A Review of Pangenome Tools and Recent Studies

With the advance of sequencing technologies, the landscape of genomic analysis has been transformed, by moving from single strain to species (or even higher taxa)-wide genomic resolution, toward the direction of capturing the “ totality ” of life diversity; from this scienti ﬁ c advance and curiosity, the concept of “ pangenome ” was born. Herein we will review, from practical and technical implementation, existing projects of pangenome analysis, with the aim of providing the reader with a snapshot of useful tools should they need to embark on such a pangenomic journey.


Introduction
Almost 15 years ago, Tettelin et al. (2005) conceived the concept of pangenome, in an attempt to describe and model the genomic totality of a taxa (species, serovar, phylum, kingdom, etc.) of interest. Since then the nomenclature of this concept became fairly wide to accommodate words like pangenome, core and dispensable genes, strain-specific genes Tettelin et al. 2005), supragenome, distributed and unique genes (Lapierre and Gogarten 2009), and flexible regions (Rodriguez-Valera and Ussery 2012). Simply put, using the original definition, the core-genome describes the set of sequences shared by all members of the taxa of interest, the dispensable genome captures a subset of sequences shared by some members of the group (dictating the diversity of the group: alternative biochemical pathways, niche adaptation, antibiotic resistance, etc.) while the pangenome is simply the union of core and dispensable genomes (describing the totality of taxa at the level of sequence datasets).
The exponential growth of genomic databases started in 1995 with Haemophilus influenzae being the first complete genome project (Fleischmann et al. 1995). Today, as of August 2018, 110,660 complete whole-genome sequencing projects-of which 87% are bacteria-and 15,066 finished whole-genome sequencing projects (Mukherjee et al. 2017) are available in the public domain. These fueled the interest of many researchers to carry out pangenome analysis at every conceivable phylogenetic resolution level (Table 1), exploiting various modeling frameworks, assumptions, and underlying homology search engines.
A pivotal work in terms of phylogenetic resolution was carried out by Lapierre and Gogarten (2009), showing that on average in the largest bacterium group analyzed so far, the core gene set accounts only for 8% of the pangenome.
The pangenome concept can be implemented either in reverse or in forwardthinking approaches; in the first case, we are interested to capture the genomic diversity of the group of interest, while in the second case we are more interested in exploring and predicting from a pragmatic perspective what is the minimum number of genome sequences required to capture the totality of the group. Obviously, limited or sparse datasets might lead to erroneous conclusions; therefore, it was recommended (Vernikos et al. 2015) that the minimum number of genomes to analyze be at least five.
The lifestyle of the species of interest is one of the parameters strongly dictating the distribution shape of the pangenome; for example, if by recurring addition of group members, the pangenome continues to grow, we are analyzing an open pangenome (such examples include human pathogens and environmental bacteria) (Hiller et al. 2007;Tettelin et al. 2008). On the other hand, if the group complexity is exhausted very fast even from the analyses of a handful of group members then we are dealing with a closed pangenome whereby we only need few representatives to describe the totality of the sequence variability.

Technical Implementation
In pangenome analysis, the sequence unit for the modeling can be anything from ORFs, genes, clusters of orthologous groups COGs (Tatusov et al. 1997), coding sequences (CDS), proteins, arbitrary sequence chunks, concatenated gene or protein entities, etc.
Practical aspects of consideration that directly influence the validity of the conclusions drawn, include how quickly is expected a pangenome to grow and reach a plateau (open or close pangenome), the parameters that determine in the search engine the orthologous sequences and thereby directly affect the pool of core and dispensable sequence entities, the mathematical model and the applied distribution of forecasting the evolution of the pangenome and core-genome size. Another limiting factor, as the number of genomes becomes higher and higher, is the scalability of all possible genome addition permutations, since the total number of comparisons needed is described from the following function: where C is the total number of comparisons, and N is the total number of genomes. A workaround to an exhaustive approach is a method of subsampling (Vernikos et al. 2015) the total number of comparisons needed; comparisons are randomly selected making sure that each genome undergoes the same number of comparisons; the trick here is to set the number of possible comparisons to a number that will optimally balance the existing computational power and the target dataset size. Indeed, observations from limited in size datasets, showed that even extreme sampling is still able to model reliably the pangenome bypassing the need to follow an exhaustive all-against-all comparison ( Fig. 1) (Vernikos et al. 2015). Additional optimizations can be achieved by exploiting alternative (to the original exponential  Tettelin et al. (2008), Eppinger et al. (2010Eppinger et al. ( , 2011, Mongodin et al. (2013) and Riley et al. (2012).
Recently several stand-alone or server-based suites have become available for pangenome analysis; in the next paragraphs, we will review the most promising and interesting initiatives. See also Table 2 for additional details. (a) Number of new genes detected for adding a genome g to g À 1 genomes. Red bubbles: 1016 points for the total number of comparisons (no subsampling). Blue bubbles: 600 points (subsampling, multiplicity of 15). Green bubbles: 248 points (subsampling, multiplicity of 5). (b) Regression curve on averages (the subsampling method has limited impact on the outcome)  (2014) designed a methodology based on Bayesian decision model, able to analyze directly next-generation sequencing (NGS) data. The model defines the core-genome of bacterial populations allowing also the identification of novel genes. A nice caveat of this approach is that it can analyze even strains without a subset of genes since the model does not assume that all sequences have the entire core gene dataset present. The model has been benchmarked analyzing Streptococcus pneumoniae sequences.

