BACKGROUND
cattle are the most common type of large domesticated animals and have consequently played an important role in recent history of humankind since their domestication  <dig>  to  <dig>  years ago  <cit> .
cattle have enhanced human civilizations through their varied uses as livestock for meat, milk, and draft power.
accordingly, there are ~ <dig>  billion cattle in the world today providing a significant source of nutrition and livelihood to the human population.
domestic cattle comprise more than  <dig> breeds and are grouped taxonomically into two species—bos taurus  and b. indicus —which were evolved from the ancestral species of b. primigenius.
given this large and venerable resource of cattle breeds, cattle research efforts have also greatly contributed to our knowledge of genetics, endocrine function, fertilization, growth, lactation and mammalian biology.
as such, there are still many unsolved questions regarding cattle adaptation to diverse terrestrial environments since domestication that center on how cattle convert low-grade forage to energy-rich fat, milk and meat, and, more fundamentally, how genetic underpinnings define economically important traits.
the cattle genome was originally selected for sequencing due to its unique biology and economic importance, virtues that are also strengthened by its role as a model organism for understanding other ruminants, or mammals.
the bovine genome sequencing and analysis consortium published the first draft sequence for the bos taurus genome in 2009--a sizable effort costing $ <dig> million and involving nearly  <dig> investigators from  <dig> countries  <cit> .
the initial sequence assembly  was constructed by the baylor college of medicine human genome sequencing center using ~ <dig> -fold sanger sequencing coverage of the genome.
their genome assembly approach combined a bac  clone-by-clone approach with whole genome shotgun  reads, and yielded an n <dig> contig size of  <dig>  kb and a n <dig> scaffold size of  <dig>  mb .
89 % of these assembled contigs and scaffolds were anchored onto the  <dig> bovine autosomes and the x chromosome based on the integrated fpc physical map  <cit> , which combined a series of complementary mapping resources:  <dig>  fingerprinted bacs, the human-cattle comparative map, the genetic map, and the radiation hybrid  map .
the center for bioinformatics and computational biology, university of maryland, using a different strategy, constructed another bovine assembly in  <dig> based on the same raw sequence and map data .
their strategy leveraged paired-end bac sequence information, mapping data and, most notably, syntenic relationships to the human genome that allowed 91 % of the umd <dig> contigs to be anchored to bovine chromosomes, based on the integrated bovine genome map  <cit> .
comparisons between these two assemblies revealed substantial differences that appear as assembly errors, genome segmental inversions, chromosomal placements, sequence gap numbers, and discrepancies of the sequence coverage across the bovine genome .
two updated bovine genome sequence assemblies  were released from these groups featuring additional bac sequence data, corrected assembly errors and additional gap filling.
although comprehensive analyses of these recent releases have yet to be done, significant differences between these updated assemblies are generally expected to be encountered.
indeed, this article reports on notable disparities.
consequently, discrepancies between these assemblies engender ambiguities when using reference sequence data, which significantly impacts almost any type of genomic study in cattle.
the cattle genome, as discussed, enjoys a broad range of map resources that include: genetic linkage maps using microsatellite markers; markers comprising aflp, est, and bac end sequences; a radiation hybrid map, and a bac physical map .
despite this, these resources fall a bit short in several ways.
the genetic linkage and radiation hybrid maps lack sufficient levels of unambiguous markers, but, more troubling, the linkage map is a composite constructed across many separate bovine populations and thus doesn’t reflect a single bovine genome.
the bovine bac physical map is a composite map that was constructed from three different bac libraries developed from three different cattle breeds   <cit> .
understandably, such haplotype and/or breed-specific variability in these map resources could translate into ambiguities evidenced by sequence-map comparisons, which may have impacted the fine-scale assembly, or previous validations of the bovine reference sequence.
we constructed a comprehensive optical map spanning the bovine genome, using genomic dna from just one animal  in order to circumvent this array of issues.
this new resource will provide the bovine community with a highly accurate and comprehensive physical map that enables direct and independent comparisons amongst sequence builds, with goals pointed at sequence finishing and discovery of genomic differences.
briefly, optical mapping is a single-molecule system that constructs high-resolution physical scaffolds, covering entire genomes to guide many stages of genome sequence assembly and validation .
since it assembles genome-wide ordered restriction maps from massive datasets comprising randomly sheared genomic dna molecules , artefacts associated with cloning and amplification are completely obviated.
furthermore, very long dna molecules span complex genomic regions that are rife with repeats that generally hinder accurate sequence assembly without optical mapping analysis.
as such, our optical map offers an uniquely effective means for resolving and mediating the differences between the two different bovine genome sequence assemblies in several ways: 1) recruiting new orphan sequence contigs that fill sequence gaps; 2) providing an independent resource that potentiates finishing through sequence gap characterization, and 3) enabling independent validations of sequence assemblies.
