View source for Plasmodium analysis workflow 1: Tools that evaluate genomic properties and amino acid usage

=== ''Outline'' ===
<span style="color:#006F00">'''RETURN TO THE MAIN PAGE:'''</span> [[Using_CoGe_for_the_analysis_of_Plasmodium_spp]]

<span style="color:#006F00">'''CONTINUE TO THE NEXT WORKFLOW:'''</span> [[Plasmodium analysis workflow 2: Tools for the syntenic analysis of whole genomes and microsyntenic regions]]

<span style="color:#006F00">'''CONTINUE TO THE FINAL WORKFLOW:'''</span> [[Plasmodium analysis workflow 3: Tools useful on the study of multigene families]]


=== ''Abstract'' ===

In a short evolutionary time, GC content has varied significantly within the genus ''Plasmodium''. The change is most evident in the differences in average GC content and the distribution of GC-right regions in ''P. vivax'' (40.57%) and ''P. falciparum'' (21.8%). GC-poor regions are restricted to the sub-telomeres in ''P.vivax''’s, whereas they are ubiquitous across the ''P. falciparum'' <ref>Carlton JM, Adams JH, Silva JC, Bidwell SL, Lorenzi H, Caler E, Crabtree J, Angiuoli SV, Merino EF, Amedeo P, Cheng Q, Coulson RM, Crabb BS, Del Portillo HA, Essien K, Feldblyum TV, Fernandez-Becerra C, Gilson PR, Gueye AH, Guo X, Kang'a S, Kooij TW, Korsinczky M, Meyer EV, Nene V, Paulsen I, White O, Ralph SA, Ren Q, Sargeant TJ, Salzberg SL, Stoeckert CJ, Sullivan SA, Yamamoto MM, Hoffman SL, Wortman JR, Gardner MJ, Galinski MR, Barnwell JW, Fraser-Liggett CM. 2008. Comparative genomics of the neglected human malaria parasite ''Plasmodium vivax''. Nature. 455:757-63. https://www.ncbi.nlm.nih.gov/pubmed/18843361</ref>. Species closely related to ''P. falciparum'' (''Laveranian'' subgenus) have equally GC-poor genomes <ref>Hamilton WL, Claessens A, Otto TD, Kekre M, Fairhurst RM, Rayner JC, Kwiatkowski D. 2016. Extreme mutation bias and high AT content in Plasmodium falciparum. Nucleic Acids Res. https://www.ncbi.nlm.nih.gov/pubmed/27994033</ref>. The current model is that AT-rich genomes represent the ancestral state and GC-rich genomes the derived state <ref>Nikbakht H, Xia X, Hickey DA. 2014. The evolution of genomic GC content undergoes a rapid reversal within the genus Plasmodium. Genome. 9:507-511. https://www.ncbi.nlm.nih.gov/pubmed/25633864</ref>. Here, we use CoGes tools to evaluate the general trends of GC content variation across the genus ''Plasmodium'' and their putative influence on amino acid usage.

=== ''Analyzing GC content and other genomic properties (GenomeList)'' ===

[[File:Genomelistnew.png|thumb|250px|'''Figure 1. Genome List''' upload window as seem from '''OrganismView'''. Twelve ''Plasmodium'' genomes have been included. Analysis can be run following this link:  https://genomevolution.org/r/lys1]]

