BACKGROUND
in the thirteen years since the announcement of the first complete organism genome  <cit> , there has been a rapid accumulation of sequence data from complete and draft genomes.
the number of complete or almost-complete projects is in the range of  <dig>   <cit> , but this number is a "moving target," and improvements in sequencing technologies over the past decade ensure continued rapid expansion in the number and diversity of organisms that are analyzed by complete genome sequencing.
despite these significant advances in data acquisition, there have not been commensurate improvements in data-quality assessment and refinement during this period.
individual miscalled bases are assumed to be present in practically all completed genome sequences, and their frequency has been suggested to be between 1– <dig> errors per  <dig> kb  <cit>  and has been measured in some instances to be at most  <dig> error per  <dig> kb  <cit> .
errors in microbial genomes are believed to be generally restricted to point miscalls, with large-scale rearrangements rarely occurring  <cit> .
to identify and correct errors, recent studies have utilized microarray-based detection, in which errors in a subject genome are identified by comparison to a reference genome which served as the basis for array construction.
for example, this method has been employed successfully in escherichia coli  <cit>  and bacillus anthracis  <cit> .
however, these analyses are unidirectional: "errors" are defined as sequence distinct from that of the reference genome, and therefore errors in the reference genome cannot be detected.
as small nucleotide changes in a genome model often manifest as large protein errors – for instance, due to introduction of frameshift and nonsense errors – multiple approaches have capitalized on this protein signal to detect dna errors in complete genomes  <cit> .
by comparing protein-coding sequences in a subject strain to those in a closely-related strain or to closely-related proteins in molecular databases, one can identify those that are potentially truncated inappropriately in the subject strain and target those regions for resequencing.
targeted resequencing has been applied successfully in b. subtilis  <cit>  and mycobacterium smegmatis  <cit> , and in both cases the errors were restricted to changes in 1– <dig> nucleotides.
importantly, perrodou et al.
<cit>  generalized this method in silico to make it available to any subject organism of interest.
targeted resequencing is efficient and available to a wide range of investigators because:  the initial steps are completed in silico prior to proceeding to the wet laboratory; and  when a closely-related strain is available targeted resequencing provides an efficient means to identify discrepancies that alter coding sequence predictions.
in this study, we focus on the genome of the luminous gram-negative bacterium vibrio fischeri es <dig>  v. fischeri forms symbiotic associations with squid and fish, and the association between v. fischeri and the hawaiian bobtail squid euprymna scolopes represents one of the most powerful natural models for the study of mutualistic animal-microbe relationships.
specific strains of symbiotic v. fischeri colonize a dedicated "light organ" in the squid host, multiply to high density, and exhibit luminescence in a density-dependent manner  <cit> .
the light produced by the bacteria is believed to aid the squid host by providing protection from predators: the shadow revealed from the nocturnal-foraging squid in moonlight is camouflaged by the downward-welling light of the host-associated v. fischeri  <cit> .
in return, the bacterium benefits from a protected, nutrient-rich environment.
this was the first system in which it was shown that a specific symbiont directs normal animal development  <cit> , and now represents an emerging model for cross-kingdom genomics-based studies.
the genomic potential for this system is based on a strong history of molecular inquiry on both the symbiont and host sides of the interaction.
first, the complete genome sequence of squid symbiont v. fischeri es <dig> has been published and studied, and the sequence revealed novel insights into pilin gene diversity and the distribution of toxin genes in beneficial bacteria  <cit> .
second, based on the genome sequence a number of global studies have been initiated; the first sets to be published yield novel results about how chemical communication among v. fischeri strains regulates bacterial behavior  <cit>  and how two-component signal transduction affects host-interaction  <cit> .
third, an est library of the squid host  <cit>  has provided novel insight into cephalopod genetic capabilities and widely conserved signaling pathways such as the nf-κb pathway  <cit> .
fourth, the phenomenon we now call quorum sensing – autoinduced density-dependent cell-cell communication – was first described in v. fischeri  <cit> , and a number of evolutionary and modeling studies of this process have focused on the well-characterized systems in v. fischeri.
fifth, by having access to the natural host – a rarity among systems in which high-throughput genetic and genomics approaches are applicable – we can exploit the high information content in the coevolved squid-vibrio relationship to learn how closely-related pathogenic marine microbes interact with natural hosts that have yet to be identified.
sixth, the draft genome of a second strain of v. fischeri, the fish symbiont mj <dig>  is being completed and will provide a strong platform for applying comparative genomic approaches to the study of host-specificity.
while undertaking such a comparative study among v. fischeri strains, we detected a high incidence of suspected genomic anomalies in the published sequence of v. fischeri es <dig>  we resequenced these suspect regions and identified 91% of these loci to be in error.
notably, in fourteen of the cases we detected misincorporation of extraneous sequence in the published assembly, leading to the appearance of duplicated dna where none existed.
in five other cases, the sequence in the suspect region was correct in the published sequence and the resulting gene product would be predicted to be nonfunctional; we therefore designated these features as pseudogenes in es <dig>  in addition to correcting these features, we completed a full genomic update of es <dig> gene annotations, and incorporated the addition of  <dig> genes that were previously unannotated into release  <dig>  of the es <dig> annotation.
together these updates advance v. fischeri as a platform for functional and comparative genomic studies, and demonstrate how a targeted set of approaches may yield high impact on genomic quality improvement.
RESULTS
identification of suspect genomic regions
we obtained the draft genome sequence of v. fischeri strain mj <dig> and, as part of our initial analysis, we conducted a number of reciprocal blast analyses to compare its predicted proteome with that of the completely sequenced conspecific strain es <dig>  <cit> .
we used blastp  <cit>  to identify orthologs between the two strains, using a modified reciprocal best-hit approach as outlined in the methods.
a surprising outcome from this analysis was the occurrence of over seventy protein-coding genes in mj <dig> with reciprocal best-hits to two neighboring genes in es <dig>  at the time that we were performing this analysis, a handful of cases were being identified empirically in which neighboring genes in es <dig> were actually one gene, and that the appearance of two genes resulted from frameshift or nonsense errors in the original sequence data.
examples that were identified independent of this work include ptsi  <cit> , fnr , and acs .
analysis of the suspect regions supported the hypothesis that there were a large number of loci in es <dig> in which sequencing errors had led to the miscalling of one gene as multiple orfs.
in support of this hypothesis, we identified a number of genes that are essential in escherichia coli and other bacteria, but that were split in version  <dig>  of the es <dig> sequence.
these included dnag, ftsq, mukb, nusg, rplc, rpln, rplo, rpob, rpoc, thrs, and tils  <cit> , and the conditionally-essential rpoh  <cit> .
second, we identified eleven ambiguous bases  that had been called in the original sequence, and the incidence of these bases correlated with the presence of suspect orfs.
in addition to suspected frameshifts and substitutions, we also identified fourteen regions in which it appeared that extraneous sequence had been incorporated that was highly similar to neighboring sequence, with the size of the duplicated/extraneous region ranging from  <dig> bp to  <dig> bp.
