BACKGROUND
the kinetoplastida  are unicellular flagellates that include the trypanosomatid parasites, most notably trypanosoma brucei, t. cruzi and leishmania spp.
these organisms cause substantial mortality and morbidity in humans and their livestock worldwide as the causative agents of african sleeping sickness, chagas disease and leishmaniasis respectively.
bodo saltans is a free-living heterotroph found worldwide in freshwater and marine habitats.
it possesses the diagnostic kinetoplastid features, such as flagella sited within a specialised flagellar pocket, glycolytic processes confined to a dedicated organelle , and the characteristic concentration of mitochondrial dna at the base of the flagellum   <cit> .
when comparing trypanosomatid parasites with each other, or collectively with other eukaryotes, the value of b. saltans is as a non-parasitic near relative, , that can illuminate their key evolutionary transitions.
five draft genome sequences exist for trypanosoma spp.
and four for leishmania spp.
<cit> ; these will be augmented with further strains and other non-human parasites in the coming years  <cit> .
with such excellent comparative resources in place or in development, there is a critical need for a non-trypanosomatid outgroup.
in effect, it will provide a model of the ancestral trypanosomatid to distinguish those derived parts of the parasite genomes  from those which are a legacy of the free-living ancestor.
for instance, such a model will help to resolve whether trypanosomatids previously possessed an algal plastid from which 'plant-like' genes in trypanosomatid genomes are derived  <cit> .
as a prelude to a complete b. saltans genome sequencing effort, this study sought to establish an initial understanding of the bodonid genome, its structure and content relative to the trypanosomatids.
the most recent kinetoplastid phylogeny has shown that trypanosomatid parasites are just one of many independent acquisitions of parasitism, indeed, a relatively minor component of total diversity  <cit> .
nonetheless, they are, naturally, the most important aspect of kinetoplastid diversity.
many features of their completed genome sequences emphasised the common ancestry of t. brucei, t. cruzi and leishmania spp., especially with respect to gene repertoire and order  <cit> , but their critical pathological differences were also evident at the genomic level.
the three human parasites cause distinct diseases; their genomes contain enigmatic adaptations related to pathogenesis and immune evasion, for instance the bloodstream expression site in t. brucei from which its variant surface glycoproteins  are expressed  <cit> , and surface antigen families in general  <cit> .
without an historical dimension, these features cannot be compared, nor understood in an evolutionary context.
as it is among the closest bodonid relatives of the trypanosomatids  <cit> , bodo saltans is a suitable outgroup to address three principal comparative issues: i) understanding how human trypanosomatid parasites acquired their distinct pathological strategies; ii) understanding how the ancestral trypanosomatid became parasitic in terms of derived innovations  and loss of genomic repertoire; iii) understanding how typical kinetoplastid features  evolved and how these might have been modified for parasitism.
quite what to expect from a bodonid genome sequence is an open question.
beyond the basic kinetoplastid features named above, the biological differences between bodonids and trypanosomatids are striking.
while b. saltans is a bacteriovore, especially prevalent in polluted waters or other environments with high bacterial densities  <cit> , trypanosomatids are obligate parasites inhabiting a nutrient-rich, but ultimately hostile, host environment, and adept at exploiting their eutrophic environment to maximise proliferation and transmission.
by contrast, b. saltans preys on bacterial cells  <cit>  and is probably adapted for resource acquisition within its relatively oligotrophic environment.
although bodonids and trypanosomatids are all flagellates, trypanosomatids attach their single flagellum to the cell surface to generate motile force, whereas the anterior flagellum in b. saltans is modified with hair-like mastigonemes, which may assist prey location during feeding  <cit> .
there are wider cytoskeletal differences also; the subpellicular microtubular cortex is instrumental in maintaining the numerous cell forms adopted by trypanosomatids  <cit> , but is reduced in bodonids, , to the region around the cytostome  <cit> .
perhaps most importantly for understanding the evolution of parasitism, we can expect substantial differences between trypanosomatid cell surfaces that function primarily to manipulate and frustrate the host immune response and bodonid membranes that are perhaps largely concerned with cellular homeostasis.
rather than providing definitive answers to these questions, the preliminary sequence data presented here provides an initial insight into a few comprehensively resolved locations in the b. saltans genome, indicating what to expect from gene content and arrangement, and testing the feasibility of a complete sequence project.
the sequence contigs were compared with corresponding regions in trypanosomatids , to examine gene content and the conservation of gene order  and, therefore, the potential for using trypanosomatid genome sequences as scaffolds to assist assembly and annotation of the b. saltans sequence.
