BACKGROUND
brucella are gram-negative bacteria and the causative agent of brucellosis in domesticated animals, wildlife, and humans.
although the bacteria exhibit relatively strong host preference, separating the various brucella species has proven extremely difficult due to minimal genetic differentiation  <cit> .
based on dna-dna hybridization, brucellae have previously been proposed to comprise a single species, with a series of biovars  <cit> .
however, phylogenetic approaches explicitly incorporating host preference and virulence have upheld the six classical brucella species: b. abortus , b. melitensis , b. suis , b. canis , b. neotomae , and b. ovis   <cit> .
several new species have been recently described, including at least two species in marine mammals   <cit>  and an additional species b. microti in the common vole   <cit> .
other brucella species undoubtedly exist within known and novel hosts  <cit> .
the limited genetic differentiation and conservation within brucella genomes has made genotyping a challenge.
a promising approach that is capable of being incorporated into high-throughput assays is the use of single nucleotide polymorphisms .
comparisons of brucella genomes have revealed hundreds of snps that distinguish various strains  <cit> .
although the era of next-generation sequencing [reviewed in  <cit>  is rapidly increasing available data for microbial genomic comparisons, full genome sequencing is currently not cost effective for genotyping large numbers of isolates and requires intensive bioinformatic efforts.
furthermore, in low diversity organisms such as brucella only a small fraction of the nucleotides are polymorphic, suggesting that once rare polymorphisms are discovered, methods other than whole genome sequencing are more efficient for most purposes.
molecular inversion probe  assays are an efficient and relatively inexpensive method of interrogating thousands of snps in large numbers of samples  <cit> .
although typically applied to research on human disease, the mip assay can be readily applied to genotype snps in bacterial genomes.
we compared four genomes from b. abortusb.
melitensis, and b. suis to discover snps.
we created a mip assay to genotype  <dig> diverse samples and to discover canonical snps  <cit>  that define brucella species, strains, or isolates.
we then created snp-specific assays that use a capillary electrophoresis universal-tailed mismatch amplification mutation assay  approach for major branch points in the phylogeny and screened them against a large and diverse collection of isolates .
finally, we compared these results to  <dig> brucella whole genomes in silico to place our genotyping into context with all major biovars and isolates.
RESULTS
a total of  <dig> mip probes consistently amplified their target sites across  <dig> samples.
among these probes,  <dig> identified truly polymorphic sites.
this dataset contained only 4% missing data , where no snp was determined at a particular locus for a sample.
comparisons to snps from  <dig> whole genomes yielded  <dig> snps in our phylogenetic analysis , allowing for placement of these genomes within the mip tree and in silico determination of their snp alleles.
the full mip tree with all  <dig> loci and  <dig> samples, excluding the whole genomes in the comparisons, is also given .
our mip assay distinguished b. abortus, b. melitensis, and b. suis; the three prominent brucella species .
a total of  <dig> snp loci had complete allele calls  across all  <dig> samples.
the assay strongly differentiated b. melitensis and b. suis into two clades each.
within b. melitensis, at least  <dig> snps on branch h separate strain 16 m and its related isolates in biovar  <dig> from isolate 63– <dig> and related isolates of biovars  <dig>  subsequent analyses  group biovar  <dig> with biovar  <dig> isolates.
based on these data, the assay for branch h appears to be specific to b. melitensis biovar  <dig>  the two clades in b. suis, denoted by branches i and j, included all isolates of in the species except for biovar  <dig>  which was distantly related to other members of this species.
some isolates from b. suis are more closely related to b. canis isolates  than to other b. suis isolates , indicating that b. suis is a paraphyletic species.
of the snps with complete genotyping data, at least  <dig> snps on branch i separate b. suis  <dig> and related isolates from b. canis and related b. canis and b. suis isolates.
however, no snps uniquely identified b. canis.
brucella abortus was even more differentiated, and can be divided into at least four distinct clades.
samples from b. abortus biovar  <dig>  which contains the two snp discovery strains, plus the type strain for biovar  <dig> , make up the majority of samples and diversity within the b. abortus clade.
all were found on branch e, which was further divided into branches a-d. samples from the other b. abortus biovars are more distantly related and form distinct branches.