BGDMdocker
BGDMdocker (Cheng et al. 2017) relies on docker technology to analyze and visualize bacterial pangenome and biosynthetic gene clusters. The pipeline consists of three stand-alone tools, namely Prokka v1.11 (Seemann 2014) for rapid prokaryotic genome annotation, panX (Ding et al. 2018) for pangenome analysis, and antiSMASH3.0 (Weber et al. 2015) for automatic genomic identification and analysis of biosynthetic gene clusters. The visualization supports several options, including alignment, phylogenetic trees, mutations mapped on the phylogenetic branches, and gene loss and gain mapping on the core-genome phylogeny. Benchmarking took place on 44 Bacillus amyloliquefaciens strains.

Bacterial PanGenome Analysis
Bacterial Pangenome Analysis (BPGA) (Chaudhari et al. 2016), comes with a handful of new options and features most notably that of optimizing the speed of execution. In addition, it offers various entity (core-, pangenome, and MLST) phylogeny, phyletic profile analysis (gene presence/absence), subset analysis, atypical sequence composition analysis, orthologous, and functional annotation for all gene datasets, user-selection of gene clustering algorithm, command line interface, and nice graphics. It runs both in Windows and in Linux as executables files (source code in Perl). BPGA has dependencies with other tools that require installation. In terms of input files, BPGA can "digest" the following file formats: GenBank (.gbk) files, protein sequence file (e.g.,.faa or .fsa or fasta format), binary (0,1) matrix (tab-delimited) file as output of other tools. The seven functional modules of BPGA algorithm include: Pangenome profile analysis, pangenome sequence extraction, exclusive gene family analysis, atypical GC content analysis, pangenome functional analysis, species phylogenetic analysis, and subset analysis.

ClustAGE
ClustAGE (Ozer 2018) suite (both online and stand-alone) clusters noncore accessory sequences within a collection of bacterial isolates implementing the BLAST algorithm. It is therefore focused on the accessory genomic dimension of pangenome; Benchmarking of this tool has taken place on Pseudomonas aeruginosa genome sequences.

DeNoGAP
DeNoGAP (Thakur and Guttman 2016) does many more than pure pangenome analysis, including functional annotation, gene prediction, protein classification, and orthology search; therefore, it is applicable both for complete and draft genomic data. To do this, it implements a big set of existing analysis algorithms. In terms of scalability, it runs linearly due to implementation of iteratively refined Hidden Markov models. Its modular structure supports easy updates and addition of new tools.

EDGAR
Implementing phylogenetic concepts like average amino acid and nucleotide identity indices, an online application namely "EDGAR" (Blom et al. 2009(Blom et al. , 2016 was developed to support comparative genomic analyses of related isolates. Strong utilities of the suite include Venn diagrams and interactive synteny plots, as well as ease of access to taxa of interest and quick analyses like pangenome vs. core plot, the core-genome and singletons.

EUPAN
EUPAN (Hu et al. 2017) is one of the first concrete attempts to analyze eukaryotic pangenomes, even at a relatively low sequencing depth supporting gene annotation of pangenomic dataset, genome assembly, and identification of core and accessory gene datasets exploiting read coverage. The tool has been benchmarked using 453 rice genomes.

GET_HOMOLOGUES
GET_HOMOLOGUES (Contreras-Moreira and Vinuesa 2013) is a customizable and detailed pangenome analysis platform (open source written in Perl and R) for microorganisms addressed to non-bioinformaticians. GET_HOMOLOGUES can cluster homologous gene families using bidirectional best-hit clustering algorithms. The cluster granularity can be adjusted by the user based on various filtering strategies (e.g., by controlling key blast parameters such as percentage overlap and identity of pairwise alignments and E-score cutoff value). To estimate the size of the core-and pangenome, the tool supports both exponential and binomial mixture models to fit the data.