RESULTS
optical map dataset
genomic dna was prepared from l <dig> dominette  <dig> blood samples, after separation of white blood cells, and then bamhi restriction mapped using our optical mapping pipeline .
this raw dataset holds  <dig> , <dig> rmaps  ≥ 300 kb, with an average size of  <dig>  kb  and a total mass of  <dig> .97 mb .
one rmap is the restriction map of a single genomic dna molecule; it represents the most fundamental unit of map data in functional ways akin to a sequence read.
a second map dataset was contributed by prof. juan f. medrano and after size filtering  it added another  <dig> , <dig> rmaps, bringing the total raw dataset up to  <dig> , <dig> .
initial evaluation of the genome builds umd <dig>  and btau <dig>  via pairwise alignments of the rmap dataset
the umd <dig>  and btau <dig>  references were first evaluated for large-scale errors by inspection of the pairwise alignments  <cit>  of the entire rmap dataset against bamh <dig> in silico restriction maps  of both sequence builds .
these map vs. reference alignments produce files, similar to sequence sam/bam files, which note the location of each aligned rmap .
such alignments allow us to quickly filter-out marginal rmaps from the raw dataset and provide an initial assessment of the completeness of a given sequence build  <cit> .
the average rmap coverage after alignment varies considerably between the two builds, with  <dig> x for btau <dig> , while umd <dig>  boasts  <dig> x. additional file 1: figure s <dig> and additional file 2: figure s <dig> also show a specific example of disparate rates of rmap coverage, focusing on a  <dig>  mb region on chromosome  <dig>  highlighted by a green box, where  <dig> rmaps  are aligned to btau <dig> , compared to  <dig> rmaps  aligned to umd <dig> .
given these vastly different overall alignment rates and patterns, we chose umd <dig>  to serve as our reference sequence build for assembling the optical map.
optical map assembly
our optical map assembly strategy used a two-pronged approach involving iterative assembly, requiring a sequence reference  <cit> , and de novo assembly for dealing with large-scale discordances  and gaps in the umd <dig>  build .
many of these problematic regions are sparsely populated by rmaps as evidenced from inspection of additional file 1: figure s <dig> .
accordingly, the workflow  shows how iterative assembly selectively shunts uncontiged rmaps, mostly originating from these problematic regions, into “germinate and grow”  for de novo assembly.
resulting optical map contigs from both sides of the workflow were then curated and combined for finishing the optical map.
details follow in the next two subsections.fig.
<dig> strategy and workflow used for assembling the optical map-- btom <dig> .
left side describes reference-based iterative map assembly: exhaustive pairwise alignments of the complete rmap dataset  against the umd <dig>  sequence reference maps  generated in silico produces “piles” of rmaps.
such alignments are then divided into overlapping bins , which are then independently assembled into updated reference maps  that are used for  <dig> subsequent cycles  of alignment  and assembly, all performed without using the sequence reference.
right side depicts de novo map assembly, using those rmaps from the entire dataset not recruited for optical map contig formation during iterative map assembly, which are used to construct a de bruijn graph via k-mer hashing.
“seed” maps are then assembled from rmaps in each each confident node within graph and used as an optical reference for pairwise alignments  of rmaps during iterative assembly .
bottom shows the merged assembly of updated optical contigs from each bin  and contigs assembled from “seed” maps  into the final optical map-- btom <dig> , which was used to tabulate map vs. sequence discordances
iterative assembly
we published a workflow in  <dig>  <cit> , termed “iterative assembly” , which embedded genome assembly algorithms , originally designed to deal with small bacterial or fungal genomes, within a new pipeline.
this pipeline supports the assembly and analysis of large mammalian and plant optical maps by distributing the computation into large numbers of independent jobs that can be executed on a high-throughput computing network.
briefly, iterative assembly uses an in silico restriction map of available genome sequence resources--contigs, scaffolds, pseudomolecules, etc.--as a reference for exhaustive pairwise alignment  <cit>  of entire rmap datasets.
both sequence data  and actual genomic dna molecules  are “cut” with the same restriction enzyme.
thusly placed rmaps, termed “piles,” covering an entire genome, are then divided into 1 mb overlapping bins along each chromosome for assembly; each bin is independently assembled into contigs.
each optical contig bears a consensus map, which now becomes the updated, independent reference; sequence information is no longer used in the assembly process.
repeatedly iterating this workflow increases optical contig length, number and depth.
eight iteration cycles were performed using a bamh <dig> in silico map constructed from the umd <dig>  sequence build as the initial reference and with a minimum depth of  <dig> rmaps.
<dig>  contigs emerged after the first iteration ranging 404– <dig>  kb in size; averaging  <dig>  kb.
however, after  <dig> iterations the number of contigs increased to  <dig> , and their average span was boosted to  <dig>  kb .
contigs presenting very long tandem repeats were removed from this process.
these  <dig>  optical contigs were then grouped by chromosome, using alignments to umd <dig>  and each grouping was independently assembled into a total of just  <dig> contigs spanning  <dig>  % of the umd <dig>  build.
de novo assembly
we have previously reported on the map assembler, a de novo optical map assembler capable of assembling bacterial maps  <cit> .
however, the map assembler algorithm has polynomial complexity  and exceeds feasible memory and time constraints for genomes of size >10 mb.
to face the challenge of assembling larger genomes, we’ve implemented germinate and grow , a new de novo assembly algorithm that will be fully described elsewhere.
the conceptual basis for g & g is an extension of the de bruijn graph approach to sequence assembly  <cit> .
simply put, a whole genome optical map can be represented by the traversals of a certain graph, and the assembly problem is to discover those traversals from the input data set of rmaps.