as expected, the other species in the assay, including b. neotomae, b. ceti, b. pinnipedialis, and b. ovis were poorly distinguished from each other.
missing data for snp loci caused the differences in branch lengths that are seen between figure  <dig> and figure s <dig>
cuma assays verified the snp alleles for all  <dig> of the samples run in the mip assay.
in addition, the  <dig> snps from the cuma assays allowed for placement of a larger panel of  <dig> isolates within the mip phylogeny .
nine of these snps were located on the same branch as another assay, or in one instance the same branch as two other assays, so did not provide any additional genotyping information.
in the final tally there were assays for eight branches of the phylogeny, with assays specific to the following prominent isolates/clades and related isolates: b. abortus  <dig>  b. abortus 2308 + s <dig>  b. melitensis 16 m, b. melitensis biovar  <dig>  and b. suis  <dig>  from our diverse isolate collection we had the following distribution of calls for the branches, with the most derived call taking precedence over more ancestral calls: a =  <dig>  b =  <dig>  c =  <dig>  d =  <dig>  e =  <dig>  f =  <dig>  g =  <dig>  h =  <dig>  i =  <dig>  j =  <dig>  no derived call  =  <dig>  no call for any assay =  <dig>  ancestral within b. abortus =  <dig>  ancestral within b. melitensis =  <dig>  ancestral within b. suis =  <dig>
discussion
our assays show clear distinctions within and among b. abortusb.
melitensis, and b. suis.
our cuma assays targeted clade-specific snps that can be incorporated into most other genotyping assays such as taqman real-time pcr for increased sensitivity  <cit> .
we have identified several important targets that should prove useful for clinical, epidemiological, and forensic purposes.
for example, the assays targeting branches a, d, and i are specific to isolates closely related to b. abortus  <dig> and b. abortus 9– <dig>  and b. suis  <dig>  respectively.
the assays for f and g target the same branch and identify b. melitensis 16 m and closely related isolates.
isolates from b. abortus  <dig> and 9– <dig>  b. suis  <dig>  and b. melitensis 16 m are from common, genetically monomorphic clades of brucella and the snp assays developed here are a reliable and useful way of identifying these four common groups.
branch e is particularly interesting in terms of brucella taxonomy.
the clade that this branch defines includes isolates from b. abortus biovars  <dig>   <dig>  and  <dig>  potential issues with biovar and phylogenetic correspondence in b. abortus have been noted previously  <cit> .
upon closer evaluation of the whole genomes used in our analyses, the apparent paraphyly within b. abortus biovar  <dig>  since isolates from biovar  <dig> are within the biovar  <dig> clade, does not hold true when all the genomes are included.
however, cuma assays indicate that at least four isolates from other b. abortus biovars  fall onto the b/c branch.
this would suggest that either biovar  <dig> is paraphyletic or there have been issues with biovar determination.
snp-based approaches also enable assessment of errors in genome sequences.
whole genome comparisons of the region associated with snp <dig>  which were intended to target branch j in b. suis/ b. canis, also share a snp allele with b. abortus 9– <dig>  taken at face value, this would suggest homoplasy at this locus.
yet, in our cuma assays b. abortus 9– <dig> did not group with b. suis, likely indicating sequencing error.
finding nucleotide polymorphisms that differentiate clades, species, or isolates is dependent on the genomes used for snp discovery.
in general, one will only find those snps that exist among the genomic samples used in the comparisons and novel snps will remain undiscovered  <cit> .
this discovery bias can strongly affect taxonomic interpretation of results  <cit> .
although discovery bias is often less consequential for genotyping efforts, the effects of our choice of strains for snp discovery are clearly apparent in our phylogenetic tree.
the discovery strains are distinguished by their positions at terminal branches in the phylogeny.
there is greater diversity observed in b. abortus simply because two strains were part of the discovery panel.
furthermore, although isolates on a branch will be grouped by the snps they share , additional structure exists in the “true” phylogeny that is not apparent in the genotype tree.
branch lengths are also highly affected by the snp discovery process.
species that are basal within this phylogeny, such as b. cetib.
pinnipedialisb.
