BACKGROUND
genome assembly, the process of combining short sequences to represent a consensus sequence of a genome, is always a compromise.
assembly methods are chosen that can be applied to the entire genome for a consistent result.
the sequences can be aggressively merged creating false joins in some cases but at the same time producing a statistically more contiguous assembly.
or, sequences can be conservatively merged, leaving many contigs and scaffolds unjoined but creating fewer false joins.
random sequences assemble more consistently than genomic sequence where the complications are due to the non-random nature of genomic sequence, such as repetitive sequences and polymorphisms.
there have been few main methods used for genome assembly.
the human genome <cit>  was assembled using a hierarchical approach, where bacterial artificial chromosomes  were isolated and mapped to the genome and then individually sequenced.
the advantage of this method is that the individual bacs contain a single haplotype and the assembly within a bac avoids conflicts due to polymorphisms and as a result is more contiguous and correct for a given level of sequence coverage.
the main disadvantage of this method is the cost associated with mapping the bacs and generating individual sequence libraries for each bac.
to reduce the cost of bac cloning and library construction, the whole genome shotgun  method has been used for a number of genomes.
while there are many advantages to the method, a wgs approach has difficulties dealing with repetitive sequences in the genome that tend to collapse in assembly, and in resolving regions of polymorphisms between the two haplotypes in the genome that may be sufficiently different to assemble as two copies rather than as one.
the first mouse genome used the wgs approach, as did the macaque <cit> , dog <cit> , opossum <cit> , platypus, chimpanzee <cit>  and the low coverage genome sequences including cat <cit> .
some of these assemblies benefited from comparison to closely related species to improve the assembly.
while this is a powerful approach, it can hide true differences between the species that are only seen in the new genome.
the bovine genome sequence reported here, like the rat genome sequence <cit>  employed a combined wgs plus bac approach.
like the sea urchin <cit> , many of the bacs for the bovine project were sequenced in pools rather than individually, as a cost saving measure.
in contrast to previous assemblies, the bovine assembly leveraged the benefit of local assembly provided by the bacs by tuning the assembly parameters for each bac to address local differences in sequence characteristics  to produce the best assembly within each enriched bac .
the bovine project was fortunate to have many sets of markers from different sources available to place the assembly on chromosomes.
a challenge in using these was the difficulty in merging the multiple marker sets into a single consistent map.
new software  assembly components were developed to solve the conflicts in the merged marker sets and maximize their usage for scaffold placement and correction.
RESULTS
the bovine genome was assembled at the baylor college of medicine human genome sequencing center using a combined method similar to that used for the rat genome <cit>  and more recently the sea urchin genome <cit> .
the combined strategy is a hybrid of the whole genome shotgun  approach used for the mouse genome and the hierarchical  approach used for the human genome.
the sequencing combines bac shotgun reads with whole-genome-shotgun  reads from small insert libraries as well as bac end sequences .
the dna for the small insert wgs libraries was from white blood cells from the hereford cow l <dig> dominette  <dig>  the source of the bac library dna was hereford bull l <dig> domino  <dig>  the sire of the former animal.
two early assembly versions  were prepared using only whole genome shotgun  reads from small insert clones and bes.
contigs from btau_ <dig>  were used in the subsequent assembly.
btau_ <dig>  was produced using the atlas genome assembly system with a combination of wgs and bac sequence <cit> .
the assembly process consisted of multiple phases .
sequences from each bac were assembled with phrap, first with just the bac generated sequences, then in combination with the wgs reads that overlapped the bac as an enriched bac .
bacs were sequenced as either individual clone libraries or as pools of arrayed clones .
bac reads from individual libraries or from deconvoluted pools were assembled as individual bacs.
<dig>  bac projects  were sequenced and assembled.
details of bac assembly methods are provided below.
contigs from the btau_ <dig>  wgs assembly were used to fill in the gaps in the bac-based assembly , creating the combined assembly, btau_ <dig> .
the assembled contigs and scaffolds of the btau_ <dig>  assembly were placed on the chromosomes using a version of the integrated bovine map that represents merged data from several independent maps <cit> .
btau_ <dig>  is the latest assembly.
this assembly added relatively little new sequence data, and thus contigs and scaffolds were not significantly changed, but used the iltx <cit>  and bac finger-print contig  <cit>  maps and split scaffolds based on consistent bovine and sheep bes data  <cit>  to place contigs and scaffolds in the genome, instead of the integrated bovine map, resulting in more accurate chromosome structures.
overall, 90% of the total genome was placed on chromosomes in the btau_ <dig>  assembly .
this assembly was tested against available bovine sequence data sets .
of the  <dig>  million est sequences,  <dig> % were contained in the assembled contigs.
assuming the ests are uniformly distributed throughout the genome, the estimated genome size is  <dig>  gb .
the quality of the assembly was also tested by alignment to  <dig> finished bacs.
the genomic coverage in these bacs was high, between  <dig> % and  <dig> %  of the bac sequence in the assembly.
the assembled contigs and scaffolds were aligned linearly to the finished bacs, suggesting that misassemblies are rare.