RESULTS
gene structure
clones were selected from the b. saltans fosmid library according to random end-sequences and positive results for specific pcr probes.
inserts from  <dig> fosmid clones were shotgun sequenced, comprising  <dig>  mbp in total and an average size of  <dig>  kbp.
table  <dig> describes the composition of the  <dig> contigs in terms of the affinity shown by each putative coding sequence to sequence databases.
<dig> putative coding sequences are specified; genes could be predicted by eye because of a definite elevation in gc content in coding regions.
subsequent matches to sequence databases showed these features to be correct.
the boundaries between coding and flanking regions are marked by a transition from gc-rich to at-rich signatures; the sequences shown in figure  <dig> clearly demonstrate the gc troughs that appear between coding sequences.
this pattern is repeated in other contigs, as shown in subsequent figures.
gene density is high relative to corresponding regions in the l. major and t. brucei genome sequences, reflected by the consistently short intercoding sequences across all contigs .
figure  <dig> compares the gene order of one region  with positionally orthologous regions in l. major  and t. brucei ; this, like most fosmid inserts, contains more genes in bodo than in trypanosomatids.
note: † clones for which no probe is stated were selected after end-sequencing.
* genes were classified according to their affinity: kinetoplastid , other eukaryotic , prokaryotic  or unknown .
gene content
coding sequences without trypanosomatid homologs were compared to sequence and structural databases .
many of the gene products are homologous to proteins beyond the kinetoplastida, suggesting that they are core eukaryotic proteins subsequently lost from trypanosomatids; for example, contiguous genes in figure 1a homologous to a gpi-anchored protein in plants and fungi  and a hypothetical gene in metazoa .
gene products in other regions  contain protein domains known elsewhere, for example an abc transporter protein  and a nucleotide-sugar transporter protein , strongly indicating that these are bodo-specific members of ubiquitous gene families.
some otherwise uncharacterised hypothetical proteins are predicted to expressed on the cell surface.
the region shown in figure 3a is notable not only for the base composition of coding regions and admixture of trypanosomatid and bodo-specific genes mentioned above, but also for a hypothetical protein  with  <dig> predicted transmembrane helices and a signal peptide.
table  <dig> contains other examples of bodo-specific hypothetical genes predicted to be surface expressed, including those shown in figure 1a  and b .
colinearity
the extent of conserved gene order, or colinearity, between bodonid and trypanosomatid genome sequences was assessed using the artemis comparison tool .
one region of excellent colinearity is shown in figure  <dig> and, despite disruption by some eukaryotic genes not seen in trypanosomatids, this contig corresponds unmistakably with chromosome  <dig> in l. major and chromosome  <dig> in t. brucei.
conversely, the presence of so many non-trypanosomatid genes meant that colinearity disappears entirely in some locations, as shown in figure  <dig>  across the  <dig> genomic regions however, both patterns were atypical; most regions shows brief patches of colinearity, perhaps  <dig> or  <dig> genes with conserved synteny, set among larger regions of bodo-specific genes or homologs to trypanosomatid genes from elsewhere in the genome.
in this sense, the sequence presented in figure  <dig> is representative because several coding sequences are homologs of trypanosomatid genes on chromosomes  <dig>  and  <dig> ; these are roughly colinear but the order is disrupted by genes present on other chromosomes or by bodo-specific genes.
discussion
in this study, various locations in b. saltans genome, amounting to ~ <dig>  mbp, were sequenced.
assuming that the bodonid genome is approximately the same size as a trypanosomatid haploid genome, i.e., 35– <dig> mbp  <cit> , these sequences comprise ~1% of the complete genome sequence, which will therefore contain roughly  <dig>  genes.
the success and utility of a b. saltans genome project will depend on its relationship with existing trypanosomatid genome sequences.
this study shows that coding regions of the b. saltans genome share several structural features with trypanosomatids, indicating that the project is both feasible and likely to provide a useful comparative resource.
putative b. saltans genes lack introns, as in most trypanosomatid genes  <cit> .