in one case, pre-genomic sequencing of a suspect region did not identify any repeated sequence  <cit> .
therefore, we hypothesized that these regions represented assembly errors in which the same stretch of dna was mistakenly incorporated twice into the genome's sequence.
these regions typically contained a few unique base pairs at either end – likely due to low-coverage sequencing – that led to the misincorporation, but were otherwise essentially a direct repeat of dna that had the effect of introducing extra and/or truncated orfs.
a list of the loci targeted for resequencing was assembled and each was assigned a "target number"; that number is used consistently in tables and figures so that the primer sequences used to analyze the data may be correlated with the resulting sequence and analysis.
in addition to the blastp-based identification of potential errors, we undertook a full-length visual comparison of the chromosomes of v. fischeri es <dig> and mj <dig>  given the prevalence of errors detected by identifying adjacent orfs that likely represented a single orf, we hypothesized that there were probably other cases of errors that would not have manifest themselves in this way.
examples of other suspected errors that warranted investigation included situations in which one of the fragmented orfs was too small to be detected as an ortholog candidate by the blastp filters, or in which the second fragment did not lead to a predicted open reading frame.
using the program mauve  <cit> , we analyzed orfs along the length of the chromosomes, identifying candidates that had suspect 5' or 3' ends.
in some cases, these appeared to result solely from annotation differences, in which identical sequences had predicted translational start sites  that were called at distinct points in the two annotations.
in other cases, sequence differences underlay the unique orf boundaries, and we targeted those for our analysis.
furthermore, there were three cases in which putative extraneous sequence was visually identified in intergenic sequence, which could not have been detected by blastp analysis in the absence of annotated orfs .
these cases were added to the list of targeted loci.
finally, any remaining ambiguous bases in the sequence were targeted for resequencing.
sequence clarification
we examined a total of  <dig> targets for resequencing.
our general approach involved amplifying across the target, and then sequencing the amplified product with the pcr primers.
in cases where we were clarifying the sequence following a large detected "deletion" , we amplified a larger product and sequenced from a set of sequencing primers across both strands.
for the oligonucleotide primers used for pcr and sequencing see additional file  <dig>  with one exception, all of the primer pairs amplified products in which there was a clear, predominant band, and thus served as satisfactory templates for sequencing.
the primer pair that failed to amplify  included a primer that was in a region that does not exist in the true es <dig> sequence, as clarified by our analysis of target no.
<dig>  therefore the absence of a band in this case supports the deletion that emerged from target no.
<dig>
seventy-five of the  <dig> sets  of resequencing targets examined were found to be in error in the published es <dig> sequence.
the errors, subsequent changes, new locus tags, and new annotations, are listed in table  <dig>  conceptual diagrams of representative sequencing and other annotation changes discussed during this report are illustrated in figure  <dig>  note that with this update, the locus tag format has been modified to the new ncbi format for locus tags .
as a convention, in cases of gene fusion, the locus tag of the 5'-proximal  fragment retained its locus tag identifier, while the identifier for the remaining gene fragment were deaccessioned.
correction types: dl, large deletion; fs, frameshift; ms, missense; ns, nonsense; n, ambiguous nucleotide.
s/m indicates whether ingle or ultiple nucleotides were affected by the sequence change.
it is thought that the creation of false large-scale genomic rearrangements such as insertions rarely occurs in microbial genome projects  <cit> ; however, we confirmed the presence of all fourteen predicted insertions by amplifying from the respective unique flanking regions, and demonstrating that the bands obtained are inconsistent with the previous sequence model .
in each case, the bands observed were smaller than predicted, and the sequence obtained led to the precise deletion of the extraneous repeated dna in the new model.
most of the resulting changes led to the fusion of two – or in some cases three – neighboring orfs, and/or the extension of orfs at the 5' or 3' end .
in one case , the deletion affected only an intergenic region that contains no annotated features.
in another case  the deletion identified by visual analysis in mauve affected what was believed to be a 1498-bp intergenic region between rlue and vf_ <dig>  the corrected sequence revealed this region to be only  <dig> bp in length, but also that it contains a predicted lipoprotein conserved in v. fischeri mj <dig>  the new release reflects the sequence deletion, as well as the added annotation for this gene .
the resulting sequence corrections led us to propose a number of protein annotations that were consistent with our predictions.
based on the corrected sequence, many conserved genes now more closely resemble their orthologs in other species.
in other cases, the domain structure of even poorly characterized proteins supported the accuracy of the corrections.
for example, target no.
<dig> extended the 3' end of vf_ <dig> by correcting a frameshift mutation.
analysis of protein domains by conserved domain search  identified an incomplete ggdef  domain in the protein's c-terminus, and correction of the frameshift led to inclusion of the entire domain.
pseudogenes and degeneration in umuc
in some of the cases we confirmed the published es <dig> sequence to be correct, and that the orf boundaries  were correct in es <dig> version  <dig> .
table  <dig> lists those five cases that we can now more confidently assume to be pseudogenes in es <dig> because they appear to be nonfunctional given their predicted amino acid sequence.
in each case, the indicated defect is predicted to interrupt a significant portion of the coding sequence required for function in well-characterized homologs.
the n-acetylglucosaminyltransferase vf_a <dig> has two  paralogs in the genome, and es <dig> is capable of utilizing n-acetylglucosamine as a sole n+c source : therefore, the appearance of a pseudogene at this location does not have obvious functional consequences for the cell.
there is little information about the remaining four pseudogenes, except for umuc.
the transcriptional organization between the genes encoding the dna polymerase v subunits umud and umuc is conserved between es <dig> and mj <dig> .
however, umuc has uniquely degenerated in es <dig>  with both a nonsense codon and a 5-bp repeat expansion following the nonsense codon.
dna polymerase v is responsible for error-prone translesion synthesis , which allows dna synthesis to proceed despite a high rate of error incorporation  <cit> , yet there are organisms, including v. cholerae el tor, that apparently do not encode these functions  <cit> .
whether the situation in es <dig> represents an evolutionary transition state, or instead this arrangement  has relevant functional implications remains to be determined.
annotation of previously uncalled protein-coding genes, regulatory rnas, and operon leader peptides
because examination of the intergenic region corrected by target no.