ovis, and b. neotomae have short branch lengths merely because these genomes were not part of snp discovery.
it must also be noted that b. suis biovar  <dig> is part of this basal group.
snps that should group it with the rest of the b. suis clade were not present in our mip assay, which is not surprising since this branch is extremely short, even with whole genome analysis [jtf unpubl.
data,  <cit> .
we did not observe differentiation of these and the other brucella species, nor did we expect it because genomes from these groups were not a part of snp discovery.
whole genome resequencing at the broad institute of mit/harvard recently generated genomes for over  <dig> additional brucella strains and these genomes should provide a broad basis for future genotyping efforts, with canonical snps developed for each of the important isolates and clades.
future genotyping efforts should include snps from all of the recognized species and biovars.
comparative work using some of these genomes has already been fruitful, demonstrating the emergence of the marine brucella from within the terrestrial brucella and showing a methodology for whole genome analysis  <cit> .
a trade-off exists in current genotyping efforts between throughput and genomic sampling.
does one aim for a maximum amount of potentially informative loci through approaches such as whole genome sequencing but having to sacrifice the number of isolates that can be evaluated?
or does one aim for more complete sampling of large numbers of isolates but with a limited set of loci using individual snp assays such as cuma?
of course the ultimate answer depends on your research interest or clinical application as well as the amount of resources at hand.
mip assays provide phylogenetic resolution for an intermediate number of samples and intermediate number of snps.
nonetheless, mip assays, or any assays based on previously discovered snps, will always have their inference limited by the genomes used in snp discovery  <cit> .
mip assays do however allow for a focus on resolving branches of specific interest.
data from these assays then allows for targeted down selection of loci so that focal branches and isolates on them can be thoroughly interrogated using individual snp assays.
identifying canonical snps and verifying their ability to differentiate clades by screening large numbers of isolates is the essential part of genotyping  <cit> .
less important is the type of assay used for snp differentiation because it is highly dependent on the numbers of snps and samples one wants to screen.
the mip and cuma snp screening techniques are just two of many methods that can be used for snp genotyping in brucella and other bacteria.
CONCLUSIONS
we developed and evaluated two different snp-based genotyping systems for three well studied species of brucella: b. abortus, b. melitensis, and b. suis.
the first genotyping approach, using molecular inversion probes, divided the species into its three respective groups and allowed for finer scale genetic resolution.
notably, this resolution occurred almost entirely within the lineages of the four strains that were used for snp discovery: b. abortus  <dig>  b. abortus 9– <dig>  b. melitensis 16 m, and b. suis  <dig>  this is to be expected since the choice of genomes for snp discovery has a pervasive effect on the phylogenetic patterns that can be determined.
we followed the mip assay with development of capillary electrophoresis universal-tailed mismatch amplification mutation assays that targeted major branch points in the mip phylogeny.
we then genotyped a large and diverse collection of isolates.
the main result is the development of fine scale genotyping assays that target among the most important and widespread lineages of brucella.
moreover, these and closely related isolates can be easily and quickly distinguished from all other brucella isolates.
despite the era of whole genome sequencing being upon us, snp-based genotyping and other targeted assays will remain relevant.
sequencing technology is advancing rapidly and costs per genome are quickly diminishing such that whole genome genotyping is the future of phylogenetics, forensics, and diagnostics.
in fact, whole genome genotyping will soon be cost competitive with most other genotyping strategies and will have the advantage of capturing nearly all of the genetic variation with no issues of discovery bias.
nonetheless, targeted assays will remain a viable option for such goals as rapidly and easily characterizing large strain collections, clinical samples, and samples containing only trace amounts of dna.
concerted efforts must be made to incorporate data from earlier genotyping strategies into genomic databases so this wealth of genetic information is not lost in the rush to sequence everything.