specifically, we use geometric k-mer hashing  <cit>  to identify nodes in the de bruijn graph that are very likely error-free and then traverse the “read” paths implied by the rmaps containing instances of those nodes.
this traversal allows us to localize the assembly; we then use the map assembler on the subsets of rmaps that are near each other on this graph.
we call the resulting consensus maps seed maps.
the seed maps typically cover most of the genome and they reliably approximate highly confident paths in the graph.
the seed maps are then extended and refined using the iterative assembly engine , producing another set of consensus maps.
the error rate for these consensus maps is sufficiently low for resolving the corresponding euler path and assembling all but the most repetitive regions of the genome.
we then fill gaps in the assembly by repeating the process, generating another set of seed maps and extending and refining them.
for this set of seed maps, we use a lower stringency  and use only those rmaps not already represented in the genome reference-based iterative assembly.
we used g & g to assemble just those rmaps  that escaped assembly within the iterative assembly pipeline, which yielded  <dig>  optical contigs, with most recapitulating those constructed by iterative assembly.
as such, these de novo optical maps were largely used to augment and cross-validate optical map assemblies constructed by iterative assembly.
the final bovine optical map--termed, “btom <dig> ”--comprises  <dig> contigs spanning of  <dig> .30 mb across the genome , at an average depth of  <dig> rmaps and an average contig size of  <dig>  mb .table  <dig> btom <dig>  contigs and their chromosome assignments
note, abbreviations: chr.
= chromosome, pooledsd = pooled standard deviation, avefragsize = average fragment size, and ave. = average, chr.end = chromosome end or telomere, rend = right hand side of the contig is telomere, lend = left hand side of the contig is telomere
the haploid bovine genome harbors  <dig> acrocentric, autosomal chromosomes, and one sex chromosome, or  <dig> telomeric ends.
accordingly, 20/ <dig> optical contigs  present sharply demarcated ends , which indicate that they’ve spanned into the repetitive sequences near telomeres.
the remaining 40/ <dig> chromosome ends are not, or, are partially spanned by optical maps because the short arms of these acrocentric chromosomes are densely populated by repeats, making them intractable to our analysis.
interestingly, we find that btomcontig_ <dig> has ~6 kb tandem repeats at one end, which also shows alignment to chromosome  <dig>  in addition, btomcontig_ <dig> presents tandem repeats with a repeat unit consisting of multiple bamhi fragments with a total unit mass of ~290 kb and is anchored on bovine chromosome  <dig> .
lastly, an additional  <dig> optical map contigs, spanning  <dig>  mb show evidence of heterozygosity, manifested as indels  on chromosomes  within btom <dig>  as illustrated by the examples in fig.
2c.
all the contiged rmaps for each chromosome, and all the optical map contig consensus maps are available at github fig.
<dig> examples of optical contigs, within btom <dig> , reveal a telomeric region, complex tandem repeats and heterozygous structural variants.
a: telomeric regions are defined through assembly when rmap contigs present sharply defined edges ; example shown is chr <dig>  btomcontig_ <dig> ; horizontal tracks depict rmaps  with boxes representing individual restriction fragments, color keyed as:  based on comparisons against the consensus map .
b: an optical map contig, showing the structure of a complex tandem repeat.
c: consensus maps of optical contigs  aligned to btom <dig>   showing heterozygous structural variants distinguished by deletions and insertions; numbers on each fragment bar show restriction fragment size .
the first haplotype deletion/insertion is in the region corresponding to umd <dig> _chr <dig>  <dig> ,591– <dig> , <dig>  and this region encoded an atpase and an aminophospholipid transporter .
the second case is corresponding to umd <dig>  chr <dig>  <dig> ,042– <dig> , <dig>  there is a ~27 kb missing sequence in umd <dig>  chr <dig> at this region, and the other part of this region encoded a solute carrier organic anion transporter
fig.
<dig> btom <dig>  optical map  comprehensively aligned to umd <dig> .
optical contigs  extensively span across the umd <dig>  sequence assembly  revealing a minimal number of gaps in the optical map
<dig>
<dig>
construction of chromosome-wide optical maps and their comparison to umd <dig>  and btau <dig>
the optical contigs generated by iterative and de novo map assembly  were merged through assembly of their consensus maps into  <dig> final optical contigs.
they were then ordered and oriented, through alignment against a bamhi in silico restriction map constructed from the umd <dig>  sequence build .
chromosome-wide optical maps, btom <dig> _chr1- <dig> and btom <dig> _chrx, were constructed with 500 kb gaps inserted between any two of optical map contigs anchored on the umd <dig>  sequence assembly .
this workflow constructed  <dig> chromosome-wide optical maps that were aligned to both btau <dig>  and umd <dig>  sequences using local alignment .
a series of contiguous restriction fragments that align between btom <dig>  and the in silico maps of a sequence build is called a “map segment.” tabulations describing these aligned map segments are listed in table  <dig>  in total,  <dig> map segments  present a total of aligned sequence segment mass of  <dig> .08 mb, with an average size of  <dig>  mb, or ~86 % of umd <dig>  are covered by optical maps.
map coverage of umd <dig>  ranges from  <dig> to 95 %.