Harvest
Harvest (Treangen et al. 2014) is suitable for the analysis of (up to thousands of) microbial genomes. It hosts three modules, namely Parsnp (core-genome analysis), Gingr (output visualization), and HarvestTools (meta-analysis). Parsnp exploits jointly whole-genome alignment and read mapping to optimize accuracy and scalability aspects of sequence alignment; this approach can accommodate scalability for up to thousands of genomic datasets. For indexing purposes, it implements directed acyclic graph improving the identification of unique matches (anchors). The input of Parsnp is a directory of MultiFASTA files; the output includes core-genome alignment, variant calls, and a SNP tree, all of which can be visualized via Gingr. Broadly speaking, this tool represents a compromise between whole-genome alignment and read mapping. Parsnp performance has been evaluated on simulated and real data.

ITEP
ITEP (Benedict et al. 2014) is a suite of BASH scripts and Python libraries that interface with an SQLite database backend and a large number of tools for the comparison of microbial genomes. ITEP hosts several de novo prediction tools such as sequence alignment, metabolic, clustering, and protein prediction. Users can develop their own customized comparative analysis workflows.

LS-BSR
LS-BSR (large-scale BLAST score ratio) (Sahl et al. 2014), calculates a score ratio (BSR value ¼ query/reference bit score) per coding sequence (matrix) within a pangenome dataset using BLAST (Altschul et al. 1997) or BLAT (Kent 2002) for all-against-all alignment purposes. The output (bit score per CDS) can be visualized as a heatmap. Benchmarking has taken place on Escherichia coli and Shigella datasets.
14 micropan micropan (Snipen and Liland 2015) is an R package for the pangenome study of prokaryotes. The R computing environment supports several options of statistical analyses (e.g., principal component analysis), pangenome models (e.g., Heaps' law), and graphics. External free software (e.g., HMMER3) is used for the heavy computations involved. Benchmarking has been carried out on 342 Enterococcus faecalis genomes.

NGSPanPipe
NGSPanPipe (Kulsum et al. 2018) supports microbial pangenome analysis directly from experimental reads. Benchmarking has been carried out using simulated reads of Mycobacterium tuberculosis. The pipeline expects as input experimental reads and outputs three files, one of which is a binary matrix showing the presence/absence of genes in each strain; this matrix can be used as input to other pangenome tools like PanOCT (Fouts et al. 2012) and PGAP (Zhao et al. 2012).

PanACEA
PanACEA (Clarke et al. 2018) is an open source stand-alone computer program written in Perl that supports users to create an interconnected set of html, javascript, and json files visualizing prokaryotic pan-chromosomes (core and variable regions) generated by PanOCT (Fouts et al. 2012) or other pangenome clustering tools.
PanACEA was developed to serve as an intuitive, easy-to-use, stand-alone viewer.
Regions and genes can be functionally annotated to allow for visual identification of regions of interest. PanACEA's memory and time requirements are within the capacities of standard laptops. Benchmarking took place on 219 Enterobacter hormaechei genomes.

Panaconda
Panaconda (Warren et al. 2017) creates whole-genome multiple sequence comparisons and provides a model for representing the relationship among sequences as a graph of syntenic gene families, by discovering collision points within a group of genomes. The first step is to create a de Bruijn graph and use its traversal to build a pan-synteny graph; the alphabet used is based on gene families (instead of nucleotide alphabet). This approach is novel in the context of generating a graph, wherein all sequences are fully represented as paths.

PanCake
PanCake (Ernst and Rahmann 2013) is another tool for pangenome analysis (core and unique regions) relying exclusively on sequence data and pairwise alignments (nucmer or BLAST), which makes it annotation independent (i.e., it processes pure whole-genome content). It hosts a command line interface with several subcommands, allowing to add chromosomes, to specify a genome for each chromosome, to add alignments, to compute core and unique regions, and to output selected regions of the analyzed chromosomes. Benchmarking took place on three genera, namely Pseudomonas, Yersinia, and Burkholderia. PanCake is written in Python.