 brucella are gram-negative bacteria and the causative agent of brucellosis in domesticated animals, wildlife, and humans. although the bacteria exhibit relatively strong host preference, separating the various brucella species has proven extremely difficult due to minimal genetic differentiation. based on dna-dna hybridization, brucellae have previously been proposed to comprise a single species, with a series of biovars. however, phylogenetic approaches explicitly incorporating host preference and virulence have upheld the six classical brucella species: b. abortus, b. melitensis, b. suis, b. canis, b. neotomae, and b. ovis. several new species have been recently described, including at least two species in marine mammals and an additional species b. microti in the common vole. other brucella species undoubtedly exist within known and novel hosts. the limited genetic differentiation and conservation within brucella genomes has made genotyping a challenge. a promising approach that is capable of being incorporated into high-throughput assays is the use of single nucleotide polymorphisms. comparisons of brucella genomes have revealed hundreds of snps that distinguish various strains. although the era of next-generation sequencing [reviewed in is rapidly increasing available data for microbial genomic comparisons, full genome sequencing is currently not cost effective for genotyping large numbers of isolates and requires intensive bioinformatic efforts. furthermore, in low diversity organisms such as brucella only a small fraction of the nucleotides are polymorphic, suggesting that once rare polymorphisms are discovered, methods other than whole genome sequencing are more efficient for most purposes. molecular inversion probe assays are an efficient and relatively inexpensive method of interrogating thousands of snps in large numbers of samples. although typically applied to research on human disease, the mip assay can be readily applied to genotype snps in bacterial genomes. we compared four genomes from b. abortusb. melitensis, and b. suis to discover snps. we created a mip assay to genotype diverse samples and to discover canonical snps that define brucella species, strains, or isolates. we then created snp-specific assays that use a capillary electrophoresis universal-tailed mismatch amplification mutation assay approach for major branch points in the phylogeny and screened them against a large and diverse collection of isolates. finally, we compared these results to brucella whole genomes in silico to place our genotyping into context with all major biovars and isolates.  a total of mip probes consistently amplified their target sites across samples. among these probes, identified truly polymorphic sites. this dataset contained only 4% missing data, where no snp was determined at a particular locus for a sample. comparisons to snps from whole genomes yielded snps in our phylogenetic analysis, allowing for placement of these genomes within the mip tree and in silico determination of their snp alleles. the full mip tree with all loci and samples, excluding the whole genomes in the comparisons, is also given. our mip assay distinguished b. abortus, b. melitensis, and b. suis; the three prominent brucella species. a total of snp loci had complete allele calls across all samples. the assay strongly differentiated b. melitensis and b. suis into two clades each. within b. melitensis, at least snps on branch h separate strain 16 m and its related isolates in biovar from isolate 63– and related isolates of biovars subsequent analyses group biovar with biovar isolates. based on these data, the assay for branch h appears to be specific to b. melitensis biovar the two clades in b. suis, denoted by branches i and j, included all isolates of in the species except for biovar which was distantly related to other members of this species. some isolates from b. suis are more closely related to b. canis isolates than to other b. suis isolates, indicating that b. suis is a paraphyletic species. of the snps with complete genotyping data, at least snps on branch i separate b. suis and related isolates from b. canis and related b. canis and b. suis isolates. however, no snps uniquely identified b. canis. brucella abortus was even more differentiated, and can be divided into at least four distinct clades. samples from b. abortus biovar which contains the two snp discovery strains, plus the type strain for biovar, make up the majority of samples and diversity within the b. abortus clade. all were found on branch e, which was further divided into branches a-d. samples from the other b. abortus biovars are more distantly related and form distinct branches. as expected, the other species in the assay, including b. neotomae, b. ceti, b. pinnipedialis, and b. ovis were poorly distinguished from each other. missing data for snp loci caused the differences in branch lengths that are seen between figure  and figure s cuma assays verified the snp alleles for all of the samples run in the mip assay. in addition, the snps from the cuma assays allowed for placement of a larger panel of isolates within the mip phylogeny. nine of these snps were located on the same branch as another assay, or in one instance the same branch as two other assays, so did not provide any additional genotyping information. in the final tally there were assays for eight branches of the phylogeny, with assays specific to the following prominent isolates/clades and related isolates: b. abortus