarrow gene$
+ \ENDIF
+\ENDFOR
+\RETURN $geneList$
+\end{algorithmic}
+\end{algorithm}
+
+\textbf{geneChk} is a subroutine used to find the best similarity score between
+two gene sequences after applying operations like \textit{reverse}, {\it complement},
+and {\it reverse complement}. Algorithm~\ref{Alg3:genechk} gives the outline of
+geneChk subroutine.
+
+\begin{algorithm}[H]
+\caption{Find the Maximum Similarity Score between two sequences}
+\label{Alg3:genechk}
+\begin{algorithmic}
+\REQUIRE $g1,g2 \leftarrow \text{NCBI gene sequence, Dogma gene sequence}$
+\ENSURE $\text{Maximum similarity score}$
+\STATE $score1 \leftarrow needle(g1,g2)$
+\STATE $score2 \leftarrow needle(g1,Reverse(g2))$
+\STATE $score3 \leftarrow needle(g1,Complement(g2))$
+\STATE $score4 \leftarrow needle(g1,Reverse(Complement(g2)))$
+\RETURN $max(score1,score2,score3,score4)$
+\end{algorithmic}
+\end{algorithm}
+
+\subsubsection{Intersection Core Matrix (\textit{ICM})}
+
+To extract core genes, we iteratively collect the maximum number of
+common genes between genomes and therefore during this stage
+an \textit{Intersection Core Matrix} (ICM) is built. ICM is a two
+dimensional symmetric matrix where each row and each column correspond
+to one genome. Hence, an element of the matrix stores
+the \textit{Intersection Score} (IS): the cardinality of the core
+genes set obtained by intersecting one genome with another
+one. Maximum cardinality results in selecting the two genomes having
+the maximum score. Mathematically speaking, if we have $n$ genomes in
+local database, the ICM is an $n \times n$ matrix whose elements
+satisfy:
+\begin{equation}
+score_{ij}=\vert g_i \cap g_j\vert
+\label{Eq1}
+\end{equation}
+\noindent where $1 \leq i \leq n$, $1 \leq j \leq n$, and $g_i, g_j$ are
+genomes. The generation of a new core gene depends obviously on the
+value of the intersection scores $score_{ij}$. More precisely, the
+idea is to consider a pair of genomes such that their score is the
+largest element in ICM. These two genomes are then removed from matrix
+and the resulting new core genome is added for the next iteration.
+The ICM is then updated to take into account the new core gene: new IS
+values are computed for it. This process is repeated until no new core
+gene can be obtained.
+
+We can observe that the ICM is very large due to the amount of
+data. As a consequence, the computation of the intersection scores is
+both time and memory consuming. However, since ICM is a symetric
+matrix we can reduce the computation overhead by considering only its
+triangular upper part. The time complexity for this process after
+enhancement is thus $O(\frac{n.(n-1)}{2})$. Algorithm ~\ref{Alg1:ICM}
+illustrates the construction of the ICM matrix and the extraction of
+the core genes, where \textit{GenomeList} represents the database
+storing all genomes data. At each iteration, it computes the maximum
+core genes with its two genomes parents.
+
+% ALGORITHM HAS BEEN REWRITTEN
+
+\begin{algorithm}[H]
+\caption{Extract Maximum Intersection Score}
+\label{Alg1:ICM}
+\begin{algorithmic}
+\REQUIRE $L \leftarrow \text{genomes sets}$
+\ENSURE $B1 \leftarrow \text{Max Core set}$
+\FOR{$i \leftarrow 1:len(L)-1$}
+ \STATE $score \leftarrow 0$
+ \STATE $core1 \leftarrow set(GenomeList[L[i]])$
+ \STATE $g1 \leftarrow L[i]$
+ \FOR{$j \leftarrow i+1:len(L)$}
+ \STATE $core2 \leftarrow set(GenomeList[L[j]])$
+ \STATE $core \leftarrow core1 \cap core2$
+ \IF{$len(core) > score$}
+ \STATE $score \leftarrow len(core)$
+ \STATE $g2 \leftarrow L[j]$
+ \ENDIF
+ \ENDFOR
+ \STATE $B1[score] \leftarrow (g1,g2)$
+\ENDFOR
+\RETURN $max(B1)$
+\end{algorithmic}
+\end{algorithm}
+
+\subsection{Features visualization}
+
+The goal is to visualize results by building a tree of evolution. All
+core genes generated represent an important information in the tree,
+because they provide ancestor information of two or more
+genomes. Each node in the tree represents one chloroplast genome or
+one predicted core and labelled as \textit{(Genes count:Family name\_Scientific
+names\_Accession number)}. While an edge is labelled with the number of
+lost genes from a leaf genome or an intermediate core gene. Such
+numbers are very interesting because they give an information about
+the evolution: how many genes were lost between two species whether
+they belong to the same family or not. By the principle of
+classification, a small number of genes lost among species indicates
+that those species are close to each other and belong to same family,
+while a large lost means that we have an evolutionary relationship
+between species from different families. To depict the links between
+species clearly, we built a phylogenetic tree showing the
+relationships based on the distances among genes sequences. Many tools
+are available to obtain a such tree, for example:
+PHYML\cite{guindon2005phyml},
+RAxML{\cite{stamatakis2008raxml,stamatakis2005raxml}, BioNJ, and
+TNT\cite{goloboff2008tnt}}. In this work, we chose to use
+RAxML\cite{stamatakis2008raxml,stamatakis2005raxml} because it is
+fast, accurate, and can build large trees when dealing with a large
+number of genomic sequences.
+
+The procedure used to built a phylogenetic tree is as follows:
+\begin{enumerate}
+\item For each gene in a core gene, extract its sequence and store it in the database.