they display a conspicuous elevation in gc content, which will greatly assist gene finding.
no evidence of strand-switching was observed in b. saltans, corroborating the view that it operates polycistronic transcription  <cit> , i.e., transcription of many contiguous loci within a single nascent transcript  <cit> , which is subsequently trans-spliced and polyadenylated to produce mature mrna, as in trypanosomatids  <cit> .
although the arrangement of coding regions along the bodonid chromosome may be conserved with trypanosomatids, it is clear that gene order was not.
the extent of conserved synteny, or rather colinear gene order, between bodonid and trypanosomatid genomes is of particular importance to the assembly of any b. saltans genome sequence.
the coding regions presented here indicate that trypanosomatid genome sequences will be of limited value in the global assembly of a b. saltans genome sequence.
strict colinearity was not normally observed, if only because of the large number of bodo-specific genes interposed between trypanosomatid homologs.
colinearity tended to persist over a distance of 3– <dig> genes, although some regions displayed conspicuous conservation , while others showed none at all .
therefore, this initial exploration of the b. saltans genome demonstrates that it should be possible to resolve a complete genome sequence, but, while the existing trypanosomatid resources will provide some useful guides for annotation, they could not be used as scaffolds for assembly, which should proceed de novo.
the purpose of a completed b. saltans genome sequence would be for understanding the evolution of trypanosomatid genome sequences.
the mixture of familiar and novel features in the regions sequenced here indicates the value of a bodonid genome sequence in distinguishing trypanosomatid characters inherited from free-living ancestors  from characters evolved since the origin of trypanosomatids.
hence, the first application would be in determining which parts of the trypanosomatid genome reflect the genomic legacy inherited from free-living ancestors, and show how they have been co-opted and modified for parasitism.
bodonid and trypanosomatid cells share various structural features, principally those that characterise kinetoplastid cells.
bodonids arrange their mitochondrial dna in kinetoplasts, although their position within the cell differs from trypanosomatids  <cit> , and conduct their glycolytic pathways within a dedicated organelle   <cit> .
bodonids construct their flagella in a similar manner to trypanosomatids, but deploy them very differently  <cit> .
while b. saltans uses one flagellum for movement and another for feeding, trypanosomatids flagella perform their motility function within the context of their sophisticated cell forms.
one might expect these structural similarities to be reflected at the genomic level.
α- and β-tubulin, the proteins that facilitate the development of flagella in trypanosomatids, are known to be arranged in tandem gene arrays, with an alternating, heterotypic α-β array in trypanosoma spp.
and distinct, monotypic α and β arrays in leishmania spp.
<cit> .
bodonids were shown to share the alternating conformation, suggesting that leishmania spp.
and their relatives had abolished the ancestral locus and evolved novel genomic repertoires  <cit> .
however, two b. saltans regions containing tubulin in this study show that modification of tubulin repertoire has also occurred in trypanosoma, since neither of the α-β arrays in b. saltans was found at the genomic position occupied in trypanosomes.
this demonstrates the utility of the b. saltans genome in resolving the evolutionary causes of structural or compositional differences between trypanosomatid genomes.
the second application of a b. saltans genome sequence would be to identify which components of the free-living legacy have been lost from trypanosomatids, and therefore, how reductive genome evolution has contributed to the parasite genomes.
table  <dig> describes many predicted proteins identified in b. saltans that have no trypanosomatid homologs.
among these, mostly bodo-specific, genes are membrane transporters, various protein kinases, and other proteins containing domains commonly associated with cell surfaces.
these and other bodo-specific proteins must include those metabolism pathways, intracellular transport, cellular signalling and subcellular structures that exist in free-living kinetoplastids, but which have been deleted during the evolution of parasitism.
many of these proteins will be widespread among eukaryotic lineages, as is evident in table 2; yet we should also expect to encounter a considerable genetic repertoire unique to the kinetoplastida and so entirely new.
having identified those features of trypanosomatid genomes that reflect their free-living ancestry, a b. saltans genome sequence would also reveal the additions to each parasite genome; structures derived from existing genes and co-opted for novel uses, and genuinely novel genes involved in parasite-specific adaptations.
these enigmatic genes include the numerous and diverse families of surface glycoprotein that form the protective coats around trypanosomatid parasites.
t. brucei, t. cruzi and l. major each display highly derived and complex surface coats to frustrate host immunity, yet they differ in structure and substance and it is not known how each acquired its distinct solution to their common problem.
understanding the origins of these surface architectures will only be achieved with an historical perspective; one principal objective of a b. saltans genome project would be to identify the precursors of proteins such as vsg in t. brucei, mucins and trans-sialidase in t. cruzi, and proteophosphoglycans in leishmania spp.