b. abortus 2308 + s b. melitensis 16 m, b. melitensis biovar and b. suis from our diverse isolate collection we had the following distribution of calls for the branches, with the most derived call taking precedence over more ancestral calls: a =  b =  c =  d =  e =  f =  g =  h =  i =  j =  no derived call  =  no call for any assay =  ancestral within b. abortus =  ancestral within b. melitensis =  ancestral within b. suis =  discussion our assays show clear distinctions within and among b. abortusb. melitensis, and b. suis. our cuma assays targeted clade-specific snps that can be incorporated into most other genotyping assays such as taqman real-time pcr for increased sensitivity. we have identified several important targets that should prove useful for clinical, epidemiological, and forensic purposes. for example, the assays targeting branches a, d, and i are specific to isolates closely related to b. abortus and b. abortus 9– and b. suis respectively. the assays for f and g target the same branch and identify b. melitensis 16 m and closely related isolates. isolates from b. abortus and 9– b. suis and b. melitensis 16 m are from common, genetically monomorphic clades of brucella and the snp assays developed here are a reliable and useful way of identifying these four common groups. branch e is particularly interesting in terms of brucella taxonomy. the clade that this branch defines includes isolates from b. abortus biovars and potential issues with biovar and phylogenetic correspondence in b. abortus have been noted previously. upon closer evaluation of the whole genomes used in our analyses, the apparent paraphyly within b. abortus biovar since isolates from biovar are within the biovar clade, does not hold true when all the genomes are included. however, cuma assays indicate that at least four isolates from other b. abortus biovars fall onto the b/c branch. this would suggest that either biovar is paraphyletic or there have been issues with biovar determination. snp-based approaches also enable assessment of errors in genome sequences. whole genome comparisons of the region associated with snp which were intended to target branch j in b. suis/ b. canis, also share a snp allele with b. abortus 9– taken at face value, this would suggest homoplasy at this locus. yet, in our cuma assays b. abortus 9– did not group with b. suis, likely indicating sequencing error. finding nucleotide polymorphisms that differentiate clades, species, or isolates is dependent on the genomes used for snp discovery. in general, one will only find those snps that exist among the genomic samples used in the comparisons and novel snps will remain undiscovered. this discovery bias can strongly affect taxonomic interpretation of results. although discovery bias is often less consequential for genotyping efforts, the effects of our choice of strains for snp discovery are clearly apparent in our phylogenetic tree. the discovery strains are distinguished by their positions at terminal branches in the phylogeny. there is greater diversity observed in b. abortus simply because two strains were part of the discovery panel. furthermore, although isolates on a branch will be grouped by the snps they share, additional structure exists in the “true” phylogeny that is not apparent in the genotype tree. branch lengths are also highly affected by the snp discovery process. species that are basal within this phylogeny, such as b. cetib. pinnipedialisb. ovis, and b. neotomae have short branch lengths merely because these genomes were not part of snp discovery. it must also be noted that b. suis biovar is part of this basal group. snps that should group it with the rest of the b. suis clade were not present in our mip assay, which is not surprising since this branch is extremely short, even with whole genome analysis [jtf unpubl. data,. we did not observe differentiation of these and the other brucella species, nor did we expect it because genomes from these groups were not a part of snp discovery. whole genome resequencing at the broad institute of mit/harvard recently generated genomes for over additional brucella strains and these genomes should provide a broad basis for future genotyping efforts, with canonical snps developed for each of the important isolates and clades. future genotyping efforts should include snps from all of the recognized species and biovars. comparative work using some of these genomes has already been fruitful, demonstrating the emergence of the marine brucella from within the terrestrial brucella and showing a methodology for whole genome analysis. a trade-off exists in current genotyping efforts between throughput and genomic sampling. does one aim for a maximum amount of potentially informative loci through approaches such as whole genome sequencing but having to sacrifice the number of isolates that can be evaluated? or does one aim for more complete sampling of large numbers of isolates but with a limited set of loci using individual snp assays such as cuma? of course the ultimate answer depends on your research interest or clinical application as well as the amount of resources at hand. mip assays provide phylogenetic resolution for an intermediate number of samples and intermediate number of snps. nonetheless, mip assays, or any assays based on previously discovered snps, will always have their inference limited by the genomes used in snp discovery. mip assays do however allow for a focus on resolving branches of specific interest. data from these assays then allows for targeted down selection of loci so that focal branches and isolates on them can be thoroughly interrogated using individual snp assays. identifying canonical snps and verifying their ability to differentiate clades by screening large numbers of isolates is the essential part of genotyping. less important is the type of assay used for snp differentiation because it is highly dependent on the numbers of snps and samples one wants to screen. the mip and cuma snp screening techniques are just two of many methods that can be used for snp genotyping in brucella and other bacteria.  