| segment_duplication | segment duplication | DNA sequences present in multiple locations within a genome that share high level of sequence identity |
| subfunctionalization | subfunctionalization | Fate of a duplicate gene which gets a part of the original gene function, the function being shared among multiple duplicates |
[[latex:lettrine][D]]uplicate genes represent an important fraction of Eukaryotic genes: It is estimated that between 46% and 65.5% of human genes could be considered as duplicate[fn:: The estimate vary strongly depending on the criteria in use, because ancient duplication event may be hard to detect.] [cite:@correaTransposableElementEnvironment2021].
#+caption[Different types of duplication]: Different types of duplication. (A) Whole genome duplication. (B) An unequal crossing-over leads to a duplication of a fragment of a chromosome. (C) In tandem duplication, two (set of) genes are duplicated one after the other. (D) Retrotransposon enables retroduplication: a RNA transcript is reverse transcribed and inserted back without introns and with a polyA tail in the genome. (E) A DNA transposon can acquire a fragment of a gene. (F) Segmental duplication corresponds to long stretches of duplicated sequences with high identity. Adapted from [cite:@lallemandOverviewDuplicatedGene2020] (fig. 1)
\caption[Different types of duplication]{\label{fig:gene-duplication-mechanisms}Different types of duplication. (A) Whole genome duplication. (B) An unequal crossing-over leads to a duplication of a fragment of a chromosome. (C) In tandem duplication, two (set of) genes are duplicated one after the other. (D) Retrotransposon enables retroduplication: a RNA transcript is reverse transcribed and inserted back without introns and with a polyA tail in the genome. (E) A DNA transposon can acquire a fragment of a gene. (F) Segmental duplication corresponds to long stretches of duplicated sequences with high identity. (Adapted from \textcite{lallemandOverviewDuplicatedGene2020} (fig. 1)).}
Multiple mechanisms may lead to a gene duplication. Their effect ranges from the duplication of the whole genome to the duplication of a fragment of a gene.
A striking example is probably /Triticum aestivum/ (wheat) which is hexaploid due to hybridisation events [cite:@golovninaMolecularPhylogenyGenus2007a].
We distinguish two kinds of glspl:polyploidisation, based on the origin of the duplicate genome: (i) Gls:allopolyploidisation occurs when the supplementary chromosomes come from a divergent species. This is the case for the /Triticum aestivum/ hybridisation, which consisted in the union of the chromosome set of a /Triticum/ species with that of an /Aegilops/ species. (ii) Gls:autopolyploidisation consists in the hybridisation or duplication of the whole genome within the same species.
Another source of gene duplication relies on unequal crossing-over. During cell division, a crossing-over occurs when two chromatids exchange fragments of chromosome. If the cleavage of the two chromatids occurs at different positions, the shared fragments may have different lengths. Homologous recombination of such uneven crossing-over leads to the incorporation of a duplicate region, as depicted in cref:fig:gene-duplication-mechanisms (B, C).
This mechanism leads to the duplication of the whole set of genes present in the fragment. These duplicate genes locate one set after the other: we call them gls:TAG. Gls:TAG are the kind of gene duplication we will be particularly interested in during this internship.
These transposons typically contain a reverse transcriptase gene. This enzyme proceeds in the reverse transcription of an mRNA transcript into its reverse complementary DNA sequence which can then insert elsewhere in the genome.
More generally, gls:retroduplication refers to the duplication of a sequence through reverse transcription of a RNA transcript. Genes duplicated through retroduplication lose their intronic sequences and bring a polyA tail with them in their new locus (cref:fig:gene-duplication-mechanisms (D)).
DNA transposons are another kind of transposable elements whose transposition mechanism can also lead to gene duplication.
This type of transposable element moves in the genome through a mechanism known as "cut-and-paste".
A typical DNA transposon contains a transposase gene. This enzyme recognizes two sites surrounding the donnor transposon sequence in the chromosome resulting in a DNA cleavage and an excision of the transposon. The transposase can then insert the transposon at a new genome locus. A transposon may bring a fragment of a gene during its transposition in the new locus (cref:fig:gene-duplication-mechanisms (E)), leading to the duplication of this fragment.