<dig> revealed a likely protein-coding gene, we asked whether there were other genes present within the es <dig> sequence that were previously unannotated.
additionally, regulatory rna genes had not been previously annotated in the v. fischeri genome, yet they are known to play important roles in v. fischeri and other diverse bacteria  <cit> .
therefore, we undertook an effort to systematically identify orfs and regulatory rna genes that had not been called in the published version  <dig>  sequence.
to accomplish this search we took advantage of the annotations present in the j. craig venter institute's comprehensive microbial resource , which include ab initio gene-calls that can differ from those in the deposited genbank flatfiles  <cit> .
we examined the list of approximately  <dig> novel genes identified in the cmr.
we excluded candidates that were unlikely to be biologically significant – generally, short orfs that were encoded on the opposite strand against much larger orfs – and were left with  <dig> likely novel orfs .
we also took advantage of the presence of the closely-related mj <dig> strain as a source for novel gene annotations.
of the mj <dig> proteins that did not have an ortholog in es <dig>  we examined those in which a tblastn query of mj <dig> proteins against the es <dig> genome yielded a percent amino acid identity value of at least 85%.
we excluded candidates in which there was low biological basis for the assignment , or in which the open reading frame was not conserved in es <dig>  there were  <dig> novel es <dig> orfs assigned by comparison with mj <dig> .
<dig> orfs were called by both methods , whereas  <dig> genes were called by only one method, for a total of  <dig> uncalled chromosomal protein-coding genes that we predict to be uncalled in es <dig> .
numbers in parentheses indicate subset of features that have an annotation other than "hypothetical."
a includes csrb <dig> and csrb <dig>  <cit> .
b includes two genes predicted from  <cit> .
in all of these cases, no sequence was changed in the genomic model, but annotations imposed on the sequence were added.
included in the list of new genes predicted from both approaches is biotin synthase .
because es <dig> grows on minimal medium lacking biotin  <cit> , biob is likely expressed by the organism.
another example of a gene predicted from both approaches is the oxaloacetate decarboxylase gamma subunit , which is predicted to be encoded in an operon with the already-predicted alpha and beta subunits .
in addition to genes that were identified from both the mj11-comparative and jcvi cmr approaches, we believe that genes identified by only one of the approaches are still worthy of inclusion, subject to the filters imposed above.
genes that were identified by comparison with mj <dig> have the support that the open reading frame is conserved in at least these two strains.
a similar measure has been used to call genes in saccharomyces cerevisiae for genome inclusion  <cit> .
genes that were identified solely from the jcvi cmr annotation include a number of regions that are unique to es <dig>  such as a prophage that is present in es <dig> and absent in mj <dig> , and therefore would not be expected to be called by comparison with mj <dig>  the coding density of the prophage was markedly increased due to the addition of the novel gene annotations, consistent with phage genome organization and supporting the assignments predicted by the cmr.
it is clear that the consolidated results from both methods, though partially overlapping, identify a significant number of novel, bona fide gene annotations in es <dig>
we added regulatory rna genes to the annotation as identified from multiple sources .
prediction of csrb regulatory rnas has been pioneered using v. fischeri  <cit> , and additional regulatory rnas were added based on motifs found in the rfam database  <cit> .
these methods identified a total of  <dig> regulatory rnas and  <dig> operon leader peptide.
although operon leader peptide predictions are not typically found in databases, we speculated that additional such genes were present in es <dig> and, using the ecocyc database  <cit>  as a guide, we manually searched for operon leader peptides in es <dig> and identified five additional high-confidence members in the genome .
in total, we called  <dig> new protein-coding genes,  <dig> regulatory rnas, and  <dig> operon leader peptides, and we incorporated  <dig> protein-coding genes and  <dig> regulatory rnas that were published previously, for a total of  <dig> new annotations incorporated into es <dig> version  <dig>  .
comprehensive annotation update
in the process of correcting sequence errors and adding missing annotations, we additionally took the opportunity to update the annotations of the genes in the es <dig> genome and to establish a framework for future genomic and genetic studies in v. fischeri.
to update the product annotations of v. fischeri, we assembled a database of v. fischeri genetic and genomic analyses from the the pubmed database  <cit> .
our initial curated v. fischeri list included  <dig> unique gene-publication associations from  <dig> publications, encompassing  <dig> distinct genes represented in strain es <dig>  this list served as the core of the reannotation effort, which further gave us the opportunity to update a number of genes whose functions have been discovered since the initial genome publication.
for all genes in es <dig>  we additionally compared protein annotations from multiple sources:  orthologous protein annotations in the recently reannotated escherichia coli k- <dig> mg <dig> sequence, and updates made subsequent to sequence publication through the asap and ecocyc databases  <cit> ;  orthologous protein annotations in v. cholerae  <cit> ;  the jcvi cmr  <cit> ; and  uniprotkb  <cit> .
these comparisons allowed us to update the annotations and to make the annotations more consistent with current practice and ncbi guidelines.
we found the annotations of e. coli – though most distant phylogenetically – to be the most valuable empirically.
the timeliness of the update and the availability of curated, referenced descriptions in the ecocyc entries allowed us to improve a number of entries that appeared to lag behind the other data sources.
as one example, we point to the case of yihy .
previously annotated as encoding the ribonuclease bn  <cit> , this annotation has been propagated through numerous sources, including most of the vibrionaceae genomes.
a subsequent report identified the e. coli rbn/elac gene  as the gene that encodes rnase bn, and the most recent genome annotation for b <dig> has been updated as yihy, "predicted inner membrane protein"  <cit> .
we compared data from the sources described above, as well as the literature described, and captured this update by calling vf_ <dig> as yihy with a product of "predicted inner membrane protein".
in fact, v. fischeri, like most sequenced vibrio spp., does not contain an rbn ortholog, and therefore having any product labeled as "ribonuclease bn" would have been misleading from the perspective of predicting genome capabilities.
we note that the old annotation persists in major databases  <cit>  and in most of the vibrionaceae genomes available at the time of data submission.
this example highlights the value and relevance of the e. coli k- <dig> update to this and related annotation projects, as well as our ability to capture the latest information about genes encoded in the es <dig> genome.
fine-scale annotation changes, such as those shown in figure 1f, are detailed both in that figure and in the methods.
we also wish to highlight the updated entry for prfb , the peptide chain release factor rf- <dig>  the programmed frameshift in prfb is not called correctly by machine-call algorithms, and this gene is improperly entered in the genbank flatfiles of all of the previously submitted vibrio spp.
genomes.
with the blossoming number of sequencing projects, utilization of locus tags  as identifiers for both genes and their products has become commonplace as the increase in genomic characterization has outpaced genetic and biochemical characterization of gene products.
nonetheless, biological analysis in a genomic context depends on understanding gene function, and proper nomenclature has been adopted in a number of species to facilitate meaningful communication about genes and their products.
in fact, we  repeatedly refer to genes by their identifiers and, without tracking in a database, this practice can lead to incorrect conclusions  <cit> .
therefore, whereas the previous es <dig> version did not contain 3– <dig> character "gene" identifiers, we added those for approximately  <dig>  genes in which the identity of the gene could be identified or inferred from published work in v. fischeri, or by orthologous genes in other organisms.
due to the availability of well-curated database resources, most of the names were derived from their orthologs in e. coli mg <dig>  <cit> .
we demanded that unique gene identifiers be a minimum of three lowercase letters , with an optional uppercase letter , and/or an optional numeral , for a maximum of five characters total.
such numeric suffixes were assigned to distinguish among members of paralogous families or genes of related function.