PanFunPro
PanFunPro (Lukjancenko et al. 2013) exploits functional information (profiles) for pangenome analysis. The suite supports among others calculation of core, and accessory gene datasets, homology search (all-against-all and pairwise sub-querying), functional annotation (HMM-based), and gene-ontology information analysis. PanFunPro is available both as a standalone (Perl) tool and as a web server. Benchmarking took place on 21 Lactobacillus genomes.

PanGeT
PanGeT (Yuvaraj et al. 2017) can digest both genomic and proteomic data in order to construct the pangenome for a selection of taxa, exploiting BLASTN or BLASTP, respectively. In terms of performance, it has been benchmarked using a set of 11 Streptococcus pyogenes strains. The output is given in the form of a flower plot (core, dispensable, and strain-specific genes).
PanGFR-HM (Chaudhari et al. 2018), is putting an interesting view point on the "table" of pangenome, by analyzing exclusively microbes from the Human Microbiome Project; it is a web-based platform integrating functional and genomic analysis for a collection of~1300 complete human-associated microbial genomes exploiting a novel dimensionality of analysis that of body site (location of the bug in the human body) when comparing different groups of organisms.

PanGP
PanGP (Zhao et al. 2014) supports scalable pangenome analysis by analyzing clusters of orthologs pre-computed by OrthoMCL (Li et al. 2003), PGAP (Zhao et al. 2012), Mugsy-Annotator ), or PanOCT (Fouts et al. 2012. In order to predict core and accessory gene datasets, the suite implements random or distance-guided sampling; in the latter, the genomic diversity (GD) drives the sampling of strain permutations. GD is modeled relying on three alternative assumptions: GD is determined by the evolutionary distance on phylogenetic trees, the difference in gene numbers per strain, or by the discrepancy among gene clusters; among the three models the third seems more reliable (preferred model for PanGP).

PANINI
PANINI (Abudahab et al. 2018) is a web browser implementation for rapid online visualization and analysis of the core and accessory genome content, implementing unsupervised machine learning with stochastic neighbour embedding based on the t-SNE (t-distributed stochastic neighbour embedding) algorithm; this algorithm calculates first the similarities between the data (in high dimensional space) and then it minimizes the divergence between the two probability matrices over the embedding coordinates. PANINI expects as input the output of Roary (Page et al. 2015).

PANNOTATOR
PANNOTATOR (Santos et al. 2013) supports the efforts of automatic annotation transfer onto related unannotated genomes exploiting the existing annotation of a curated genome. From this perspective, it is not a main pangenome analysis tool, but rather as a side-product of cross-comparison it provides pangenomic-related information. Its main contribution though to pangenome analysis is to accelerate the functional annotation of closely related isolates. For this task, it implements a relational database, interactive tools, several SQL reports, and a web-based interface. The expected input is the DNA strand, the gene prediction plus the reference annotated genome.

PanOCT
PanOCT (Fouts et al. 2012) is a graph-based ortholog clustering tool for pangenome analysis of closely related prokaryotic genomes exploiting conserved gene neighborhood information to separate recently diverged paralogs into distinct clusters of orthologs where homology-only clustering methods cannot. PanOCT is utilizing BLAST (Altschul et al. 1997) and conserved gene neighborhood information. Four input files are expected including a tabular file of all-versus-all BLASTP searches and the actual protein fasta sequences. PanOCT is specifically designed for pangenome analysis of closely related taxa (in order to be able to distinguish groups of paralogs into separate clusters of orthologs). In terms of memory requirements, PanOCT is greedier than other tools used to benchmark its performance; the memory usage is unchanged until the sixth genome, with a usage of 0.25 GB per genome, maxing out at 0.5 GB per genome by the 25th genome.

Panseq
Panseq (Laing et al. 2010) builds pangenomes and identifies single nucleotide polymorphisms (SNPs) using genomic data as input. In addition, it produces files for further phylogenetic analysis exploiting both the information of SNPs as well as the phyletic profile of accessory sequences; all these wrapped-up with a user-friendly graphical user interface.

Pan-Tetris
Pan-Tetris (Hennig et al. 2015) is a Java-based tool that exploits an aggregation technique inspired by the Tetris game, to provide an interactive and dynamic visualization of the gene content in a pangenome table with the option of editing and on-the-fly modification of user-defined (pan) gene groups. The suite has been tested on 32 Staphylococcus aureus genomes. Pan-Tetris is one of the first attempts that enable modification of the computed pangenome. The computation of whole genome alignment exploits progressiveMAUVE (Darling et al. 2010) algorithm.
PanTools (Sheikhizadeh et al. 2016) suite supports the construction and visualization of pangenomes hosting online tools and algorithms; the visual representation of the pangenome is based on generalized De Bruijn graphs. The pangenome construction algorithm scales nicely even with large eukaryotic datasets. In addition to the basic pangenome tasks (construction and visualization), the suite supports other handy utilities such as adding, retrieving and grouping of sequences as well as annotating, reconstructing, and comparing genomes or pangenomes. Overall, it can easily support multi-genome read mapping, pangenome browsing, structure-based variation detection and comparative genomics. It has been benchmarked on E. coli, yeast, and Arabidopsis thaliana genomes.