Finally, glspl:segment_duplication, also called /low copy repeats/ are long stretches of DNA with high identity score ([[cref:fig:gene-duplication-mechanisms]] (F)). Their exact duplication mechanism remains unclear [cite:@lallemandOverviewDuplicatedGene2020]. They may come from an accidental replication, distinct from an uneven cross-over or a double stranded breakage.
Transposable elements may well be involved in the mechanism, as a high enrichment of transposable elements is found next to duplicate segment extremities, in /Drosophila/ [cite:@lallemandOverviewDuplicatedGene2020].
,#+caption[Fate of duplicate genes]: Fate of duplicate genes. An original gene with four functions is duplicated. Its two copies may both keep the original functions (functional redoundancy). The original functions may split between the different copies (subfunctionalization). One of the copy may acquire a new function (neofunctionalization). It may also degenerate and lose its original functions (pseudogenization). Adapted from [[https://commons.wikimedia.org/wiki/File:Evolution_fate_duplicate_genes_-_vector.svg][Smedlib]], [[https://creativecommons.org/licenses/by-sa/4.0][CC BY-SA 4.0]] via Wikimedia Commons.
\caption[Fate of duplicate genes]{\label{fig:fate-duplicate-genes} Fate of duplicate genes. An original gene with four functions is duplicated. Its two copies may both keep the original functions (functional redoundancy). The original functions may split between the different copies (subfunctionalization). One of the copy may acquire a new function (neofunctionalization). It may also degenerate and lose its original functions (pseudogenization). (Adapted from \href{https://commons.wikimedia.org/wiki/File:Evolution_fate_duplicate_genes_-_vector.svg}{Smedlib}, \href{https://creativecommons.org/licenses/by-sa/4.0}{CC BY-SA 4.0}, via Wikimedia Commons).}
In his book /Evolution by Gene Duplication/, Susumu [[latex:textsc][Ohno]] proposed that gene duplication plays a major role in species evolution [cite:@ohnoEvolutionGeneDuplication1970], because it provides new genetic materials to build on new phenotypes while keeping a backup gene for the previous function.
Indeed, duplicate genes evolve after duplication: they may be inactivated, and become glspl:pseudogene; they may be deleted or conserved, and if conserved, the may or may not acquire a new function.
As genome evolves, duplicate genes may be inactivated and become pseudogenes. These pseudogenes keep a gene-like structure which degrades as and when further genome modifications occur but they are no longer expressed.
# *** Neofunctionalization
After duplication, the new gene copy may gain a new function. We call this possible outcome gls:neofunctionalization.
For instance, the current set of olfactory receptor genes result from several duplication and deletion events (for /Drosophila/, see: [cite/t:@nozawaEvolutionaryDynamicsOlfactory2007]), after which each duplicate olfactory gene specialized in the detection of a particular chemical compound.
# *** Subfunctionalization
Two duplicate genes with the same original function may encounter a gls:subfunctionalization: each gene conserves only one part of the function.
# *** Functional redundancy
Another possibility is that the two gene copies keep the ancestral function, resulting in a functional redoundancy. In this case the quantity of gene product may increase.
The underlying question of FTAG Finder is the study of the evolutionary fate of duplicate genes in Eukaryotes.
Duplicate genes are
** Extend the existing FTAG Finder Galaxy pipeline
Galaxy is a web-based platform for running accessible data analysis pipelines, first designed for use in genomics data analysis [cite:@goecksGalaxyComprehensiveApproach2010].
Last year, Séanna [[latex:textsc][Charles]] worked on the Galaxy version of the FTAG Finder pipeline during her M1 internship [cite:@charlesFinalisationPipelineFTAG2023]. I will continue this work.
FTAG Finder is currently deployed on the server of the /Laboratoire de Mathématiques et Modélisation d'Évry/[fn:: [[http://stat.genopole.cnrs.fr/galaxy]] ].
** Port FTAG Finder pipeline on a workflow manager
Another objective of my internship will be to port FTAG Finder on a workflow manager better suited to larger and more reproducible analysis.
We will have to make a choice for the tool we will use.