There are significant variations in average GC content and GC content distribution between the two main human malaria agents: ''P. vivax'' and ''P. falciparum''. The average GC content is 40.57% ''P. vivax'' compared to 19.4% in ''P. falciparum''. GC-poor regions are restricted to the subtelomeric regions of ''P.vivax''’s genome, whereas they are ubiquitous across the ''P. falciparum'' genome <ref>Carlton JM, Adams JH, Silva JC, Bidwell SL, Lorenzi H, Caler E, Crabtree J, Angiuoli SV, Merino EF, Amedeo P, Cheng Q, Coulson RM, Crabb BS, Del Portillo HA, Essien K, Feldblyum TV, Fernandez-Becerra C, Gilson PR, Gueye AH, Guo X, Kang'a S, Kooij TW, Korsinczky M, Meyer EV, Nene V, Paulsen I, White O, Ralph SA, Ren Q, Sargeant TJ, Salzberg SL, Stoeckert CJ, Sullivan SA, Yamamoto MM, Hoffman SL, Wortman JR, Gardner MJ, Galinski MR, Barnwell JW, Fraser-Liggett CM. 2008. Comparative genomics of the neglected human malaria parasite ''Plasmodium vivax''. Nature. 455:757-63. https://www.ncbi.nlm.nih.gov/pubmed/18843361</ref>. The current model is that AT-rich genomes represent the ancestral state and GC-rich genomes the derived state in specific ''Plasmodium'' lineages <ref>Nikbakht H, Xia X, Hickey DA. 2014. The evolution of genomic GC content undergoes a rapid reversal within the genus Plasmodium. Genome. 9:507-511. https://www.ncbi.nlm.nih.gov/pubmed/25633864</ref>. Here, we will evaluate the patterns of GC content variation across three of the four main ''Plasmodium'' clades. 

[[File:Genomelistmakedwithcorrectcolors.png|thumb|250px|'''Figure 2. Genome List''' output window shows the analysis of 12 ''Plasmodium'' genomes. Clades are indicated with colors: simian clade (brown), rodent clade (red), and ''Laveranian'' subgenus (blue). The number of columns on display has been modified.]]

CoGe can display a genome’s GC content in '''[[GenomeInfo]]'''. To calculate GC content, click on '''%GC''' under the Length and/or Noncoding sequence sections on the ''Statistics tab''. You can compare and contrast GC content (and other genomic features) across several species and/or strains using '''[[GenomeList]]'''. This tool creates a list of genomes selected by the user and calculates features such as: 
*Amino acid usage. 
*Codon usage. 
*Coding sequence (CDS) GC content. 
*Number of genes. 
*Number of introns. 

'''GenomeList''' also summarizes some of the genomes’s metadata including: 
*[[Sequence type]]. 
*[[Sequence origin]].
*Taxonomy. 
*Provenance. 
*Genome version.

[[File:Genoemlistresultsonphylogeny.png|thumb|250px|'''Figure 3.''' GC content is written in color text next to each analyzed ''Plasmodium'' genome. Species are colored according to their clade: simian (brown), rodent (red), ''Laveranian'' subgenus (blue), and reptile-birds (green/purple). Figure modified from Hayakawa et al. (2008) <ref>Hayakawa T, Culleton R, Otani H, Horii T, Tanabe K. 2008. Big bang in the evolution of extant malaria parasites. Mol Biol Evol. 10:2233-9. https://www.ncbi.nlm.nih.gov/pubmed/18687771</ref>]]

{| class=wikitable align=center style="background: #F5FFF5;"

|The following steps indicate how to perform comparative analyses using the '''GenomeList''' tool in CoGe:


'''1.''' 	Go to: https://genomevolution.org/coge/ and login to CoGe

'''2.''' 	Click on '''OrganismView''' or follow this link: https://genomevolution.org/coge/OrganismView.pl

'''3.''' 	Type the scientific name of any organism of interest on the ''Search'' box. Then, select a genome version. 

'''4.''' 	Find the '''Tools''' section under '''Genome Information''' and click on '''Add to GenomeList'''. The first genome added to '''GenomeList''' will appear in a new window. 

'''5.''' 	Without closing this window, type the scientific name of another organism on the ''Search'' box. Select the genome version and click on '''Add to GenomeList'''. 

'''6.''' 	Once you have added all genomes click on '''Send to GenomeList''' ('''Figure 1'''). 

'''7.''' 	'''GenomeList''' will generate a table including all the selected genomes. You can use '''GenomeList''' to select and compare different genomic features and attributes. The analyses can be run on specific genomes or on all the included genomes. You can also select the display columns by clicking on '''Change Viewable Columns'''. 

'''8.''' 	Click on "''Send Selected Genomes to''" to download the genomes included on '''GenomeList'''. 