50/ <dig> chromosome ends,  <dig>  mb in total, within umd <dig> , are extended by optical maps .
for btau <dig> ,  <dig> map segments align to btom <dig> , with a total mass of  <dig> .74 mb  and an aligned map segment size averaging  <dig>  mb.
the optical map coverage of btau <dig>  by optical maps is less than that tabulated for umd <dig>  and it ranges from ~ <dig> to 82 % for all  <dig> bovine chromosomes.
lastly, 55/ <dig> of the btau <dig>  chromosome ends are extended by optical maps with a mass totalling  <dig>  mb .fig.
<dig> genome-wide alignment of btom <dig>  to umd <dig>  and btau <dig>  sequence assemblies.
tracks show btom <dig>   alignments to umd <dig>   and btau <dig>   for each chromosome.
red highlights  restriction fragments aligned to both in silico maps of umd <dig>  and btau <dig> ; cyan  highlights alignment of btom <dig>  to umd <dig> , or btau <dig> ; and white or black highlights no alignments to either sequence build.
inset  shows a zoomed view of chr <dig> detailing a large-scale discordance with the optical map: an inverted sequence assembly within btau <dig> .
also note transposed sequence assemblies, flagged by black lines running between separate chromosomes
fig.
<dig> examples of optical map coverage within telomeric, or sub-telomeric regions.
a: btom <dig> _chr <dig> aligned to chr <dig> of both umd <dig>  and btau <dig> .
lines show bamhi restriction sites with red highlighting those btom <dig> _chr <dig> track  restriction fragments aligned to both umd <dig> _chr <dig> and btau <dig> _chr <dig>  white highlights unaligned btom <dig> _chr <dig> restriction fragments and extend 517 kb past both the ummd <dig> _chr <dig> and btau <dig> _chr <dig> sequence.
b: btom <dig> _chr <dig> aligned to chr <dig> of both umd <dig>  and btau <dig> .
the white region on btom <dig> _chr <dig>  is unaligned to both sequence builds and extends ~625 kb past umd <dig> _chr22
discordance calling between optical maps and sequence assemblies
discordances between btom <dig>  and the umd <dig>  were called based on the alignments between the consensus maps that were trimmed and stripped off from the last  cycle of iterative assembly  and then manually curated.
complex discordances required directed alignment and assembly steps, complemented by additional curation, for their complete characterization.
there are, in total,  <dig>  discordances called between btom <dig>  and the umd <dig>  based on only confident alignments and these discordances are tabulated as six categories :  large segments of inverted/translocated sequence ,  complex-events/misassembly/inversion ,  ins-insertion/missing sequence ,  del-deletion/extra sequence included ,  ec-extra restriction site , and  mc-missing restriction site .table  <dig> statistics for the six categories of discordances between sequence builds and btom <dig>
fig.
<dig> circos plot of discordances found between btom <dig>   and umd <dig>  .
the tracks a, b, c, d, e, and f represents the six categories of discordances: insertion , implying missing sequence; deletions  implying extra sequence; compound multiple events including insertion/deletion/inversion etc.
; missing restriction sites ; extra restriction sites ; and large inversions or translocations
fig.
<dig> examples of large-scale map vs. sequence discordances .
a: gps  viewer shows the btom <dig> _chr <dig> optical map  alignments to umd <dig> _chr <dig> and btau <dig> _chr <dig> revealing 759 kb translocated region  and a  <dig>  kb inversion .
color scheme is the same as fig.
<dig>  with cyan highlighting btom <dig> _chr <dig> portions aligning to umd <dig> _chr <dig> or btu <dig> _chr <dig>  b: ~1 mb region of chromosome 17: the in silico maps  of umd <dig> _chr <dig> perfectly align to the btom <dig> _chr <dig>  but btau <dig> _chr17/btom <dig> _chr <dig> alignment reveals two misassembled regions in the btau <dig>  sequence build: an inversion  and extraneous sequence.
restriction fragments  are color keyed as: gold ; red ; cyan ; and purple .
c: ~500 kb region on chromosome 19: alignment of btom <dig> _chr <dig> and btau <dig> _chr <dig>  suggest that the btau <dig> _chr <dig> sequence  was probably misplaced and should be inverted and placed nearby.
btom <dig> _chr <dig> and umd <dig> _chr <dig>  alignment suggests that sequence here also presents misassemblies similar to btau <dig> _chr19
similarly, btau <dig>  discordances were tabulated as just described for umd <dig> , but relied on the same optical consensus maps created from umd <dig> .
these efforts identified  <dig>  discordances in the btau <dig>  sequence assembly .
such tabulations include  <dig> large segments of inverted/translocated  ,  <dig>  complex-complex events/misassembly/inversion ,  <dig> ins-insertion/missing sequences ,  <dig> del-deletion/extraneous sequence ,  <dig>  ec-extra restriction sites, and  <dig> mc-missing restriction sites.
discussion and
CONCLUSIONS
a whole genome optical map, btom <dig> , of the b. taurus hereford breed, l <dig> dominette  <dig> was constructed using the same animal employed for whole genome shotgun sequencing, which was also the daughter of the hereford bull l <dig> domino  used for the construction of the previously analyzed bac library  <cit> .
the optical map spans  <dig> .30 mb across the b. taurus genome and comprises  <dig> optical contigs, which provide accurate size estimations for  <dig>  bamhi restriction fragments.
alignments between btom <dig>  and in silico restriction maps of umd <dig>  and btau <dig>  revealed numerous discordances at genomic length scales reaching from a restriction site to portions of a chromosomal arm.