The two main options being Snakemake and Nextflow. Snakemake is a python powered workflow manager based on rules /à la/ GNU Make [cite:@kosterSnakemakeScalableBioinformatics2012]. Nextflow is a groovy powered workflow manager, which rely on the data flows paradigm [cite:@ditommasoNextflowEnablesReproducible2017]. Both are widely used in the bioinformatics community. Their use have been on the rise since they came out in 2012 and 2013 respectively [cite:@djaffardjyDevelopingReusingBioinformatics2023].
# #+begin_export latex
# \fladdtab{
# \begin{tabular}{ccc}
# \toprule
# & List ref & List $L$ \\
# \midrule
# related to $go$ & $a$ & $b$ \\
# unrelated & $c$ & $d$ \\
# \bottomrule
# \end{tabular}
# \caption{\label{tab:fisher-test-contigency-table}Contingency table for a Fisher exact test on gene lists}
# }
# #+end_export
* Methodological approaches
** Duplicate gene detection method used in FTAG Finder
#+caption[Schematic representation of TAG definitions]: Schematic representation of TAG definitions. Several genes are represented on a linear chromosome. The red box represent a singleton gene. Orange boxes represent a TAG with two duplicate genes seperated by 7 other genes ($\\mathrm{TAG}_7$). Four green boxes constitute a TAG, the gene at the extremities are seperated by three genes ($\\mathrm{TAG}_3$). The two blue boxes represents a TAG with two genes next to each other ($\\mathrm{TAG}_0$). The bended edges represents the homology links between each pair of genes within a TAG.
\caption[Schematic representation of TAG definitions]{\label{fig:tag-definitions} Schematic representation of TAG definitions. Several genes are represented on a linear chromosome. The red box represent a singleton gene. Orange boxes represent a TAG with two duplicate genes seperated by 7 other genes ($\mathrm{TAG}_7$). Four green boxes constitute a TAG, the gene at the extremities are seperated by three genes ($\mathrm{TAG}_3$). The two blue boxes represents a TAG with two genes next to each other ($\mathrm{TAG}_0$). The bended edges represents the homology links between each pair of genes within a TAG.}}
Different methods exists to detect duplicate genes. These methods depend on the type of duplicate genes they target and vary on computation burden as well as in the ease of use (for a review, see [cite/t:@lallemandOverviewDuplicatedGene2020]).
Paralogs are homologous genes derived from a duplication event. We can identify them as homologous genes coming from the same genome, or as homologous genes between different species once we filtered out gls:orthologues (homologous genes derived from a speciation event).
The sequence similarity can be tested with a sequence alignment tool, such as =BLAST= [cite:@altschulBasicLocalAlignment1990], =Psi-BLAST=, and =HMMER3= [cite:@johnsonHiddenMarkovModel2010], or =diamond= [cite:@buchfinkSensitiveProteinAlignments2021]. These tools are heuristic algorithms, which means they may not provide the best results, but do so way faster than exact algorithms, such as the classical Smith and Waterman algorithm [cite:@smithIdentificationCommonMolecular1981] or its optimized versions =PARALIGN= [cite:@rognesParAlignParallelSequence2001] or =SWIMM=.
Developed in the LaMME laboratory, the FTAG Finder (Families and Tandemly Arrayed Genes Finder) pipeline is a simple pipeline targeting the detection of gls:TAG based on the sequence of the proteome of single species [cite:@bouillonFTAGFinderOutil2016].
The pipeline proceeds in three steps. First, it estimates the homology links between each pair of genes. Then, it deduces the gene families. Finally, it searches for gls:TAG, relying on the position of genes belonging to the same family.
In this step, FTAG Finder uses =BLAST= (Basic Local Alignment Search Tool) [cite:@altschulBasicLocalAlignment1990] with an "all against all" search on the proteome.
Several =BLAST= metrics can be used as an homology measure, such as bitscore, identity percentage, E-value or a variation on these. The choice of metrics can affect the results of graph clustering in the following step, and we should therefore chose them carefully [cite:@gibbonsEvaluationBLASTbasedEdgeweighting2015].
Based on the homology links between each pair of genes, we construct an undirected weighted graph whose vertices correspond to genes and whose edges corresponds to homology links between them.