<span style="color:#8B008B">'''You can follow a link to an example analysis here:'''</span> https://genomevolution.org/r/lys1

|}

We used '''GenomeList''' to compare 12 fully sequenced ''Plasmodium'' genomes ('''Figure 1'''). Our results show that species closely related to ''P. falciparum'' (subgenus ''Laverania'') have similarly AT rich genomes. GC content was higher in ''Plasmodium'' species of the simian and rodent clades ('''Figure 2''' and '''Figure 3'''). The highest GC content values were observed in species of the simian clade (''P. vivax'', ''P. cynomolgi'' and ''P. knowlesi''). Tellingly, these species all share a common ancestor and diverged from one another recently. GC content varied widely across ''Plasmodium'' species infecting humans (''P. vivax'', ''P. ovale'', ''P. malariae'', and ''P. falciparum'') but not on species infecting rodents (''P. berghei'', ''P. chabaudi'', and ''P. yoelii''). GC content also varied in human-infecting ''Plasmodium'' within the simian clade (''P. vivax'' =  46.89%, ''P. ovale'' = 32.83%, and ''P. malariae'' = 25.12%). Our results suggest that GC-richness (> 30%) evolved recently and is a derived state within the genus. Our results suggest that a correlation between GC-content and evolutionary relatedness, but not with host-related selective pressures. 

AT-richness as an ancestral state for the ''Plasmodium'' genus is unusual since closely related genera within the phylum ''Apicomplexas'' frequently have GC-rich genomes (''Toxoplasma gondii'' = 52.28%, ''Cryptosporidium parvum'' = 30.4%, ''C. muris'' = 28.5%, ''Theileria orientalis'' = 41.58%, ''T. equii'' = 39.47%, ''Babesia bovis'' = 36.3%, ''Eimeria tenella'' = 51.07%, etc.). Our data suggests that ''Plasmodium'' GC content may be in the process of being reinstated to values that can be considered typical for the phylum. The implications of and mechanisms behind the extreme variability in GC-content within ''Plasmodium'' are currently being investigated <ref>Bensch S, Canbäck B, DeBarry JD, Johansson T, Hellgren O, Kissinger JC, Palinauskas V, Videvall E, Valkiūnas G. 2016. The Genome of Haemoproteus tartakovskyi and Its Relationship to Human Malaria Parasites. Genome Biol Evol. 8:1361-73.https://www.ncbi.nlm.nih.gov/pubmed/27190205</ref>.


=== ''Identifying codon and amino acid substitution frequencies (CodeOn)'' ===

[[File:Sco.png|thumb|250px|'''Figure 4.''' Amino acid usage tables of simian clade ''Plasmodium'' species. '''Upper row:''' sister species ''P. vivax'' and ''P. cynomolgi''. '''Bottom row:''' sister species ''P. knowlesi'' and ''P.coatneyi''. See steps section (green box) to find links to rerun the analyses.]]

Codon and amino acid usage are significantly shaped by two factors: selection for translational efficiency and genome composition. The significance of translational selection on genome evolution varies across the genus ''Plasmodium''. It is believed that usage of less energetically expensive amino acids provides an evolutionary advantage by decreasing energetic costs during infection <ref>Peixoto L, Fernández V, Musto H. 2004. The effect of expression levels on codon usage in Plasmodium falciparum. Parasitology. 128:245-51. https://www.ncbi.nlm.nih.gov/pubmed/15074874</ref>.  
In ''P. falciparum'' many highly expressed genes are predominantly composed of C-ended codons despite the AT-rich genome. In the GC-rich ''P. vivax'' genome, translational selection and codon usage bias are not strongly related <ref>Yadav MK, Swati D. 2012. Comparative genome analysis of six malarial parasites using codon usage bias based tools. Bioinformation. 8:1230-9. https://www.ncbi.nlm.nih.gov/pubmed/23275725</ref>. Genome composition is also a powerful force in protein evolution. 

Here, we will use '''[[CodeOn]] ''' to calculate amino acid usage across a range of GC-rich to GC-poor genomes. We will measure the effects of genome composition bias on amino acid usage across 7 ''Plasmodium'' genomes from two major clades (''Laveranian'' and simian). 