we developed and evaluated two different snp-based genotyping systems for three well studied species of brucella: b. abortus, b. melitensis, and b. suis. the first genotyping approach, using molecular inversion probes, divided the species into its three respective groups and allowed for finer scale genetic resolution. notably, this resolution occurred almost entirely within the lineages of the four strains that were used for snp discovery: b. abortus b. abortus 9– b. melitensis 16 m, and b. suis this is to be expected since the choice of genomes for snp discovery has a pervasive effect on the phylogenetic patterns that can be determined. we followed the mip assay with development of capillary electrophoresis universal-tailed mismatch amplification mutation assays that targeted major branch points in the mip phylogeny. we then genotyped a large and diverse collection of isolates. the main result is the development of fine scale genotyping assays that target among the most important and widespread lineages of brucella. moreover, these and closely related isolates can be easily and quickly distinguished from all other brucella isolates. despite the era of whole genome sequencing being upon us, snp-based genotyping and other targeted assays will remain relevant. sequencing technology is advancing rapidly and costs per genome are quickly diminishing such that whole genome genotyping is the future of phylogenetics, forensics, and diagnostics. in fact, whole genome genotyping will soon be cost competitive with most other genotyping strategies and will have the advantage of capturing nearly all of the genetic variation with no issues of discovery bias. nonetheless, targeted assays will remain a viable option for such goals as rapidly and easily characterizing large strain collections, clinical samples, and samples containing only trace amounts of dna. concerted efforts must be made to incorporate data from earlier genotyping strategies into genomic databases so this wealth of genetic information is not lost in the rush to sequence everything. 
we then genotyped a diverse collection of brucella strains at these snp loci and generated a phylogenetic tree of relationships. first, we developed a microarray-based assay based on single nucleotide polymorphisms that were identified from whole genome comparisons of two b. abortus isolates, one b. melitensis, and one b. suis. our snp microarray readily distinguished b. abortus, b. melitensis, and b. suis, differentiating b. melitensis and b. suis into two clades each. our assays represent snp genotyping approaches that can reliably determine the evolutionary relationships of bacterial isolates without the need for whole genome sequencing of all isolates. second, we developed a selective primer-extension assay system using capillary electrophoresis that targeted high value snps across major branches of the phylogeny and determined their genotypes in a large collection of diverse isolates. we have identified clade-specific snps in brucella that can be used for rapid assignment into major groups below the species level in the three main brucella species. brucella abortus was divided into four major clades.
 brucellosis is a worldwide disease of mammals caused by alphaproteobacteria in the genus brucella. the genus is genetically monomorphic, requiring extensive genotyping to differentiate isolates. we utilized two different genotyping strategies to characterize isolates. first, we developed a microarray-based assay based on single nucleotide polymorphisms that were identified from whole genome comparisons of two b. abortus isolates, one b. melitensis, and one b. suis. we then genotyped a diverse collection of brucella strains at these snp loci and generated a phylogenetic tree of relationships. second, we developed a selective primer-extension assay system using capillary electrophoresis that targeted high value snps across major branches of the phylogeny and determined their genotypes in a large collection of diverse isolates.  our snp microarray readily distinguished b. abortus, b. melitensis, and b. suis, differentiating b. melitensis and b. suis into two clades each. brucella abortus was divided into four major clades. our capillary-based snp genotyping confirmed all major branches from the microarray assay and assigned all samples to defined lineages. isolates from these lineages and closely related isolates, among the most commonly encountered lineages worldwide, can now be quickly and easily identified and genetically characterized.  we have identified clade-specific snps in brucella that can be used for rapid assignment into major groups below the species level in the three main brucella species. our assays represent snp genotyping approaches that can reliably determine the evolutionary relationships of bacterial isolates without the need for whole genome sequencing of all isolates. 