*percentage of genome sequence in each category.
scaffolds includes single contig scaffolds as well as multiple contig scaffolds.
the table gives the numbers of bacs, markers, ests or bes used for comparison, and the percentage of contigs or scaffolds that matched each set.
additional file  <dig> gives the same comparisons for the unassembled reads as well as the assembled sequences.
two groups have used snp linkage data to order scaffolds on particular chromosomes.
one group used snp linkage data to order scaffolds on chr <dig>  <cit>  and another placed scaffolds on chr <dig> and chr <dig>  <cit> .
their studies provided additional evidence for scaffold placements and independent measurements for the quality of the assembly.
scaffolds in btau_ <dig>  have an order entirely consistent with the evidence from these three chromosomes, while both btau_ <dig>  and the composite map <cit>  show misplaced scaffolds .
further assessment of the btau_ <dig>  assembly was performed by comparing dense snp linkage maps constructed from genotyping  <dig>  snps in  <dig>  bulls belonging to  <dig> half-sib families with the physical positioning of the snps on all autosomal chromosomes.
the analysis revealed that  <dig> snps were incorrectly positioned within assembly.
this relatively small number  indicates the high degree of precision in the btau_ <dig>  assembly.
these misplaced snps were relocated in the linkage map to a position corresponding to the most closely linked, correctly assigned snp.
additionally,  <dig> snps from  <dig> unplaced scaffolds were mapped to linkage groups.
discussion
the genome assembly version btau_ <dig>  is available in genbank under accession number aafc <dig> .
in addition, the individual components of the genome assembly  and the file with the component assembly instructions  are available from the bcm-hgsc ftp site <cit>  which is a link from the bovine page on the bcm-hgsc web site.)
<cit>  since the process of genome assembly involves decisions about which sequences to include and which sequences to exclude, there are sequences from this project that were omitted from the final assembly.
some of the omitted sequences are highly repetitive sequence reads, others may have enough sequencing errors that they did not match the assembled sequences, others are assembled sequence contigs that appear to be duplicates of sequences in the assembly .
these excluded sequence are also available from the bcm-hgsc ftp site.
the majority of the sequence in the project is from the female animal, the genome sequence is described for the  <dig> autosomes and the x chromosome.
however, as the bac library was prepared from a male animal, and the bac fingerprint contigs were built from random clones from that library, both the x and y chromosomes are represented in the bac fingerprint contigs.
representative bacs in all of the bac fingerprint contigs were sequenced to low coverage, including y chromosome bacs.
since the clone coverage on the sex chromosomes in the bac library is half that of the autosomes, there will be less depth of clone coverage on the sex chromosomes and this may result in more gaps in the coverage of the sex chromosomes by bac clones.
the wgs sequence was from the female animal, so there is not additional wgs sequence to assemble with the low coverage bac skim sequences for the y chromosome, unless it is pseudoautosomal sequence from the x chromosome or autosomal sequence that is similar to the y sequence.
since the bac fingerprint contigs were used to build the combined bac+wgs assemblies, there are genome sequence scaffolds from both sex chromosomes as well as the autosomes.
the y chromosome scaffolds are unlabeled in the unplaced chromosome.
the use of linkage data to identify incorrectly positioned snps has been extended to also reveal the identity of the most closely linked, and correctly positioned snps.
in addition, the identification of  <dig> snps that map to linkage groups but are found within  <dig> unplaced scaffolds can be used to suggest a position for these scaffolds within the assembly.
taken together, data associating misplaced snps and unplaced scaffolds with correctly positioned markers could be used to highlight regions that could benefit from map assisted assembly improvements.
however, the moderate number of individuals being genotyped  limits the mapping resolution.
so while this analysis is effective at resolving large distance misplacements, additional genotyping of families would be required to reveal more local rearrangements.
CONCLUSIONS
the bovine genome assembly reported here was used for the analysis of the bovine genome sequence that is being published.
most of those analyses used the gene annotation from the btau_ <dig>  assembly.
some of the analyses used the btau_ <dig>  assembly.