for approximately half of the genes, no gene identifier could be assigned at this time.
v. fischeri transcription machinery
because three rna polymerase subunit genes were affected by the resequencing , we took a genomic inventory of the corrected es <dig> transcriptional apparatus in a manner that was not possible prior to the targeted resequencing.
the subunits identified in the genome are listed in table  <dig> and include the core subunits α, β, β', and ω. classification of the eleven identified sigma factors is described below and was performed by the scheme outlined in gruber & gross  <cit> .
group  <dig> sigma factors include regions  <dig> ,  <dig> ,  <dig>   <dig>  and  <dig>  this category includes only σ <dig> in es <dig>  group  <dig> sigma factors  include the closely-related σs subunits; as mentioned above, v. fischeri curiously contains two of these sigma subunits.
in addition to a clear ortholog of rpos , the gene encoding the stationary-phase sigma subunit , es <dig> also contains a gene that is expected to encode a σs-like subunit .
transcript levels of this second σs-family subunit increase upon c8-homoserine-lactone  quorum-sensing  <cit> , so we have called the product σq  to designate this as a quorum-responsive sigma factor and to distinguish it from the σs paralog.
group  <dig> sigma factors  include the specialized sigma factors σh and σf.
group  <dig> sigma factors , also called ecf sigma factors because they perform an extra cytoplasmic function in responding to envelope stresses, are the most divergent.
es <dig> contains five of these subunits, with the corresponding genes named rpoe, rpoe <dig>  rpoe <dig>  rpoe <dig>  and rpoe <dig>  although these have not yet been studied in v. fischeri, their genomic context suggest function in some cases.
unlike the other group  <dig> sigma factors, the product of the gene called rpoe is a close homolog of e. coli σe  and is organized transcriptionally with regulatory genes homologous to its e. coli counterparts .
we assigned this subunit σe, and this subunit may respond to outer membrane protein misfolding in a similar manner as in e. coli  <cit> .
rpoe <dig> is predicted to be transcribed in an operon with chrr, which likely encodes an anti-σe <dig> factor based on homology with the reactive oxygen-sensing σe/chrr system of rhodobacter sphaeroides  <cit> .
additionally, the two genes flanking rpoe <dig>  though their functions are unknown, share a similar phylogenetic distribution as rpoe <dig>  because group  <dig> sigma factors are typically cotranscribed with cognate anti-sigma factors  <cit> , evolutionary co-inheritance of this three-gene cassette supports a role for the surrounding genes in regulating the levels and activation of σe <dig>
discussion
we initiated this study to clarify the status of a number of suspect orfs in a completed genome sequence of an organism that is of value for studies on bacterial communication and host interaction.
of the  <dig>  mbp of chromosomal dna in the original, version  <dig>  release,  <dig> % of the sequence was in error, mostly due to fourteen regions  in which unique dna was incorporated in tandem in the version  <dig>  release.
a total of  <dig> individual sites were corrected – by insertion, deletion , or substitution – leading to changes in  <dig> protein-coding loci of the version  <dig>  release , and one newly-annotated orf.
salzberg and yorke  <cit>  describe "compressions" that can occur in eukaryotic genome assemblies when errors compress multiple, repeated sequences and exclude intervening unique regions.
the type of error that we have detected is an "expansion" in that sequence is illegitimately repeated and the resulting region has been expanded.
the initial shotgun sequencing and assembly for es <dig> were conducted circa  <dig>  however, sequence quality varies by project and center/investigator, and the problems encountered here are neither unique to  older genomes.
for example, as we identified resequencing targets in es <dig>  we additionally targeted regions in the draft v. fischeri mj <dig> sequence as part of our ongoing effort to close an accurate mj <dig> sequence.
of fifteen loci targeted for resequencing, nine were found to contain point mutations that led to frameshifts .
these data will be incorporated into the complete mj <dig> sequence.
additionally, a systematic analysis to identify possible gene errors of the type we describe here identified interrupted genes in other complete genomes from gram-negative γ-proteobacteria, and in some of those cases the interrupted genes are orthologs of essential genes in e. coli  <cit> .
in cases where essential genes may be predicted from related organisms, we propose that measuring the proportion of such genes that are interrupted could serve as a measure of quality assessment for newly-assembled genomes.
in the case of v. fischeri es <dig>  identifying interrupted essential genes was a significant clue that the sequence model required refinement, and those genes represent  <dig> of the  <dig>  loci corrected .
the comparative approach that we describe was necessary to identify the additional affected loci.
in addition to the e. coli and m. smegmatis resequencing/reannotation projects discussed earlier, a comparative-based reannotation project has been reported in s. cerevisiae  <cit> .
our approaches and results are similar to the yeast study in that both relied heavily on comparison with a single additional genome as a basis for gene discovery and clarification.
in addition to a draft genomic sequence of a closely-related sequence, we relied solely on publicly-available data to compile our annotations and complete this update.
thus, the methods described here are generally applicable in any case in which there is updated genbank data for closely-related organisms.
the timeliness of any genome update is necessarily transient, and therefore it is of community interest to optimize the data quality and usability of annotation systems.
to track future sequence and annotation changes in v. fischeri, we have established a web site that will assist in coordinating v. fischeri annotation resources and genome projects into the future  <cit> .
because genbank functions mainly as a deposition library – and not as a dynamic annotation interface – development of such an interface would enhance the ability to keep genome data current.
if such a resource were to be developed, annotations could be propagated in a manner that can be intelligently curated by individual genome owners, and could be managed through a user-friendly interface.
the development of bioinformatics resource centers   has advanced the annotation pipelines for pathogenic microorganisms, but this system is insulated from many of the investigators who work on the vast majority of organisms represented in the database, and requires consistent deposition in genbank for there to be a "paper trail" that can be accessed by outside investigators.
additionally, establishing a distinction between human-pathogenic and human-non-pathogenic organisms in genome annotation is artificial when the organisms overlap phylogenetically and significant work is underway to characterize similar  gene products and pathways in related organisms.
it would be beneficial to the prokaryotic genome community to find a way to integrate genomic data into a dynamic resource without regard to the human-pathogenic phenotype of the organism.
CONCLUSIONS
as dna sequencing technology accelerates, it is critical to ensure that high-fidelity sequence assemblies are obtained, and that large-scale errors be readily detected.
by using publicly-available resources and the sequence  of a close relative, errors that lead to miscalling of orf boundaries in one strain relative to that of another may be identified for regions syntenous between the strains.
such analyses can identify regions that were assembled incorrectly, or even point mutations that were miscalled in one strain.
this analysis in v. fischeri es <dig> identified over  <dig> kb of sequence that required adjustment, including  <dig> regions requiring deletion from the annotation, and overall errors affecting  <dig> orfs across  <dig> loci.
it is similarly important to maintain accurate and updated annotations for genes in sequenced genomes.
although some extensively-studied model organisms have systematic programs for their annotation, organisms with sparser genomic resources – and often, fortunately for this purpose, fewer data generated from direct studies in that organism – can benefit from a streamlined reannotation pipeline using recently updated annotations from publicly-available databases.
by applying such an approach, we updated the complete annotation of v. fischeri es <dig> and included regulatory rnas and operon leader peptides, important regulatory features that are commonly missed by automated gene calling.
although community-based updates are desirable when they can be accomplished, our individual approach is generally applicable across microbial genomics and demonstrates a straightforward way to achieve a high-yield update with resources that are common in hundreds of laboratories.