on average, there is a bamhi site every  <dig>  kb, and such “marker” density is far greater than the bovine genetic map  and the composite map  which combines linkage and radiation hybrid resources  <cit> .
the size of the b. taurus genome, estimated by optical mapping, is similar to those estimates provided by the umd <dig>  and btau <dig>  genome sequence assemblies , but is ~17 % smaller than the  <dig>  mb size estimated by bac fingerprinting  <cit> .
genome size differences may stem, in part, from genome analysis efforts that employed dna samples from three different cattle breeds: hereford, holstein and angus  <cit> , whose separate bac ibraries were used for previous mapping efforts.
our analysis, through alignments to btom <dig> , showed that btau <dig>  presented far more discordances as compared to umd <dig> .
overall, we found that btau <dig>  presented almost double the number of discordances across most of its  <dig> categories of sequence vs. map discrepancies.
they include complex  discordances as compared to umd <dig>  , which more than doubled the amount of affected sequence .
further comparisons of btau <dig>  to umd <dig>  also showed a doubled count of dels  discordances  and the amount of affected sequence .
similarly, btau <dig>  presented a doubled rate of called ins  and inverted/translocated discordances .
comparisons of btau <dig>  and umd <dig>  to btom <dig>  also revealed large-scale difference between these sequence assemblies.
such issues became most apparent through our analysis of optical map alignments at telomeric regions, or ends of chromosomes.
table  <dig> shows that btau <dig>  is missing more sequence at chromosome ends  as compared to umd <dig>  .
also, the btau <dig>  assembly of the x chromosome excluded ~60 mb of sequence relative to btom <dig>  .
therefore, our comparative analysis results of umd <dig>  and btau <dig>  based on alignments to btom <dig>  are in line with previous reports  <cit> , and affirm the ncbi’s current designation of umd <dig>  sequence assembly as the “reference” assembly and the btau <dig> .
<dig> assembly as the “alternate” assembly  <cit> .fig.
<dig> map alignments among btom <dig>  chromosome x and umd <dig>  and btau <dig> .
gps   reveals numerous and very large-scale discordances as a series of misassemblies.
zoomed portion shows details of such misassembled regions
there are numerous sequence gaps in the two b. taurus genome sequence assemblies .
however, most of the sequence gaps are small, in that there are only  <dig> sequence gaps ≥ 2 kb in umd <dig>  and  <dig>  in btau <dig> .
importantly, greater than 96 % of these gaps in umd <dig>  and btau <dig>  were successfully bridged by btom <dig>  .
accordingly, this analysis begs the question: are the discordances called in umd <dig>  and btau <dig> , through alignments to btom <dig> , largely due to sequence gaps inserted into the two sequence assemblies?
we explored this question by intersecting the sequence gap and discordance coordinates from both sequence assemblies, and identified within umd <dig>  only 162/ <dig>  discordances: , which intersected just 167/ <dig> sequence gaps .
for btau <dig>  we found  <dig> / <dig> : , which intersected  <dig> / <dig>  sequence gaps .
thus,  <dig>  % of the large sequence gaps in umd <dig>  contribute to only  <dig>  % of the discordances called in umd <dig> , while  <dig>  % of the large sequence gaps in btau <dig>  are responsible for  <dig>  % of the called discordances in this assembly.
as such, this simple analysis further substantiates the superior quality of umd <dig>  vs. btau <dig> , which in part, is reflected by the high rate of parsimoniously inserted sequence gaps.
our systematic tabulation and curation of discordances found through comparison of btom <dig>  vs. umd <dig> , or btau <dig>  will greatly facilitate future improvements of b. taurus genome sequence assemblies in order to build a more accurate and unified version of the reference sequence.
because btom <dig>  was constructed from dna derived from the very same animal that was sequenced, this physical map provides direct comparisons to these other resources that are not affected by genotype differences manifested by other breeds, or even animals of the same breed.
although there are many map resources available for the b. taurus genome, which include genetic linkage maps , radiation hybrid maps  <cit> , bac physical maps  <cit> , cytogenetic maps  <cit>  and comparative maps between cattle and human  <cit> , the resolution of these maps can be modest.
consider that the b. taurus composite map of integrated linkage/radiation hybrid maps  <cit>  and bac physical maps  <cit>  features the greatest number of markers , but with a density of only ~180 kb/marker.
in comparison, btom <dig>  boasts an average restriction site density of  <dig>  kb, which fostered resolution of difficult-to-discern errors in sequence assembly.
for example, fig.
<dig> shows a 79 kb region that was inverted and misplaced based on alignment to btom <dig> , which was also substantiated by new sequence data and pcr.fig.