.
a glimpse of this potential is seen in figure  <dig>  which includes a predicted protein with a complex  <dig> amino acid repeat .
the protein had a high affinity  with a gene family on chromosome  <dig> in leishmania spp., , and a more distant affinity with proteophosphoglycans.
figure 3b shows a sequence alignment of the repeat domain from the b. saltans protein and its leishmanial homologs, where the level of amino acid identity rises to 50%.
CONCLUSIONS
thorough sequencing of a few locations in the b. saltans genome has revealed clear similarities with trypanosomatids, but has also shown that trypanosomatid genome sequences will not be effective guides for any complete bodonid project, due to significant differences in content and gene order.
this mixture of familiar and novel features suggests that b. saltans will indeed provide an effective outgroup for comparisons of trypanosomatid parasites, and, as with the evolution of tubulin repertoire, the historical perspective to understand which aspects of trypanosomatid biology have been retained from their common ancestry, which have been lost, and what has been uniquely derived since.

 the kinetoplastida are unicellular flagellates that include the trypanosomatid parasites, most notably trypanosoma brucei, t. cruzi and leishmania spp. these organisms cause substantial mortality and morbidity in humans and their livestock worldwide as the causative agents of african sleeping sickness, chagas disease and leishmaniasis respectively. bodo saltans is a free-living heterotroph found worldwide in freshwater and marine habitats. it possesses the diagnostic kinetoplastid features, such as flagella sited within a specialised flagellar pocket, glycolytic processes confined to a dedicated organelle, and the characteristic concentration of mitochondrial dna at the base of the flagellum. when comparing trypanosomatid parasites with each other, or collectively with other eukaryotes, the value of b. saltans is as a non-parasitic near relative,, that can illuminate their key evolutionary transitions. five draft genome sequences exist for trypanosoma spp. and four for leishmania spp.; these will be augmented with further strains and other non-human parasites in the coming years. with such excellent comparative resources in place or in development, there is a critical need for a non-trypanosomatid outgroup. in effect, it will provide a model of the ancestral trypanosomatid to distinguish those derived parts of the parasite genomes from those which are a legacy of the free-living ancestor. for instance, such a model will help to resolve whether trypanosomatids previously possessed an algal plastid from which'plant-like' genes in trypanosomatid genomes are derived. as a prelude to a complete b. saltans genome sequencing effort, this study sought to establish an initial understanding of the bodonid genome, its structure and content relative to the trypanosomatids. the most recent kinetoplastid phylogeny has shown that trypanosomatid parasites are just one of many independent acquisitions of parasitism, indeed, a relatively minor component of total diversity. nonetheless, they are, naturally, the most important aspect of kinetoplastid diversity. many features of their completed genome sequences emphasised the common ancestry of t. brucei, t. cruzi and leishmania spp., especially with respect to gene repertoire and order, but their critical pathological differences were also evident at the genomic level. the three human parasites cause distinct diseases; their genomes contain enigmatic adaptations related to pathogenesis and immune evasion, for instance the bloodstream expression site in t. brucei from which its variant surface glycoproteins are expressed, and surface antigen families in general. without an historical dimension, these features cannot be compared, nor understood in an evolutionary context. as it is among the closest bodonid relatives of the trypanosomatids, bodo saltans is a suitable outgroup to address three principal comparative issues: i) understanding how human trypanosomatid parasites acquired their distinct pathological strategies; ii) understanding how the ancestral trypanosomatid became parasitic in terms of derived innovations and loss of genomic repertoire; iii) understanding how typical kinetoplastid features evolved and how these might have been modified for parasitism. quite what to expect from a bodonid genome sequence is an open question. beyond the basic kinetoplastid features named above, the biological differences between bodonids and trypanosomatids are striking. while b. saltans is a bacteriovore, especially prevalent in polluted waters or other environments with high bacterial densities, trypanosomatids are obligate parasites inhabiting a nutrient-rich, but ultimately hostile, host environment, and adept at exploiting their eutrophic environment to maximise proliferation and transmission. by contrast, b. saltans preys on bacterial cells and is probably adapted for resource acquisition within its relatively oligotrophic environment. although bodonids and trypanosomatids are all flagellates, trypanosomatids attach their single flagellum to the cell surface to generate motile force, whereas