 in the thirteen years since the announcement of the first complete organism genome, there has been a rapid accumulation of sequence data from complete and draft genomes. the number of complete or almost-complete projects is in the range of, but this number is a "moving target," and improvements in sequencing technologies over the past decade ensure continued rapid expansion in the number and diversity of organisms that are analyzed by complete genome sequencing. despite these significant advances in data acquisition, there have not been commensurate improvements in data-quality assessment and refinement during this period. individual miscalled bases are assumed to be present in practically all completed genome sequences, and their frequency has been suggested to be between 1– errors per kb and has been measured in some instances to be at most error per kb. errors in microbial genomes are believed to be generally restricted to point miscalls, with large-scale rearrangements rarely occurring. to identify and correct errors, recent studies have utilized microarray-based detection, in which errors in a subject genome are identified by comparison to a reference genome which served as the basis for array construction. for example, this method has been employed successfully in escherichia coli and bacillus anthracis. however, these analyses are unidirectional: "errors" are defined as sequence distinct from that of the reference genome, and therefore errors in the reference genome cannot be detected. as small nucleotide changes in a genome model often manifest as large protein errors – for instance, due to introduction of frameshift and nonsense errors – multiple approaches have capitalized on this protein signal to detect dna errors in complete genomes. by comparing protein-coding sequences in a subject strain to those in a closely-related strain or to closely-related proteins in molecular databases, one can identify those that are potentially truncated inappropriately in the subject strain and target those regions for resequencing. targeted resequencing has been applied successfully in b. subtilis and mycobacterium smegmatis, and in both cases the errors were restricted to changes in 1– nucleotides. importantly, perrodou et al. generalized this method in silico to make it available to any subject organism of interest. targeted resequencing is efficient and available to a wide range of investigators because: the initial steps are completed in silico prior to proceeding to the wet laboratory; and when a closely-related strain is available targeted resequencing provides an efficient means to identify discrepancies that alter coding sequence predictions. in this study, we focus on the genome of the luminous gram-negative bacterium vibrio fischeri es v. fischeri forms symbiotic associations with squid and fish, and the association between v. fischeri and the hawaiian bobtail squid euprymna scolopes represents one of the most powerful natural models for the study of mutualistic animal-microbe relationships. specific strains of symbiotic v. fischeri colonize a dedicated "light organ" in the squid host, multiply to high density, and exhibit luminescence in a density-dependent manner. the light produced by the bacteria is believed to aid the squid host by providing protection from predators: the shadow revealed from the nocturnal-foraging squid in moonlight is camouflaged by the downward-welling light of the host-associated v. fischeri. in return, the bacterium benefits from a protected, nutrient-rich environment. this was the first system in which it was shown that a specific symbiont directs normal animal development, and now represents an emerging model for cross-kingdom genomics-based studies. the genomic potential for this system is based on a strong history of molecular inquiry on both the symbiont and host sides of the interaction. first, the complete genome sequence of squid symbiont v. fischeri es has been published and studied, and the sequence revealed novel insights into pilin gene diversity and the distribution of toxin genes in beneficial bacteria. second, based on the genome sequence a number of global studies have been initiated; the first sets to be published yield novel results about how chemical communication among v. fischeri strains regulates bacterial behavior and how two-component signal transduction affects host-interaction. third, an est library of the squid host has provided novel insight into cephalopod genetic capabilities and widely conserved signaling pathways such as the nf-κb pathway. fourth, the phenomenon we now call quorum sensing – autoinduced density-dependent cell-cell communication – was first described in v. fischeri, and a number of evolutionary and modeling studies of this process have focused on the well-characterized systems in v. fischeri. fifth, by having access to the natural host – a rarity among systems in which high-throughput genetic and genomics approaches are applicable – we can exploit the high information content in the coevolved squid-vibrio relationship to learn how closely-related pathogenic marine microbes interact with natural hosts that have yet to be identified. sixth, the draft genome of a second strain of v. fischeri, the fish symbiont mj is being completed and will provide a strong platform for applying comparative genomic approaches to the study of host-specificity. while undertaking such a comparative study among v. fischeri strains, we detected a high incidence of suspected genomic anomalies in the published sequence of v. fischeri es we resequenced these suspect regions and identified 91% of these loci to be in error. notably, in fourteen of the cases we detected misincorporation of extraneous sequence in the published assembly, leading to the appearance of duplicated dna where none existed. in five other cases, the sequence in the suspect region was correct in the published sequence and the resulting gene product would be predicted to be nonfunctional; we therefore designated these features as pseudogenes in es in addition to correcting these features, we completed a full genomic update of es gene annotations, and incorporated the addition of genes that were previously unannotated into release of the es annotation. together these updates advance v. fischeri as a platform for functional and comparative genomic studies, and demonstrate how a targeted set of approaches may yield high impact on genomic quality improvement.  identification of suspect genomic regions we obtained the draft genome sequence of v. fischeri strain mj and, as part of our initial analysis, we conducted a number of reciprocal blast analyses to compare its predicted proteome with that of the completely sequenced conspecific strain es. we used blastp to identify orthologs between the two strains, using a modified reciprocal best-hit approach as outlined in the methods. a surprising outcome from this analysis was the occurrence of over seventy protein-coding genes in mj with reciprocal best-hits to two neighboring genes in es at the time that we were performing this analysis, a handful of cases were being identified empirically in which neighboring genes in es were actually one gene, and that the appearance of two genes resulted from frameshift or nonsense errors in the original sequence data. examples that were identified independent of this work include ptsi, fnr, and acs. analysis of the suspect regions supported the hypothesis that there were a large number of loci in es in which sequencing errors had led to the miscalling of one gene as multiple orfs. in support of this hypothesis, we identified a number of genes that are essential in escherichia coli and other bacteria, but that were split in version of the es sequence. these included dnag, ftsq, mukb, nusg, rplc, rpln, rplo, rpob, rpoc, thrs, and tils, and the conditionally-essential rpoh. second, we identified eleven ambiguous bases that had been called in the original sequence, and the incidence of these bases correlated with the presence of suspect orfs. in addition to suspected frameshifts and substitutions, we also identified fourteen regions in which it appeared that extraneous sequence had been incorporated that was highly similar to neighboring sequence, with the size of the duplicated/extraneous region ranging from bp to bp. in one case, pre-genomic sequencing of a suspect region did not identify any repeated sequence. therefore, we hypothesized that these regions represented assembly errors in which the same stretch of dna was mistakenly incorporated twice into the genome's sequence. these regions typically contained a few unique base pairs at either end – likely due to low-coverage sequencing – that led to the misincorporation, but were otherwise essentially a direct repeat of dna that had the effect of introducing extra and/or truncated orfs. a list of the loci targeted for resequencing was assembled and each was assigned a "target number"; that number is used consistently in tables and figures so