[[File:Lco.png|thumb|250px|'''Figure 5.''' Amino acid usage tables in ''Plasmodium'' species from the ''Laveranian'' subgenus. '''Upper row:''' sister species ''P. falciparum'' and ''P. reichenowi''. '''Bottom row:''' ''P. gaboni''. See steps section (green box) to find links to rerun the analyses.]]

{| class=wikitable align=center style="background: #F5FFF5;"

|The following steps indicate how to built amino acid usage tables using '''CodeOn''': 


'''1.''' 	Go to: https://genomevolution.org/coge/ and login to CoGe.

'''2.''' 	Find the genome of interest in '''OrganismView''' or follow this link https://genomevolution.org/coge/OrganismView.pl

'''3.''' 	Click on '''CodeOn''' to start the analysis. After a couple of minutes, the output will show in a different tab. 


<span style="color:#8B008B">'''You can follow links to CodeOn example analyses for the simian clade here (Figure 4):'''</span> 

https://genomevolution.org/coge/CodeOn.pl?oid=27002 <span style="color:#8B008B">('''''P. vivax''''')</span>

https://genomevolution.org/coge/CodeOn.pl?dsgid=32770 <span style="color:#8B008B">('''''P. cynomolgi''''')</span>

https://genomevolution.org/coge/CodeOn.pl?oid=26997 <span style="color:#8B008B">('''''P. knowlesi''''')</span>

https://genomevolution.org/coge/CodeOn.pl?oid=40698 <span style="color:#8B008B">('''''P. coatneyi''''')</span>


<span style="color:#8B008B">'''You can follow links to CodeOn example analyses for the ''Laveranian'' subgenus here (Figure 5):'''</span> 

https://genomevolution.org/coge/CodeOn.pl?oid=26992 <span style="color:#8B008B">('''''P. falciparum''''')</span>

https://genomevolution.org/coge/CodeOn.pl?oid=40801 <span style="color:#8B008B">('''''P. reichenowi''''')</span>

https://genomevolution.org/coge/CodeOn.pl?oid=40696 <span style="color:#8B008B">('''''P. gaboni''''')</span>

|}

Amino acid usage trends were markedly different in species from different clades ('''Figure 4''' and '''Figure 5'''). On the other hand, closely related ''Plasmodium'' species showed similar amino acid usage patterns. 

''P. vivax'' (Salvador-1) had the highest number of CDS with 45-55% GC content. Closely related species (''P. cynomolgi'', ''P. knowlesi'', and ''P.coatneyi'') had a higher number of CDS in the 40-45% GC tier ('''Figure 4'''). Genome composition is similar between ''P. cynomolgi'', ''P. knowlesi'', and ''P. coatneyi'' ('''Figure 2''' and '''Figure 3'''). However, patterns of amino acid usage were markedly different on ''P. coatneyi'' respect to other simian species ('''Figure 4''').

In the ''Laveranian'' subgenus, the number of CDS with 20-30% GC content was significantly larger. Amino acid usage was similar in ''P. falciparum'' (3D7) and ''P. reichenowi'' (SY57), but slightly different in ''P. gaboni'' ('''Figure 5'''). This variation is noteworthy given that the three species share a similar GC-content ('''Figure 2''' and '''Figure 3'''). This result suggests that GC-content is a significant factor in amino acid usage on both the simian clade and ''Laveranian'' subgenus. However, we cannot discard the significance of additional factors not evaluated here.


===''Outline''===

<span style="color:#006F00">'''RETURN TO THE MAIN PAGE:'''</span> [[Using_CoGe_for_the_analysis_of_Plasmodium_spp]]

<span style="color:#006F00">'''CONTINUE TO THE NEXT WORKFLOW:'''</span> [[Plasmodium analysis workflow 2: Tools for the syntenic analysis of whole genomes and microsyntenic regions]]

<span style="color:#006F00">'''CONTINUE TO THE FINAL WORKFLOW:'''</span> [[Plasmodium analysis workflow 3: Tools useful on the study of multigene families]]


=='''References'''==
{{reflist}}