the anterior flagellum in b. saltans is modified with hair-like mastigonemes, which may assist prey location during feeding. there are wider cytoskeletal differences also; the subpellicular microtubular cortex is instrumental in maintaining the numerous cell forms adopted by trypanosomatids, but is reduced in bodonids,, to the region around the cytostome. perhaps most importantly for understanding the evolution of parasitism, we can expect substantial differences between trypanosomatid cell surfaces that function primarily to manipulate and frustrate the host immune response and bodonid membranes that are perhaps largely concerned with cellular homeostasis. rather than providing definitive answers to these questions, the preliminary sequence data presented here provides an initial insight into a few comprehensively resolved locations in the b. saltans genome, indicating what to expect from gene content and arrangement, and testing the feasibility of a complete sequence project. the sequence contigs were compared with corresponding regions in trypanosomatids, to examine gene content and the conservation of gene order and, therefore, the potential for using trypanosomatid genome sequences as scaffolds to assist assembly and annotation of the b. saltans sequence.  gene structure clones were selected from the b. saltans fosmid library according to random end-sequences and positive results for specific pcr probes. inserts from fosmid clones were shotgun sequenced, comprising mbp in total and an average size of kbp. table describes the composition of the contigs in terms of the affinity shown by each putative coding sequence to sequence databases. putative coding sequences are specified; genes could be predicted by eye because of a definite elevation in gc content in coding regions. subsequent matches to sequence databases showed these features to be correct. the boundaries between coding and flanking regions are marked by a transition from gc-rich to at-rich signatures; the sequences shown in figure clearly demonstrate the gc troughs that appear between coding sequences. this pattern is repeated in other contigs, as shown in subsequent figures. gene density is high relative to corresponding regions in the l. major and t. brucei genome sequences, reflected by the consistently short intercoding sequences across all contigs. figure compares the gene order of one region with positionally orthologous regions in l. major and t. brucei; this, like most fosmid inserts, contains more genes in bodo than in trypanosomatids. note: † clones for which no probe is stated were selected after end-sequencing.* genes were classified according to their affinity: kinetoplastid, other eukaryotic, prokaryotic or unknown. gene content coding sequences without trypanosomatid homologs were compared to sequence and structural databases. many of the gene products are homologous to proteins beyond the kinetoplastida, suggesting that they are core eukaryotic proteins subsequently lost from trypanosomatids; for example, contiguous genes in figure 1a homologous to a gpi-anchored protein in plants and fungi and a hypothetical gene in metazoa. gene products in other regions contain protein domains known elsewhere, for example an abc transporter protein and a nucleotide-sugar transporter protein, strongly indicating that these are bodo-specific members of ubiquitous gene families. some otherwise uncharacterised hypothetical proteins are predicted to expressed on the cell surface. the region shown in figure 3a is notable not only for the base composition of coding regions and admixture of trypanosomatid and bodo-specific genes mentioned above, but also for a hypothetical protein with predicted transmembrane helices and a signal peptide. table contains other examples of bodo-specific hypothetical genes predicted to be surface expressed, including those shown in figure 1a and b. colinearity the extent of conserved gene order, or colinearity, between bodonid and trypanosomatid genome sequences was assessed using the artemis comparison tool. one region of excellent colinearity is shown in figure and, despite disruption by some eukaryotic genes not seen in trypanosomatids, this contig corresponds unmistakably with chromosome in l. major and chromosome in t. brucei. conversely, the presence of so many non-trypanosomatid genes meant that colinearity disappears entirely in some locations, as shown in figure across the genomic regions however, both patterns were atypical; most regions shows brief patches of colinearity, perhaps or genes with conserved synteny, set among larger regions of bodo-specific genes or homologs to trypanosomatid genes from elsewhere in the genome. in this sense, the sequence presented in figure is representative because several coding sequences are homologs of trypanosomatid genes on chromosomes and; these are roughly colinear but the order is disrupted by genes present on other chromosomes or by bodo-specific genes. discussion in this study, various locations in b. saltans genome, amounting to ~ mbp, were sequenced. assuming that the bodonid genome is approximately the same size as a trypanosomatid haploid genome, i.e., 35– mbp, these sequences comprise ~1% of the complete genome