that the primer sequences used to analyze the data may be correlated with the resulting sequence and analysis. in addition to the blastp-based identification of potential errors, we undertook a full-length visual comparison of the chromosomes of v. fischeri es and mj given the prevalence of errors detected by identifying adjacent orfs that likely represented a single orf, we hypothesized that there were probably other cases of errors that would not have manifest themselves in this way. examples of other suspected errors that warranted investigation included situations in which one of the fragmented orfs was too small to be detected as an ortholog candidate by the blastp filters, or in which the second fragment did not lead to a predicted open reading frame. using the program mauve, we analyzed orfs along the length of the chromosomes, identifying candidates that had suspect 5' or 3' ends. in some cases, these appeared to result solely from annotation differences, in which identical sequences had predicted translational start sites that were called at distinct points in the two annotations. in other cases, sequence differences underlay the unique orf boundaries, and we targeted those for our analysis. furthermore, there were three cases in which putative extraneous sequence was visually identified in intergenic sequence, which could not have been detected by blastp analysis in the absence of annotated orfs. these cases were added to the list of targeted loci. finally, any remaining ambiguous bases in the sequence were targeted for resequencing. sequence clarification we examined a total of targets for resequencing. our general approach involved amplifying across the target, and then sequencing the amplified product with the pcr primers. in cases where we were clarifying the sequence following a large detected "deletion", we amplified a larger product and sequenced from a set of sequencing primers across both strands. for the oligonucleotide primers used for pcr and sequencing see additional file with one exception, all of the primer pairs amplified products in which there was a clear, predominant band, and thus served as satisfactory templates for sequencing. the primer pair that failed to amplify included a primer that was in a region that does not exist in the true es sequence, as clarified by our analysis of target no. therefore the absence of a band in this case supports the deletion that emerged from target no. seventy-five of the sets of resequencing targets examined were found to be in error in the published es sequence. the errors, subsequent changes, new locus tags, and new annotations, are listed in table conceptual diagrams of representative sequencing and other annotation changes discussed during this report are illustrated in figure note that with this update, the locus tag format has been modified to the new ncbi format for locus tags. as a convention, in cases of gene fusion, the locus tag of the 5'-proximal fragment retained its locus tag identifier, while the identifier for the remaining gene fragment were deaccessioned. correction types: dl, large deletion; fs, frameshift; ms, missense; ns, nonsense; n, ambiguous nucleotide. s/m indicates whether ingle or ultiple nucleotides were affected by the sequence change. it is thought that the creation of false large-scale genomic rearrangements such as insertions rarely occurs in microbial genome projects; however, we confirmed the presence of all fourteen predicted insertions by amplifying from the respective unique flanking regions, and demonstrating that the bands obtained are inconsistent with the previous sequence model. in each case, the bands observed were smaller than predicted, and the sequence obtained led to the precise deletion of the extraneous repeated dna in the new model. most of the resulting changes led to the fusion of two – or in some cases three – neighboring orfs, and/or the extension of orfs at the 5' or 3' end. in one case, the deletion affected only an intergenic region that contains no annotated features. in another case the deletion identified by visual analysis in mauve affected what was believed to be a 1498-bp intergenic region between rlue and vf_ the corrected sequence revealed this region to be only bp in length, but also that it contains a predicted lipoprotein conserved in v. fischeri mj the new release reflects the sequence deletion, as well as the added annotation for this gene. the resulting sequence corrections led us to propose a number of protein annotations that were consistent with our predictions. based on the corrected sequence, many conserved genes now more closely resemble their orthologs in other species. in other cases, the domain structure of even poorly characterized proteins supported the accuracy of the corrections. for example, target no. extended the 3' end of vf_ by correcting a frameshift mutation. analysis of protein domains by conserved domain search identified an incomplete ggdef domain in the protein's c-terminus, and correction of the frameshift led to inclusion of the entire domain. pseudogenes and degeneration in umuc in some of the cases we confirmed the published es sequence to be correct, and that the orf boundaries were correct in es version. table lists those five cases that we can now more confidently assume to be pseudogenes in es because they appear to be nonfunctional given their predicted amino acid sequence. in each case, the indicated defect is predicted to interrupt a significant portion of the coding sequence required for function in well-characterized homologs. the n-acetylglucosaminyltransferase vf_a has two paralogs in the genome, and es is capable of utilizing n-acetylglucosamine as a sole n+c source: therefore, the appearance of a pseudogene at this location does not have obvious functional consequences for the cell. there is little information about the remaining four pseudogenes, except for umuc. the transcriptional organization between the genes encoding the dna polymerase v subunits umud and umuc is conserved between es and mj. however, umuc has uniquely degenerated in es with both a nonsense codon and a 5-bp repeat expansion following the nonsense codon. dna polymerase v is responsible for error-prone translesion synthesis, which allows dna synthesis to proceed despite a high rate of error incorporation, yet there are organisms, including v. cholerae el tor, that apparently do not encode these functions. whether the situation in es represents an evolutionary transition state, or instead this arrangement has relevant functional implications remains to be determined. annotation of previously uncalled protein-coding genes, regulatory rnas, and operon leader peptides because examination of the intergenic region corrected by target no. revealed a likely protein-coding gene, we asked whether there were other genes present within the es sequence that were previously unannotated. additionally, regulatory rna genes had not been previously annotated in the v. fischeri genome, yet they are known to play important roles in v. fischeri and other diverse bacteria. therefore, we undertook an effort to systematically identify orfs and regulatory rna genes that had not been called in the published version sequence. to accomplish this search we took advantage of the annotations present in the j. craig venter institute's comprehensive microbial resource, which include ab initio gene-calls that can differ from those in the deposited genbank flatfiles. we examined the list of approximately novel genes identified in the cmr. we excluded candidates that were unlikely to be biologically significant – generally, short orfs that were encoded on the opposite strand against much larger orfs – and were left with likely novel orfs. we also took advantage of the presence of the closely-related mj strain as a source for novel gene annotations. of the mj proteins that did not have an ortholog in es we examined those in which a tblastn query of mj proteins against the es genome yielded a percent amino acid identity value of at least 85%. we excluded candidates in which there was low biological basis for the assignment, or in which the open reading frame was not conserved in es there were novel es orfs assigned by comparison with mj. orfs were called by both methods, whereas genes were called by only one method, for a total of uncalled chromosomal protein-coding genes that we predict to be uncalled in es. numbers in parentheses indicate subset of features that have an annotation other than "hypothetical." a includes csrb and csrb. b includes two genes predicted from. in all of these cases, no sequence was changed in the genomic model, but annotations imposed on the sequence were added. included in the list of new genes predicted from both approaches is biotin synthase. because es grows on minimal medium lacking biotin, biob is likely expressed by the organism. another example of a gene predicted from both approaches is the oxaloacetate decarboxylase gamma subunit, which is