PanViz
PanViz (Pedersen et al. 2017) is a pangenome visualization tool with some analysis options. It can generate dynamic visualizations supporting both pangenome subset selection as well as mapping of new genomes to existing pangenomes. The input data needed is a pangenome matrix (gene group presence/absence across the included genomes), as well as a gene ontology-based functional annotation of each gene group.

PanWeb
PanWeb (Pantoja et al. 2017) is a web application that performs pangenome analyses based on PGAP pipeline, providing in addition a user-friendly graphical interface supporting multiple user-defined analysis queries. It can be implemented by users without computational skills. As input, it receives the annotation files for each genome in EMBL format. A complete set of graphs (e.g., pangenome, accessory, core-genome, and unique genes) is provided.

panX
panX (Ding et al. 2018) identifies orthologous gene clusters in pangenomes via a user-friendly and interactive web-based visualization. The visualization consists of connected components that allow further analysis. The suite provides alignment and, phylogenetic tree, it maps mutations of each gene cluster and infers gene gain and loss in the core-genome phylogeny. The pipeline breaks annotated genomes into genes and then clusters them into orthologous groups. To identify homologous proteins, panX performs an all-against-all similarity search, while the actual clustering of orthologous genes is carried out by a Markov clustering algorithm.

PGAdb-Builder
PGAdb-builder (Liu et al. 2016), constructs a pangenome allele database (PGAdb) to empower whole genome multilocus sequence typing (wgMLST) analyses and operates as a web service suite. Two modules are implemented, namely Build_PGAdb for building a PGAdb database and Build_wgMLSTtree for constructing a wgMLST tree and determine the genetic relatedness of the input sequences; both modules "digest" genome contigs in FASTA format. PGAdbbuilder, has however dependencies with other existing suites like Prokka (Seemann 2014) and Roary (Page et al. 2015).

PGAP
PGAP (Zhao et al. 2012) supports pangenome analysis and in addition analysis of functional gene clusters, species evolution, genetic variation, and functional enrichment of query sequences. It outputs the basic pangenome structure and growth curve and in addition SNP and genomic variation information, phylogenetic, and functional annotation metadata. Benchmarking has taken place on Streptococcus pyogenes datasets.

PGAP-X
Building on PGAP, and in order to more effectively interpret and visualize the results, PGAP-X (Zhao et al. 2018) was developed. The visualization utility can intuitively lead to conclusions on pangenomic structure, conserved regions and overall on genetic variability throughout the pangenomic datasets at hand. Benchmarking has taken place on S. pneumoniae and Chlamydia trachomatis datasets. One current limitation of PGAP-X (that is not present in PGAP) is that it expects as input only complete genomes.
Piggy (Thorpe et al. 2018) is a tool for analyzing the intergenic component of bacterial genomes and it is designed to be used in conjunction with Roary (Page et al. 2015). The latter works by analyzing protein-coding sequences thus excluding nonprotein-coding intergenic regions (IGRs) which typically account for approximately 15% of the genome. Piggy matches Roary except that it is based only on IGRs. Benchmarking took place on Staphylococcus aureus and Escherichia coli using large genome datasets. In terms of input and output, Piggy uses the same format as in Roary and has similar running time requirements. Piggy provides a means to rapidly identify IGR switches, with many evolutionary applications including analysis of the role of horizontal transfer in shaping the bacterial regulome.

pyseer
pyseer (Lees et al. 2018), is geared toward genome-wide association studies in the "world" of microbes with the task at hand to identify potential genetic variation linked with certain phenotypic aspects. Pyseer is actually a python implementation of a previous initiative written in C++, namely SEER (Lees et al. 2016). The foundation of pyseer is the use of K-mers (words) of variable length (input) coming from draft assemblies, while using a generalized linear model for each word their link with a potential phenotype is evaluated. In addition, multidimensional scaling of a pairwise distance matrix is implemented in order to control for population structure (embedded in the regression analysis).