sequence, which will therefore contain roughly genes. the success and utility of a b. saltans genome project will depend on its relationship with existing trypanosomatid genome sequences. this study shows that coding regions of the b. saltans genome share several structural features with trypanosomatids, indicating that the project is both feasible and likely to provide a useful comparative resource. putative b. saltans genes lack introns, as in most trypanosomatid genes. they display a conspicuous elevation in gc content, which will greatly assist gene finding. no evidence of strand-switching was observed in b. saltans, corroborating the view that it operates polycistronic transcription, i.e., transcription of many contiguous loci within a single nascent transcript, which is subsequently trans-spliced and polyadenylated to produce mature mrna, as in trypanosomatids. although the arrangement of coding regions along the bodonid chromosome may be conserved with trypanosomatids, it is clear that gene order was not. the extent of conserved synteny, or rather colinear gene order, between bodonid and trypanosomatid genomes is of particular importance to the assembly of any b. saltans genome sequence. the coding regions presented here indicate that trypanosomatid genome sequences will be of limited value in the global assembly of a b. saltans genome sequence. strict colinearity was not normally observed, if only because of the large number of bodo-specific genes interposed between trypanosomatid homologs. colinearity tended to persist over a distance of 3– genes, although some regions displayed conspicuous conservation, while others showed none at all. therefore, this initial exploration of the b. saltans genome demonstrates that it should be possible to resolve a complete genome sequence, but, while the existing trypanosomatid resources will provide some useful guides for annotation, they could not be used as scaffolds for assembly, which should proceed de novo. the purpose of a completed b. saltans genome sequence would be for understanding the evolution of trypanosomatid genome sequences. the mixture of familiar and novel features in the regions sequenced here indicates the value of a bodonid genome sequence in distinguishing trypanosomatid characters inherited from free-living ancestors from characters evolved since the origin of trypanosomatids. hence, the first application would be in determining which parts of the trypanosomatid genome reflect the genomic legacy inherited from free-living ancestors, and show how they have been co-opted and modified for parasitism. bodonid and trypanosomatid cells share various structural features, principally those that characterise kinetoplastid cells. bodonids arrange their mitochondrial dna in kinetoplasts, although their position within the cell differs from trypanosomatids, and conduct their glycolytic pathways within a dedicated organelle. bodonids construct their flagella in a similar manner to trypanosomatids, but deploy them very differently. while b. saltans uses one flagellum for movement and another for feeding, trypanosomatids flagella perform their motility function within the context of their sophisticated cell forms. one might expect these structural similarities to be reflected at the genomic level. α- and β-tubulin, the proteins that facilitate the development of flagella in trypanosomatids, are known to be arranged in tandem gene arrays, with an alternating, heterotypic α-β array in trypanosoma spp. and distinct, monotypic α and β arrays in leishmania spp.. bodonids were shown to share the alternating conformation, suggesting that leishmania spp. and their relatives had abolished the ancestral locus and evolved novel genomic repertoires. however, two b. saltans regions containing tubulin in this study show that modification of tubulin repertoire has also occurred in trypanosoma, since neither of the α-β arrays in b. saltans was found at the genomic position occupied in trypanosomes. this demonstrates the utility of the b. saltans genome in resolving the evolutionary causes of structural or compositional differences between trypanosomatid genomes. the second application of a b. saltans genome sequence would be to identify which components of the free-living legacy have been lost from trypanosomatids, and therefore, how reductive genome evolution has contributed to the parasite genomes. table describes many predicted proteins identified in b. saltans that have no trypanosomatid homologs. among these, mostly bodo-specific, genes are membrane transporters, various protein kinases, and other proteins containing domains commonly associated with cell surfaces. these and other bodo-specific proteins must include those metabolism pathways, intracellular transport, cellular signalling and subcellular structures that exist in free-living kinetoplastids, but which have been deleted during the evolution of parasitism. many of these proteins will be widespread among eukaryotic lineages, as is evident in table 2; yet we should also expect to encounter a considerable genetic repertoire unique to the kinetoplastida and so entirely new. having identified those features of trypanosomatid genomes that reflect their free-living