<dig> optical map  reveals a 79 kb sequence assembly as being transposed and inverted in umd <dig> _chr <dig>   alignment shows an unaligned 79 kb segment  within the optical map and a corresponding gap in the sequence.
same 79 kb segment within the umd <dig>  build but apparently transposed to  <dig> ,754 bp and inverted.
accordingly, there is a 79 kb gap in the optical map.
illumina paired-end dominette l <dig> sequence data, aligned to umd <dig>  corroborates sequence misjoining points  at  <dig> ,000 bp and  <dig> ,500 bp.
pcr experiments confirms that the 79 kb segment should be placed between  <dig>  and  <dig>  on chromosome  <dig> .
paired-end reads mapped to umd <dig>  showing correct orientations with both ends mapped are named an intact pair .
when only one end is mapped, or mapping shows wrong orientation, or revealing discordant distances between mapped read pairs, these events are termed broken pairs.
reads of a broken pair that map to a unique location against the reference are colored green or red, according to whether they mapped in the forward, or reverse, orientation respectively
during the course of writing this manuscript, a reviewer questioned the extent of map errors and possible biases that may be introduced through our selective use of umd <dig>  as the reference genome for btom <dig> .
although our previous publications report a high degree of accuracy and minimal biases stemming from the iterative assembly pipeline  <cit> , we compared the iterative assembly of optical maps constructed from umd <dig>  vs. btau <dig>  using chromosomes  <dig> and  <dig> .
using btau <dig>  as the reference sequence, eight iterations  and merging of optical contigs produced three optical map contigs for chromosome  <dig> and a single optical map contig was derived for chromosome  <dig>  alignments show that these new maps are essentially identical to btom <dig>  except for a few restriction site differences .
we attribute these minor differences to heterozygosity, since our calling of discordances uses a single representation of the physical map created by optical mapping.
however, over an entire chromosome multi-mb-scale differences are apparent.
fig.
10b shows three optical map contigs aligned to btau <dig> , chromosome  <dig>  presenting a large gap  between contig <dig>  and contig <dig> , and no gap between contig <dig>  and contig <dig>  relative to btom <dig> ; while a single contig  spans the same chromosome using umd <dig>  chromosome  <dig>  in comparison, chromosome  <dig> shows a single optical map contig  generated from the btau <dig>  sequence as the starting reference, while two optical map contigs  formed using the umd <dig>  sequence for chromosome  <dig> .
the absence of the small contig <dig>  from the btau <dig>  derived optical contigs implies reduced coverage for chromosome  <dig>  as such, our analysis shows that btom <dig>  bears minimal local biases stemming from the choice of sequence build used for iterative assembly, but the overall optical map coverage varies.
fortunately, absent, or problematic genomic regions would then be covered, as required, by optical maps constructed by de novo techniques .
consequently, the need for de novo assembly steps is minimized by judicious selection of a reference genome for iterative assembly of an optical map.fig.
<dig> comparisons of optical maps assemblies seeded by umd <dig> , or btau <dig> .
.
black vertical lines demarcate restriction fragments, or map alignments.)
a: alignments of btom <dig> _chrs  <dig>   <dig>  against the in silico maps of umd <dig> --chrs  <dig> and  <dig> .
b: iterative assembly results for chrs  <dig> and  <dig> using btau <dig>  as the reference sequence.
note large inversion , not present in umd <dig> , or optical maps, revealed within the btau <dig>  chr <dig> sequence.
c: alignment of the optical maps shown in  and .
inset shows  <dig>  mb region; vertical lines locate bamhi restriction sites.
pink fragment  shows the gap between the two optical map contigs in btom <dig>  chromosome 28
we conclude that btom <dig>  will prove to be a valuable resource for advancing the state of current sequence assemblies, by serving as a largely independent physical scaffold, as shown in figs.
<dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  but perhaps, more importantly, as a platform to support future comparative studies, focusing on structural variation amongst different cattle breeds, or within populations.
lastly, errors always accompany any ambitious effort pointed at comprehensive analysis of entire genomes.
accordingly, the true merits and accuracy of a new resource, such as btom <dig> , will be comprehensively assessed over time by individual researchers in the bovine community.

 cattle are the most common type of large domesticated animals and have consequently played an important role in recent history of humankind since their domestication to  years ago. cattle have enhanced human civilizations through their varied uses as livestock for meat, milk, and draft power. accordingly, there are ~ billion cattle in the world today providing a significant source of nutrition and livelihood to the human population. domestic cattle comprise more than breeds and are grouped taxonomically into two species—bos taurus and b. indicus —which were evolved from the ancestral species of b. primigenius. given this large and venerable resource of cattle breeds, cattle research efforts have also greatly contributed to our knowledge of genetics, endocrine function, fertilization, growth, lactation and mammalian biology. as such, there are still many unsolved questions regarding cattle adaptation to diverse terrestrial environments since domestication that center on how cattle convert low-grade forage to energy-rich fat, milk and meat, and, more fundamentally, how genetic underpinnings define economically important traits. the cattle genome was originally selected for sequencing due to its unique biology and economic importance, virtues that are also strengthened by its role as a model organism for understanding other ruminants, or mammals. the bovine genome sequencing and analysis consortium published the first draft sequence for the bos taurus genome in 2009--a sizable effort costing $ million and involving nearly investigators from countries. the initial sequence assembly was constructed by the baylor college of medicine human genome sequencing center using ~ -fold sanger sequencing coverage of the genome. their genome assembly approach combined a bac clone-by-clone approach with whole genome shotgun reads, and yielded an n contig size of  kb and a n scaffold size of  mb. 89 % of these assembled contigs and scaffolds were anchored onto the bovine autosomes and the x chromosome based on the integrated