 genome assembly, the process of combining short sequences to represent a consensus sequence of a genome, is always a compromise. assembly methods are chosen that can be applied to the entire genome for a consistent result. the sequences can be aggressively merged creating false joins in some cases but at the same time producing a statistically more contiguous assembly. or, sequences can be conservatively merged, leaving many contigs and scaffolds unjoined but creating fewer false joins. random sequences assemble more consistently than genomic sequence where the complications are due to the non-random nature of genomic sequence, such as repetitive sequences and polymorphisms. there have been few main methods used for genome assembly. the human genome was assembled using a hierarchical approach, where bacterial artificial chromosomes were isolated and mapped to the genome and then individually sequenced. the advantage of this method is that the individual bacs contain a single haplotype and the assembly within a bac avoids conflicts due to polymorphisms and as a result is more contiguous and correct for a given level of sequence coverage. the main disadvantage of this method is the cost associated with mapping the bacs and generating individual sequence libraries for each bac. to reduce the cost of bac cloning and library construction, the whole genome shotgun method has been used for a number of genomes. while there are many advantages to the method, a wgs approach has difficulties dealing with repetitive sequences in the genome that tend to collapse in assembly, and in resolving regions of polymorphisms between the two haplotypes in the genome that may be sufficiently different to assemble as two copies rather than as one. the first mouse genome used the wgs approach, as did the macaque, dog, opossum, platypus, chimpanzee and the low coverage genome sequences including cat. some of these assemblies benefited from comparison to closely related species to improve the assembly. while this is a powerful approach, it can hide true differences between the species that are only seen in the new genome. the bovine genome sequence reported here, like the rat genome sequence employed a combined wgs plus bac approach. like the sea urchin, many of the bacs for the bovine project were sequenced in pools rather than individually, as a cost saving measure. in contrast to previous assemblies, the bovine assembly leveraged the benefit of local assembly provided by the bacs by tuning the assembly parameters for each bac to address local differences in sequence characteristics to produce the best assembly within each enriched bac. the bovine project was fortunate to have many sets of markers from different sources available to place the assembly on chromosomes. a challenge in using these was the difficulty in merging the multiple marker sets into a single consistent map. new software assembly components were developed to solve the conflicts in the merged marker sets and maximize their usage for scaffold placement and correction.  the bovine genome was assembled at the baylor college of medicine human genome sequencing center using a combined method similar to that used for the rat genome and more recently the sea urchin genome. the combined strategy is a hybrid of the whole genome shotgun approach used for the mouse genome and the hierarchical approach used for the human genome. the sequencing combines bac shotgun reads with whole-genome-shotgun reads from small insert libraries as well as bac end sequences. the dna for the small insert wgs libraries was from white blood cells from the hereford cow l dominette the source of the bac library dna was hereford bull l domino the sire of the former animal. two early assembly versions were prepared using only whole genome shotgun reads from small insert clones and bes. contigs from btau_ were used in the subsequent assembly. btau_ was produced using the atlas genome assembly system with a combination of wgs and bac sequence. the assembly process consisted of multiple phases. sequences from each bac were assembled with phrap, first with just the bac generated sequences, then in combination with the wgs reads that overlapped the bac as an enriched bac. bacs were sequenced as either individual clone libraries or as pools of arrayed clones. bac reads from individual libraries or from deconvoluted pools were assembled as individual bacs. bac projects were sequenced and assembled. details of bac assembly methods are provided below. contigs from the btau_ wgs assembly were used to fill in the gaps in the bac-based assembly, creating the combined assembly, btau_. the assembled contigs and scaffolds of the btau_ assembly were placed on the chromosomes using a version of the integrated bovine map that represents merged data from several independent maps. btau_ is the latest assembly. this assembly added relatively little new sequence data, and thus contigs and scaffolds were not significantly changed, but used the iltx and bac finger-print contig maps and split scaffolds based on consistent bovine and sheep bes data to place contigs and scaffolds in the genome, instead of the integrated bovine map, resulting in more accurate chromosome structures. overall, 90% of the total genome was placed on chromosomes in the btau_ assembly. this assembly was tested against available bovine sequence data sets. of the million est sequences, % were contained in the assembled contigs. assuming the ests are uniformly distributed throughout the genome, the estimated genome size is gb. the quality of the assembly was also tested by alignment to finished bacs. the genomic coverage in these bacs was high, between % and % of the bac sequence in the assembly. the assembled contigs and scaffolds were aligned linearly to the finished bacs, suggesting that misassemblies are rare. *percentage of genome sequence in each category. scaffolds includes single contig scaffolds as well as multiple contig scaffolds. the table gives the numbers of bacs, markers, ests or bes used for comparison, and the percentage of contigs or scaffolds that matched each set. additional file gives the same comparisons for the unassembled reads as well as the assembled sequences. two groups have used snp linkage data to order scaffolds on particular chromosomes. one group used snp linkage data to order scaffolds on chr and another placed scaffolds on chr and chr. their studies provided additional evidence for scaffold placements and independent measurements for the quality of the assembly. scaffolds in btau_ have an