We apply a graph clustering algorithm on the homology gene graph in order to infer the gene families corresponding to densely connected communities of vertices.
FTAG Finder proposes three graph clustering algorithm alternatives: single linkage, Markov Clustering [cite:@vandongenNewClusterAlgorithm1998] or Walktrap [cite:@ponsComputingCommunitiesLarge2005].
For a given chromosome, the tool seeks genes belonging to the same family and located close to each other. The tool allows a maximal number of genes between the homologous genes, with a parameter set by the user. Cref:fig:tag-definitions is a schematic representation of some possible gls:TAG positioning on a genome associated with their definition in this FTAG Finder step.
FTAG Finder output consist mostly in list of genes, corresponding to TAG of various definition. These list can be subsequently used as the basis of more specific statistical analysis.
The gls:GO describes biological concepts across three main classes: Cellular Component, Molecular Function and Biological Process. It describes a controlled vocabulary of concepts and the relationships between them. We can link genes with function annotation with particular GO terms. We can then perform an GO enrichment analysis to assess whether a particular GO term is over-represented in a particular gene list, compared to another. To do so, we can use a Fisher exact test, using the FDR (False Discovery Rate) control procedure of [[latex:textsc][Benjamini]] and [[latex:textsc][Hocheberg]].
# Let $go$ be a GO term. We construct a contingency matrix based on the count of genes associated with this GO term (or associated with one of its brother GO term) for the reference gene list and the list of interest (here, the list of genes in a TAG) (see cref:tab:fisher-test-contigency-table).
*** Are TAG located preferentially on specific chromosome region?
The concordance of two genes of a TAG falls in three possible cases: either both genes are on the same strand (\(\rightarrow \rightarrow\)), either they have a divergent orientation (\(\leftarrow \rightarrow\)), or a convergent one (\(\rightarrow \leftarrow\)). Graham conjectured that genes of a TAG that are close to each other would be more likely to share the same orientation, and it seems to be effectively the case [cite:@shojaRoadmapTandemlyArrayed2006].
# To test this, we can use a $\Chi^2$ test of goodness of fit or a Student $t$-test.
[cite/t:@le-hoangEtudeTranscriptomiqueGenes2017] started analyzing the impact of parameter choice on FTAG Finder results. A more detailed benchmark of FTAG Finder in comparison with other methods on some controlled test dataset might be of particular interest.
This would pose the challenge of homogenization of the outputs of the different methods.
Duplicate genes is an important feature of Eukaryotic genomes. They contribute to the plasticity of genome, hence to the capacity of species to evolve.
Several mechanisms may lead to gene duplication. Among them, an unequal crossing-over leads to the formation of Tandemly Arrayed Genes (TAG) corresponding to homologous genes located one set after the other on the same chromosome.
There are multiple methods for detecting duplicate genes from sequences. These methods vary in terms of the particular gene duplication mechanism they target, computational efficiency and ease of use.
FTAG Finder is a simple Galaxy pipeline aiming at the detection of families of duplicate genes and the identification of TAG based on the proteome of a single species. FTAG Finder is developed in the /Laboratoire de Mathématiques et Modélisation d'Évry/, where I will do my internship.
On the one hand, the aim of my internship is to extend the current Galaxy implementation of FTAG Finder with new export lists best suited to the analysis requirements of the laboratory. On the other hand, the objective of my internship will be to port the Galaxy pipeline on another scientific workflow manager better suited to reproducible analyses such as Snakemake and Nextflow.
Then, the updated version of the FTAG Finder pipeline will be used to perform an analysis on the TAG of a model species, to assess its proper behavior. A benchmark of the pipeline will probably be run to compare the FTAG Finder with alternative published methods targetting duplicate genes and TAG in particular.
MCL uses two operations on a stochastic matrix representation $M$ of the graph first derived from the adjacency matrix, namely /expansion/ and /inflation/. Expansion consists in elevating the matrix to a power $r$, and subsequently scaling its columns so that they sum to 1 again. The image of the inflation operator $\Gamma_r$ is defined as
The application of both operator iteratively eventually ends up in a partition of the initial graph's edges into clusters of closely connected nodes (corresponding, in our case to gene families).