ancestry, a b. saltans genome sequence would also reveal the additions to each parasite genome; structures derived from existing genes and co-opted for novel uses, and genuinely novel genes involved in parasite-specific adaptations. these enigmatic genes include the numerous and diverse families of surface glycoprotein that form the protective coats around trypanosomatid parasites. t. brucei, t. cruzi and l. major each display highly derived and complex surface coats to frustrate host immunity, yet they differ in structure and substance and it is not known how each acquired its distinct solution to their common problem. understanding the origins of these surface architectures will only be achieved with an historical perspective; one principal objective of a b. saltans genome project would be to identify the precursors of proteins such as vsg in t. brucei, mucins and trans-sialidase in t. cruzi, and proteophosphoglycans in leishmania spp.. a glimpse of this potential is seen in figure which includes a predicted protein with a complex amino acid repeat. the protein had a high affinity with a gene family on chromosome in leishmania spp.,, and a more distant affinity with proteophosphoglycans. figure 3b shows a sequence alignment of the repeat domain from the b. saltans protein and its leishmanial homologs, where the level of amino acid identity rises to 50%.  thorough sequencing of a few locations in the b. saltans genome has revealed clear similarities with trypanosomatids, but has also shown that trypanosomatid genome sequences will not be effective guides for any complete bodonid project, due to significant differences in content and gene order. this mixture of familiar and novel features suggests that b. saltans will indeed provide an effective outgroup for comparisons of trypanosomatid parasites, and, as with the evolution of tubulin repertoire, the historical perspective to understand which aspects of trypanosomatid biology have been retained from their common ancestry, which have been lost, and what has been uniquely derived since. 
a b. saltans genome sequence will provide a free-living comparison with parasitic genomes necessary for comparative analyses of existing and future trypanosomatid genomic resources. although synteny was typically conserved for those genes with trypanosomatid homologs, strict colinearity was rarely observed because gene order was regularly disrupted by bodo-specific genes. a complete genome sequence will provide an effective ancestral model for understanding the shared and derived features of known trypanosomatid genomes, but it will also identify those kinetoplastid genome features lost during the evolution of parasitism. structural similarities suggest that its assembly should be solvable, and, although de novo assembly will be necessary, existing trypanosomatid projects will provide some guide to annotation. various coding regions were sequenced to provide a preliminary insight into the bodonid genome sequence, relative to trypanosomatid sequences. bodo saltans is a free-living kinetoplastid and among the closest relatives of the trypanosomatid parasites, which cause such human diseases as african sleeping sickness, leishmaniasis and chagas disease. mbp of b. saltans genome was sequenced from distinct regions and contained coding sequences. the b. saltans genome contains both sequences homologous to trypanosomatids and sequences never seen before. intergenic sequences were typically short, resulting in higher gene density than in trypanosomatids.
 bodo saltans is a free-living kinetoplastid and among the closest relatives of the trypanosomatid parasites, which cause such human diseases as african sleeping sickness, leishmaniasis and chagas disease. a b. saltans genome sequence will provide a free-living comparison with parasitic genomes necessary for comparative analyses of existing and future trypanosomatid genomic resources. various coding regions were sequenced to provide a preliminary insight into the bodonid genome sequence, relative to trypanosomatid sequences.  mbp of b. saltans genome was sequenced from distinct regions and contained coding sequences. as in trypanosomatids, introns were absent and %gc was elevated in coding regions, greatly assisting in gene finding. in the regions studied, roughly 60% of all genes had homologs in trypanosomatids, while 28% were bodo-specific. intergenic sequences were typically short, resulting in higher gene density than in trypanosomatids. although synteny was typically conserved for those genes with trypanosomatid homologs, strict colinearity was rarely observed because gene order was regularly disrupted by bodo-specific genes.  the b. saltans genome contains both sequences homologous to trypanosomatids and sequences never seen before. structural similarities suggest that its assembly should be solvable, and, although de novo assembly will be necessary, existing trypanosomatid projects will provide some guide to annotation. a complete genome sequence will provide an effective ancestral model for understanding the shared and derived features of known trypanosomatid genomes, but it will also identify those kinetoplastid genome features lost during the evolution of parasitism. 