Roary
Roary (Page et al. 2015) enables the construction of large pangenomes even on a typical desktop machine, yielding fairly accurate output. For example, it can digest up to 1000 strains (13 GB of RAM) building the pangenome in~4 h. Roary achieves high accuracy which is attributable to utilization of the context of conserved gene neighborhood information. A suite of command line tools is provided to interrogate the dataset providing union, intersection, and complement.

seq-seq-pan
seq-seq-pan (Jandrasits et al. 2018) is a workflow for the sequential alignment of sequences to build a pangenome data structure and a whole-genome alignment. seqseq-pan builds a pangenome data structure allowing editing (addition or removal) of genomes from a set of aligned sequences and subsequent re-alignment of the wholegenome sequences; for whole-genome alignments it relies on progressiveMauve (Darling et al. 2010). The alignment is optimized for generating a representative linear presentation of the aligned set of genomes.

Spine and AGEnt
Spine (Ozer et al. 2014) determines the core-genome from a group of genomic sequences and AGEnt (Ozer et al. 2014) identifies the accessory genome in draft genomic sequences. They both use nucmer to align sequences. The pipeline has been tested on genome sequences of Pseudomonas aeruginosa. However, as mentioned by the authors, whole genome alignment of reference genomes and core-genome identification with Spine can be time-consuming.

SplitMEM
SplitMEM (Marcus et al. 2014) scales linearly in terms of time and space in relation to the number of genomes of interest. To do this, it traverses suffix trees (for the genomes) and builds compressed de Bruijn graphs of pangenomes. In terms of notation, nodes within the graph represent conserved or strain-specific sequences of the pangenome. Benchmarking has taken place on Bacillus anthracis and E. coli datasets.

Highlights
Pangenome analysis has today many options when it comes to practical implementation. Depending on the analysis focus, the desired input and output, the dependability on other algorithms, as well as the modeling parametrization, users have many options to choose from. In the current review, we highlight the following five tools: BPGA (Chaudhari et al. 2016) for its very fast execution time, the intuitive handling and the user-defined clustering algorithm, Roary (Page et al. 2015) due to its internal processing (clustering of high similarity sequences) that results in linear memory consumption, LS-BSR (Sahl et al. 2014) that similarly to Roary performs pre-clustering reducing substantially the running time, PanOCT (Fouts et al. 2012), which takes into account both homology and positional gene neighborhood information and PGAP (Zhao et al. 2012) that can work also with draft forms of genomic data such as annotated assemblies.

Food for Thought
The final results and conclusions of a pangenome analysis, among others, massively depend on the following aspects, that need thoughtful consideration prior to embarking any such project: Homology search algorithm, the phylogenetic sample at hand, the pangenome model implemented and the type and quality of sequence entities (e.g., DNA, protein, presence/absence-phyletic profile, and SNPs). For example, when it comes to homology definition based on sequence similarity there is a wide range of similarity thresholds used in previous attempts: i ¼ 50%, L ¼ 50% , i ¼ 70%, L ¼ 70% (Hiller et al. 2007), i ¼ 70%, L ¼ 50% (Meric et al. 2014), i ¼ 30%, L ¼ 80% (Bentley et al. 2007), where i stands for sequence identity and L for sequence length.
The starting level (ORFs, CDSs, genes, proteins, SNPs) and the quality (in silico, manual curation) of annotation as well as inherent bacterial genomic complexity at the sequence level such as low complexity repeats, recombination hot spots, horizontally acquired genomic fragments constitute other important aspects of consideration. Such information variability can massively affect the predicted conserved and unique genes in favor of the former or the latter; this might also determine the structure of pangenome (open or closed).

Conclusions
Being able algorithmically to digest the largest possible pool of data available is critical in order to approach more reliably the phylogenetic history of bacterial populations. Indeed such comparative genomic analyses started by exploiting $0.07% of a genome (16s rRNA) (Woese 1987), latter on using up to $0.2% of the genomic information (MLST) (Maiden et al. 1998), and recently up to 100% of the information exploiting the pangenome wealth of data Tettelin et al. 2005).
The recent explosion of sequencing projects replaced the limiting factor of data sparsity with the immense data dimensionality (Vernikos 2010) and we are now in the middle of a transformation moving from top-down (trying to fit the limited data to the model) to bottom-up approaches in an attempt to move from the "infant" stage of single-strain genomics to the post pangenome era of "adulthood." The model assumptions therefore become less and less pivotal as the pace of primary data generation continues to grow exponentially, asking not for modeling superpower but instead interpretation and connecting the dots super skills.