order entirely consistent with the evidence from these three chromosomes, while both btau_ and the composite map show misplaced scaffolds. further assessment of the btau_ assembly was performed by comparing dense snp linkage maps constructed from genotyping snps in bulls belonging to half-sib families with the physical positioning of the snps on all autosomal chromosomes. the analysis revealed that snps were incorrectly positioned within assembly. this relatively small number indicates the high degree of precision in the btau_ assembly. these misplaced snps were relocated in the linkage map to a position corresponding to the most closely linked, correctly assigned snp. additionally, snps from unplaced scaffolds were mapped to linkage groups. discussion the genome assembly version btau_ is available in genbank under accession number aafc. in addition, the individual components of the genome assembly and the file with the component assembly instructions are available from the bcm-hgsc ftp site which is a link from the bovine page on the bcm-hgsc web site.) since the process of genome assembly involves decisions about which sequences to include and which sequences to exclude, there are sequences from this project that were omitted from the final assembly. some of the omitted sequences are highly repetitive sequence reads, others may have enough sequencing errors that they did not match the assembled sequences, others are assembled sequence contigs that appear to be duplicates of sequences in the assembly. these excluded sequence are also available from the bcm-hgsc ftp site. the majority of the sequence in the project is from the female animal, the genome sequence is described for the autosomes and the x chromosome. however, as the bac library was prepared from a male animal, and the bac fingerprint contigs were built from random clones from that library, both the x and y chromosomes are represented in the bac fingerprint contigs. representative bacs in all of the bac fingerprint contigs were sequenced to low coverage, including y chromosome bacs. since the clone coverage on the sex chromosomes in the bac library is half that of the autosomes, there will be less depth of clone coverage on the sex chromosomes and this may result in more gaps in the coverage of the sex chromosomes by bac clones. the wgs sequence was from the female animal, so there is not additional wgs sequence to assemble with the low coverage bac skim sequences for the y chromosome, unless it is pseudoautosomal sequence from the x chromosome or autosomal sequence that is similar to the y sequence. since the bac fingerprint contigs were used to build the combined bac+wgs assemblies, there are genome sequence scaffolds from both sex chromosomes as well as the autosomes. the y chromosome scaffolds are unlabeled in the unplaced chromosome. the use of linkage data to identify incorrectly positioned snps has been extended to also reveal the identity of the most closely linked, and correctly positioned snps. in addition, the identification of snps that map to linkage groups but are found within unplaced scaffolds can be used to suggest a position for these scaffolds within the assembly. taken together, data associating misplaced snps and unplaced scaffolds with correctly positioned markers could be used to highlight regions that could benefit from map assisted assembly improvements. however, the moderate number of individuals being genotyped limits the mapping resolution. so while this analysis is effective at resolving large distance misplacements, additional genotyping of families would be required to reveal more local rearrangements.  the bovine genome assembly reported here was used for the analysis of the bovine genome sequence that is being published. most of those analyses used the gene annotation from the btau_ assembly. some of the analyses used the btau_ assembly. 
the estimated genome size is gb which represents a high degree of completeness, with 95% of the available est sequences found in assembled contigs. the assembly method combines the bac plus wgs local assembly used for the rat and sea urchin with the whole genome shotgun only assembly used for many other animal genomes including the rhesus macaque. the assembly process consisted of multiple phases: first, bacs were assembled with bac generated sequence, then subsequently in combination with the individual overlapping wgs reads. we present here the assembly of the bovine genome. the biological analysis of this bovine genome assembly is being published, and the sequence data is available to support future bovine research. the two assemblies were combined to create a more complete genome representation that retained the high quality bac-based local assembly information, but with gaps between bacs filled in with the wgs-only assembly.
 we present here the assembly of the bovine genome. the assembly method combines the bac plus wgs local assembly used for the rat and sea urchin with the whole genome shotgun only assembly used for many other animal genomes including the rhesus macaque.  the assembly process consisted of multiple phases: first, bacs were assembled with bac generated sequence, then subsequently in combination with the individual overlapping wgs reads. different assembly parameters were tested to separately optimize the performance for each bac assembly of the bac and wgs reads. in parallel, a second assembly was produced using only the wgs sequences and a global whole genome assembly method. the two assemblies were combined to create a more complete genome representation that retained the high quality bac-based local assembly information, but with gaps between bacs filled in with the wgs-only assembly. finally, the entire assembly was placed on chromosomes using the available map information. over 90% of the assembly is now placed on chromosomes. the estimated genome size is gb which represents a high degree of completeness, with 95% of the available est sequences found in assembled contigs. the quality of the assembly was evaluated by comparison to finished bacs, where the draft assembly covers between and 100% of the finished bacs. the assembly contigs and scaffolds align linearly to the finished bacs, suggesting that misassemblies are rare. genotyping and genetic mapping of snps revealed that more than % were correctly positioned within the btau_ assembly, confirming the accuracy of the assembly.  the biological analysis of this bovine genome assembly is being published, and the sequence data is available to support future bovine research. 