+\item Use multiple alignment tools such as (****to be write after see christophe****)
+to align these sequences with each others.
+\item Use an outer-group genome from cyanobacteria to calculate distances.
+\item Submit the resulting aligned sequences to RAxML program to compute
+the distances and finally draw the phylogenetic tree.
+\end{enumerate}
+
+\begin{figure}[H]
+ \centering \includegraphics[width=0.75\textwidth]{Whole_system} \caption{Overview
+ of the pipeline}\label{wholesystem}
+\end{figure}
+
+\section{Implementation}
+
+The different algorithms have been implemented using Python version
+2.7, on a laptop running Ubuntu~12.04~LTS. More precisely, the
+computer is a Dell Latitude laptop - model E6430 with 6~GiB memory and
+a quad-core Intel core~i5~processor with an operating frequency of
+2.5~GHz. Many python packages such as os, Biopython, memory\_profile,
+re, numpy, time, shutil, and xlsxwriter were used to extract core
+genes from large amount of chloroplast genomes.
+
+\begin{center}
+\begin{table}[b]
+\caption{Type of annotation, execution time, and core genes
+for each method}\label{Etime}
+{\scriptsize
+\begin{tabular}{p{2cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.25cm}p{0.5cm}p{0.2cm}}
+\hline\hline
+ Method & \multicolumn{2}{c}{Annotation} & \multicolumn{2}{c}{Features} & \multicolumn{2}{c}{Exec. time (min.)} & \multicolumn{2}{c}{Core genes} & \multicolumn{2}{c}{Bad genomes} \\
+~ & N & D & Name & Seq & N & D & N & D & N & D \\
+\hline
+Gene prediction & $\surd$ & - & - & $\surd$ & ? & - & ? & - & 0 & -\\[0.5ex]
+Gene features & $\surd$ & $\surd$ & $\surd$ & - & 4.98 & 1.52 & 28 & 10 & 1 & 0\\[0.5ex]
+Gene quality & $\surd$ & $\surd$ & $\surd$ & $\surd$ & \multicolumn{2}{c}{$\simeq$3 days + 1.29} & \multicolumn{2}{c}{4} & \multicolumn{2}{c}{1}\\[1ex]
+\hline
+\end{tabular}
+}
+\end{table}
+\end{center}
+
+\vspace{-1cm}
+
+Table~\ref{Etime} presents for each method the annotation type,
+execution time, and the number of core genes. We use the following
+notations: \textbf{N} denotes NCBI, while \textbf{D} means DOGMA,
+and \textbf{Seq} is for sequence. The first {\it Annotation} columns
+represent the algorithm used to annotate chloroplast genomes, the {\it
+Features} columns mean the kind of gene feature used to extract core
+genes: gene name, gene sequence, or both of them. It can be seen that
+almost all methods need low {\it Execution time} to extract core genes
+from large chloroplast genome. Only the gene quality method requires
+several days of computation (about 3-4 days) for sequence comparisons,
+once the quality genomes are construced it takes just 1.29~minutes to
+extract core gene. Thanks to this low execution times we can use these
+methods to extract core genes on a personal computer rather than main
+frames or parallel computers. The lowest execution time: 1.52~minutes,
+is obtained with the second method using Dogma annotations. The number
+of {\it Core genes} represents the amount of genes in the last core
+genome. The main goal is to find the maximum core genes that simulate
+biological background of chloroplasts. With NCBI we have 28 genes for
+96 genomes, instead of 10 genes for 97 genomes with
+Dogma. Unfortunately, the biological distribution of genomes with NCBI
+in core tree do not reflect good biological perspective, whereas with
+DOGMA the distribution of genomes is biologically relevant. {\it Bad
+genomes} gives the number of genomes that destroy core genes due to
+low number of gene intersection. \textit{NC\_012568.1 Micromonas
+pusilla} is the only genome which destroyed the core genome with NCBI
+annotations for both gene features and gene quality methods.
+
+The second important factor is the amount of memory being used by each
+methodology. Table \ref{mem} shows the memory usage of each
+method. We used a package from PyPI~(\textit{the Python Package
+Index}) named \textit{Memory\_profile} (located at~{\tt
+https://pypi.python.org/pypi}) to extract all the values in
+table~\ref{mem}. In this table, the values are presented in megabyte
+unit and \textit{gV} means genevision~file~format. We can notice that
+the level of memory which is used is relatively low for all methods
+and is available on any personal computer. The different values also
+show that the gene features method based on Dogma annotations has the
+more reasonable memory usage, except when extracting core
+sequences. The third method gives the lowest values if we already have
+the quality genomes, otherwise it will consume far more
+memory. Moreover, the amount of memory used by the third method also
+depends on the size of each genome.
+
+\begin{center}
+\begin{table}[H]
+\caption{Memory usages in (MB) for each methodology}\label{mem}
+{\scriptsize
+\begin{tabular}{p{2.5cm}p{1.5cm}p{1cm}p{1cm}p{1cm}p{1cm}p{1cm}p{1cm}}
+\hline\hline
+Method& & Load Gen. & Conv. gV & Read gV & ICM & Core tree & Core Seq. \\
+\hline
+Gene prediction & ~ & ~ & ~ & ~ & ~ & ~ & ~\\
+\multirow{2}{*}{Gene Features} & NCBI & 15.4 & 18.9 & 17.5 & 18 & 18 & 28.1\\
+ & DOGMA& 15.3 & 15.3 & 16.8 & 17.8 & 17.9 & 31.2\\
+Gene Quality & ~ & 15.3 & $\le$3G & 16.1 & 17 & 17.1 & 24.4\\
+\hline
+\end{tabular}
+}
+\end{table}
+\end{center}
+
+
+
+
+