predicted to be encoded in an operon with the already-predicted alpha and beta subunits. in addition to genes that were identified from both the mj11-comparative and jcvi cmr approaches, we believe that genes identified by only one of the approaches are still worthy of inclusion, subject to the filters imposed above. genes that were identified by comparison with mj have the support that the open reading frame is conserved in at least these two strains. a similar measure has been used to call genes in saccharomyces cerevisiae for genome inclusion. genes that were identified solely from the jcvi cmr annotation include a number of regions that are unique to es such as a prophage that is present in es and absent in mj, and therefore would not be expected to be called by comparison with mj the coding density of the prophage was markedly increased due to the addition of the novel gene annotations, consistent with phage genome organization and supporting the assignments predicted by the cmr. it is clear that the consolidated results from both methods, though partially overlapping, identify a significant number of novel, bona fide gene annotations in es we added regulatory rna genes to the annotation as identified from multiple sources. prediction of csrb regulatory rnas has been pioneered using v. fischeri, and additional regulatory rnas were added based on motifs found in the rfam database. these methods identified a total of regulatory rnas and operon leader peptide. although operon leader peptide predictions are not typically found in databases, we speculated that additional such genes were present in es and, using the ecocyc database as a guide, we manually searched for operon leader peptides in es and identified five additional high-confidence members in the genome. in total, we called new protein-coding genes, regulatory rnas, and operon leader peptides, and we incorporated protein-coding genes and regulatory rnas that were published previously, for a total of new annotations incorporated into es version. comprehensive annotation update in the process of correcting sequence errors and adding missing annotations, we additionally took the opportunity to update the annotations of the genes in the es genome and to establish a framework for future genomic and genetic studies in v. fischeri. to update the product annotations of v. fischeri, we assembled a database of v. fischeri genetic and genomic analyses from the the pubmed database. our initial curated v. fischeri list included unique gene-publication associations from publications, encompassing distinct genes represented in strain es this list served as the core of the reannotation effort, which further gave us the opportunity to update a number of genes whose functions have been discovered since the initial genome publication. for all genes in es we additionally compared protein annotations from multiple sources: orthologous protein annotations in the recently reannotated escherichia coli k- mg sequence, and updates made subsequent to sequence publication through the asap and ecocyc databases; orthologous protein annotations in v. cholerae; the jcvi cmr; and uniprotkb. these comparisons allowed us to update the annotations and to make the annotations more consistent with current practice and ncbi guidelines. we found the annotations of e. coli – though most distant phylogenetically – to be the most valuable empirically. the timeliness of the update and the availability of curated, referenced descriptions in the ecocyc entries allowed us to improve a number of entries that appeared to lag behind the other data sources. as one example, we point to the case of yihy. previously annotated as encoding the ribonuclease bn, this annotation has been propagated through numerous sources, including most of the vibrionaceae genomes. a subsequent report identified the e. coli rbn/elac gene as the gene that encodes rnase bn, and the most recent genome annotation for b has been updated as yihy, "predicted inner membrane protein". we compared data from the sources described above, as well as the literature described, and captured this update by calling vf_ as yihy with a product of "predicted inner membrane protein". in fact, v. fischeri, like most sequenced vibrio spp., does not contain an rbn ortholog, and therefore having any product labeled as "ribonuclease bn" would have been misleading from the perspective of predicting genome capabilities. we note that the old annotation persists in major databases and in most of the vibrionaceae genomes available at the time of data submission. this example highlights the value and relevance of the e. coli k- update to this and related annotation projects, as well as our ability to capture the latest information about genes encoded in the es genome. fine-scale annotation changes, such as those shown in figure 1f, are detailed both in that figure and in the methods. we also wish to highlight the updated entry for prfb, the peptide chain release factor rf- the programmed frameshift in prfb is not called correctly by machine-call algorithms, and this gene is improperly entered in the genbank flatfiles of all of the previously submitted vibrio spp. genomes. with the blossoming number of sequencing projects, utilization of locus tags as identifiers for both genes and their products has become commonplace as the increase in genomic characterization has outpaced genetic and biochemical characterization of gene products. nonetheless, biological analysis in a genomic context depends on understanding gene function, and proper nomenclature has been adopted in a number of species to facilitate meaningful communication about genes and their products. in fact, we repeatedly refer to genes by their identifiers and, without tracking in a database, this practice can lead to incorrect conclusions. therefore, whereas the previous es version did not contain 3– character "gene" identifiers, we added those for approximately genes in which the identity of the gene could be identified or inferred from published work in v. fischeri, or by orthologous genes in other organisms. due to the availability of well-curated database resources, most of the names were derived from their orthologs in e. coli mg. we demanded that unique gene identifiers be a minimum of three lowercase letters, with an optional uppercase letter, and/or an optional numeral, for a maximum of five characters total. such numeric suffixes were assigned to distinguish among members of paralogous families or genes of related function. for approximately half of the genes, no gene identifier could be assigned at this time. v. fischeri transcription machinery because three rna polymerase subunit genes were affected by the resequencing, we took a genomic inventory of the corrected es transcriptional apparatus in a manner that was not possible prior to the targeted resequencing. the subunits identified in the genome are listed in table and include the core subunits α, β, β', and ω. classification of the eleven identified sigma factors is described below and was performed by the scheme outlined in gruber & gross. group sigma factors include regions,, and this category includes only σ in es group sigma factors include the closely-related σs subunits; as mentioned above, v. fischeri curiously contains two of these sigma subunits. in addition to a clear ortholog of rpos, the gene encoding the stationary-phase sigma subunit, es also contains a gene that is expected to encode a σs-like subunit. transcript levels of this second σs-family subunit increase upon c8-homoserine-lactone quorum-sensing, so we have called the product σq to designate this as a quorum-responsive sigma factor and to distinguish it from the σs paralog. group sigma factors include the specialized sigma factors σh and σf. group sigma factors, also called ecf sigma factors because they perform an extra cytoplasmic function in responding to envelope stresses, are the most divergent. es contains five of these subunits, with the corresponding genes named rpoe, rpoe rpoe rpoe and rpoe although these have not yet been studied in v. fischeri, their genomic context suggest function in some cases. unlike the other group sigma factors, the product of the gene called rpoe is a close homolog of e. coli σe and is organized transcriptionally with regulatory genes homologous to its e. coli counterparts. we assigned this subunit σe, and this subunit may respond to outer membrane protein misfolding in a similar manner as in e. coli. rpoe is predicted to be transcribed in an operon with chrr, which likely encodes an anti-σe factor based on homology with the reactive oxygen-sensing σe/chrr system of rhodobacter sphaeroides. additionally, the two genes flanking rpoe though their functions are unknown, share a similar phylogenetic distribution as rpoe because group sigma factors are typically cotranscribed with cognate anti-sigma factors, evolutionary co-inheritance of this three-gene cassette supports a role for the surrounding genes in