fpc physical map, which combined a series of complementary mapping resources: fingerprinted bacs, the human-cattle comparative map, the genetic map, and the radiation hybrid map. the center for bioinformatics and computational biology, university of maryland, using a different strategy, constructed another bovine assembly in based on the same raw sequence and map data. their strategy leveraged paired-end bac sequence information, mapping data and, most notably, syntenic relationships to the human genome that allowed 91 % of the umd contigs to be anchored to bovine chromosomes, based on the integrated bovine genome map. comparisons between these two assemblies revealed substantial differences that appear as assembly errors, genome segmental inversions, chromosomal placements, sequence gap numbers, and discrepancies of the sequence coverage across the bovine genome. two updated bovine genome sequence assemblies were released from these groups featuring additional bac sequence data, corrected assembly errors and additional gap filling. although comprehensive analyses of these recent releases have yet to be done, significant differences between these updated assemblies are generally expected to be encountered. indeed, this article reports on notable disparities. consequently, discrepancies between these assemblies engender ambiguities when using reference sequence data, which significantly impacts almost any type of genomic study in cattle. the cattle genome, as discussed, enjoys a broad range of map resources that include: genetic linkage maps using microsatellite markers; markers comprising aflp, est, and bac end sequences; a radiation hybrid map, and a bac physical map. despite this, these resources fall a bit short in several ways. the genetic linkage and radiation hybrid maps lack sufficient levels of unambiguous markers, but, more troubling, the linkage map is a composite constructed across many separate bovine populations and thus doesn’t reflect a single bovine genome. the bovine bac physical map is a composite map that was constructed from three different bac libraries developed from three different cattle breeds. understandably, such haplotype and/or breed-specific variability in these map resources could translate into ambiguities evidenced by sequence-map comparisons, which may have impacted the fine-scale assembly, or previous validations of the bovine reference sequence. we constructed a comprehensive optical map spanning the bovine genome, using genomic dna from just one animal in order to circumvent this array of issues. this new resource will provide the bovine community with a highly accurate and comprehensive physical map that enables direct and independent comparisons amongst sequence builds, with goals pointed at sequence finishing and discovery of genomic differences. briefly, optical mapping is a single-molecule system that constructs high-resolution physical scaffolds, covering entire genomes to guide many stages of genome sequence assembly and validation. since it assembles genome-wide ordered restriction maps from massive datasets comprising randomly sheared genomic dna molecules, artefacts associated with cloning and amplification are completely obviated. furthermore, very long dna molecules span complex genomic regions that are rife with repeats that generally hinder accurate sequence assembly without optical mapping analysis. as such, our optical map offers an uniquely effective means for resolving and mediating the differences between the two different bovine genome sequence assemblies in several ways: 1) recruiting new orphan sequence contigs that fill sequence gaps; 2) providing an independent resource that potentiates finishing through sequence gap characterization, and 3) enabling independent validations of sequence assemblies.  optical map dataset genomic dna was prepared from l dominette blood samples, after separation of white blood cells, and then bamhi restriction mapped using our optical mapping pipeline. this raw dataset holds, rmaps  ≥ 300 kb, with an average size of  kb and a total mass of.97 mb. one rmap is the restriction map of a single genomic dna molecule; it represents the most fundamental unit of map data in functional ways akin to a sequence read. a second map dataset was contributed by prof. juan f. medrano and after size filtering it added another, rmaps, bringing the total raw dataset up to,. initial evaluation of the genome builds umd and btau via pairwise alignments of the rmap dataset the umd and btau references were first evaluated for large-scale errors by inspection of the pairwise alignments of the entire rmap dataset against bamh in silico restriction maps of both sequence builds. these map vs. reference alignments produce files, similar to sequence sam/bam files, which note the location of each aligned rmap. such alignments allow us to quickly filter-out marginal rmaps from the raw dataset and provide an initial assessment of the completeness of a given sequence build. the average rmap coverage after alignment varies considerably between the two builds, with x for btau, while umd boasts x. additional file 1: figure s and additional file 2: figure s also show a specific example of disparate rates of rmap coverage, focusing on a  mb region on chromosome highlighted by a green box, where rmaps are aligned to btau, compared to rmaps aligned to umd. given these vastly different overall alignment rates and patterns, we chose umd to serve as our reference sequence build for assembling the optical map. optical map assembly our optical map assembly strategy used a two-pronged approach involving iterative assembly, requiring a sequence reference, and de novo assembly for dealing with large-scale discordances and gaps in the umd build. many of these problematic regions are sparsely populated by rmaps as evidenced from inspection of additional file 1: figure s. accordingly, the workflow shows how iterative assembly selectively shunts uncontiged rmaps, mostly originating from these problematic regions, into “germinate and grow” for de novo assembly. resulting optical map contigs from both sides of the workflow were then curated and combined for finishing the optical map. details follow in the next two subsections.fig. strategy and workflow used for assembling the optical map-- btom. left side describes reference-based iterative map assembly: exhaustive pairwise alignments of the complete rmap dataset against the umd sequence reference maps generated in silico produces “piles” of rmaps. such alignments are then divided into overlapping bins, which are then independently assembled into updated reference maps that are used for subsequent cycles of alignment and assembly, all performed without using the sequence reference. right side