regulating the levels and activation of σe discussion we initiated this study to clarify the status of a number of suspect orfs in a completed genome sequence of an organism that is of value for studies on bacterial communication and host interaction. of the mbp of chromosomal dna in the original, version release, % of the sequence was in error, mostly due to fourteen regions in which unique dna was incorporated in tandem in the version release. a total of individual sites were corrected – by insertion, deletion, or substitution – leading to changes in protein-coding loci of the version release, and one newly-annotated orf. salzberg and yorke describe "compressions" that can occur in eukaryotic genome assemblies when errors compress multiple, repeated sequences and exclude intervening unique regions. the type of error that we have detected is an "expansion" in that sequence is illegitimately repeated and the resulting region has been expanded. the initial shotgun sequencing and assembly for es were conducted circa however, sequence quality varies by project and center/investigator, and the problems encountered here are neither unique to older genomes. for example, as we identified resequencing targets in es we additionally targeted regions in the draft v. fischeri mj sequence as part of our ongoing effort to close an accurate mj sequence. of fifteen loci targeted for resequencing, nine were found to contain point mutations that led to frameshifts. these data will be incorporated into the complete mj sequence. additionally, a systematic analysis to identify possible gene errors of the type we describe here identified interrupted genes in other complete genomes from gram-negative γ-proteobacteria, and in some of those cases the interrupted genes are orthologs of essential genes in e. coli. in cases where essential genes may be predicted from related organisms, we propose that measuring the proportion of such genes that are interrupted could serve as a measure of quality assessment for newly-assembled genomes. in the case of v. fischeri es identifying interrupted essential genes was a significant clue that the sequence model required refinement, and those genes represent of the loci corrected. the comparative approach that we describe was necessary to identify the additional affected loci. in addition to the e. coli and m. smegmatis resequencing/reannotation projects discussed earlier, a comparative-based reannotation project has been reported in s. cerevisiae. our approaches and results are similar to the yeast study in that both relied heavily on comparison with a single additional genome as a basis for gene discovery and clarification. in addition to a draft genomic sequence of a closely-related sequence, we relied solely on publicly-available data to compile our annotations and complete this update. thus, the methods described here are generally applicable in any case in which there is updated genbank data for closely-related organisms. the timeliness of any genome update is necessarily transient, and therefore it is of community interest to optimize the data quality and usability of annotation systems. to track future sequence and annotation changes in v. fischeri, we have established a web site that will assist in coordinating v. fischeri annotation resources and genome projects into the future. because genbank functions mainly as a deposition library – and not as a dynamic annotation interface – development of such an interface would enhance the ability to keep genome data current. if such a resource were to be developed, annotations could be propagated in a manner that can be intelligently curated by individual genome owners, and could be managed through a user-friendly interface. the development of bioinformatics resource centers has advanced the annotation pipelines for pathogenic microorganisms, but this system is insulated from many of the investigators who work on the vast majority of organisms represented in the database, and requires consistent deposition in genbank for there to be a "paper trail" that can be accessed by outside investigators. additionally, establishing a distinction between human-pathogenic and human-non-pathogenic organisms in genome annotation is artificial when the organisms overlap phylogenetically and significant work is underway to characterize similar gene products and pathways in related organisms. it would be beneficial to the prokaryotic genome community to find a way to integrate genomic data into a dynamic resource without regard to the human-pathogenic phenotype of the organism.  as dna sequencing technology accelerates, it is critical to ensure that high-fidelity sequence assemblies are obtained, and that large-scale errors be readily detected. by using publicly-available resources and the sequence of a close relative, errors that lead to miscalling of orf boundaries in one strain relative to that of another may be identified for regions syntenous between the strains. such analyses can identify regions that were assembled incorrectly, or even point mutations that were miscalled in one strain. this analysis in v. fischeri es identified over kb of sequence that required adjustment, including regions requiring deletion from the annotation, and overall errors affecting orfs across loci. it is similarly important to maintain accurate and updated annotations for genes in sequenced genomes. although some extensively-studied model organisms have systematic programs for their annotation, organisms with sparser genomic resources – and often, fortunately for this purpose, fewer data generated from direct studies in that organism – can benefit from a streamlined reannotation pipeline using recently updated annotations from publicly-available databases. by applying such an approach, we updated the complete annotation of v. fischeri es and included regulatory rnas and operon leader peptides, important regulatory features that are commonly missed by automated gene calling. although community-based updates are desirable when they can be accomplished, our individual approach is generally applicable across microbial genomics and demonstrates a straightforward way to achieve a high-yield update with resources that are common in hundreds of laboratories. 
a majority of the inaccurate loci involved frameshift errors, correction of which fused adjacent orfs. building on the genome project of vibrio fischeri es we used a comparative approach to identify and investigate genes that had a high likelihood of sequence error. although insertions/deletions are thought to be rare in microbial genome assemblies, fourteen of the loci contained extraneous sequence of over bp, likely due to imperfect contig ends that were misassembled in tandem rather than as overlapping segments. comparison of the v. fischeri es genome with that of conspecific strain mj identified target loci in es as containing likely errors, and thus of high-priority for resequencing. these updates further advance v. fischeri as an important model for understanding intercellular communication and colonization of animal tissue. additionally we updated the entire genome annotation with new features including previously uncalled protein-coding genes, regulatory rna genes and operon leader peptides, and we analyzed the transcriptional apparatus encoded by es 
 sequence closure often represents the end-point of a genome project, without a system in place for subsequent improvement and refinement. building on the genome project of vibrio fischeri es we used a comparative approach to identify and investigate genes that had a high likelihood of sequence error.  comparison of the v. fischeri es genome with that of conspecific strain mj identified target loci in es as containing likely errors, and thus of high-priority for resequencing. analysis of the targets identified loci in which an error had occurred, resulting in the correction of base pairs to generate the new es genomic sequence. a majority of the inaccurate loci involved frameshift errors, correction of which fused adjacent orfs. although insertions/deletions are thought to be rare in microbial genome assemblies, fourteen of the loci contained extraneous sequence of over bp, likely due to imperfect contig ends that were misassembled in tandem rather than as overlapping segments. additionally we updated the entire genome annotation with new features including previously uncalled protein-coding genes, regulatory rna genes and operon leader peptides, and we analyzed the transcriptional apparatus encoded by es  we demonstrate that errors in microbial genome sequences, thought to largely be confined to point mutations, may also consist of other prevalent large-scale rearrangements such as insertions. ongoing genome quality control and annotation programs are necessary to accompany technological advancements in data generation. these updates further advance v. fischeri as an important model for understanding intercellular communication and colonization of animal tissue. 