depicts de novo map assembly, using those rmaps from the entire dataset not recruited for optical map contig formation during iterative map assembly, which are used to construct a de bruijn graph via k-mer hashing. “seed” maps are then assembled from rmaps in each each confident node within graph and used as an optical reference for pairwise alignments of rmaps during iterative assembly. bottom shows the merged assembly of updated optical contigs from each bin and contigs assembled from “seed” maps into the final optical map-- btom, which was used to tabulate map vs. sequence discordances iterative assembly we published a workflow in, termed “iterative assembly”, which embedded genome assembly algorithms, originally designed to deal with small bacterial or fungal genomes, within a new pipeline. this pipeline supports the assembly and analysis of large mammalian and plant optical maps by distributing the computation into large numbers of independent jobs that can be executed on a high-throughput computing network. briefly, iterative assembly uses an in silico restriction map of available genome sequence resources--contigs, scaffolds, pseudomolecules, etc.--as a reference for exhaustive pairwise alignment of entire rmap datasets. both sequence data and actual genomic dna molecules are “cut” with the same restriction enzyme. thusly placed rmaps, termed “piles,” covering an entire genome, are then divided into 1 mb overlapping bins along each chromosome for assembly; each bin is independently assembled into contigs. each optical contig bears a consensus map, which now becomes the updated, independent reference; sequence information is no longer used in the assembly process. repeatedly iterating this workflow increases optical contig length, number and depth. eight iteration cycles were performed using a bamh in silico map constructed from the umd sequence build as the initial reference and with a minimum depth of rmaps. contigs emerged after the first iteration ranging 404–  kb in size; averaging  kb. however, after iterations the number of contigs increased to, and their average span was boosted to  kb. contigs presenting very long tandem repeats were removed from this process. these optical contigs were then grouped by chromosome, using alignments to umd and each grouping was independently assembled into a total of just contigs spanning  % of the umd build. de novo assembly we have previously reported on the map assembler, a de novo optical map assembler capable of assembling bacterial maps. however, the map assembler algorithm has polynomial complexity and exceeds feasible memory and time constraints for genomes of size >10 mb. to face the challenge of assembling larger genomes, we’ve implemented germinate and grow, a new de novo assembly algorithm that will be fully described elsewhere. the conceptual basis for g & g is an extension of the de bruijn graph approach to sequence assembly. simply put, a whole genome optical map can be represented by the traversals of a certain graph, and the assembly problem is to discover those traversals from the input data set of rmaps. specifically, we use geometric k-mer hashing to identify nodes in the de bruijn graph that are very likely error-free and then traverse the “read” paths implied by the rmaps containing instances of those nodes. this traversal allows us to localize the assembly; we then use the map assembler on the subsets of rmaps that are near each other on this graph. we call the resulting consensus maps seed maps. the seed maps typically cover most of the genome and they reliably approximate highly confident paths in the graph. the seed maps are then extended and refined using the iterative assembly engine, producing another set of consensus maps. the error rate for these consensus maps is sufficiently low for resolving the corresponding euler path and assembling all but the most repetitive regions of the genome. we then fill gaps in the assembly by repeating the process, generating another set of seed maps and extending and refining them. for this set of seed maps, we use a lower stringency and use only those rmaps not already represented in the genome reference-based iterative assembly. we used g & g to assemble just those rmaps that escaped assembly within the iterative assembly pipeline, which yielded optical contigs, with most recapitulating those constructed by iterative assembly. as such, these de novo optical maps were largely used to augment and cross-validate optical map assemblies constructed by iterative assembly. the final bovine optical map--termed, “btom ”--comprises contigs spanning of.30 mb across the genome, at an average depth of rmaps and an average contig size of  mb.table btom contigs and their chromosome assignments note, abbreviations: chr. = chromosome, pooledsd = pooled standard deviation, avefragsize = average fragment size, and ave. = average, chr.end = chromosome end or telomere, rend = right hand side of the contig is telomere, lend = left hand side of the contig is telomere the haploid bovine genome harbors acrocentric, autosomal chromosomes, and one sex chromosome, or telomeric ends. accordingly, 20/ optical contigs present sharply demarcated ends, which indicate that they’ve spanned into the repetitive sequences near telomeres. the remaining 40/ chromosome ends are not, or, are partially spanned by optical maps because the short arms of these acrocentric chromosomes are densely populated by repeats, making them intractable to our analysis. interestingly, we find that btomcontig_ has ~6 kb tandem repeats at one end, which also shows alignment to chromosome in addition, btomcontig_ presents tandem repeats with a repeat unit consisting of multiple bamhi fragments with a total unit mass of ~290 kb and is anchored on bovine chromosome. lastly, an additional optical map contigs, spanning  mb show evidence of heterozygosity, manifested as indels on chromosomes within btom as illustrated by the examples in fig. 2c. all the contiged rmaps for each chromosome, and all the optical map contig consensus maps are available at github fig. examples of optical contigs, within btom, reveal a telomeric region, complex tandem repeats and heterozygous structural variants. a: telomeric regions are defined through assembly when rmap contigs present sharply defined edges; example shown is chr btomcontig_; horizontal tracks depict rmaps with boxes representing individual restriction fragments, color keyed as: based on comparisons against the consensus map. b: an optical map contig, showing the structure of a complex tandem repeat. c: consensus maps of optical contigs aligned to btom showing heterozygous structural variants distinguished by deletions and insertions; numbers on each fragment bar show restriction fragment size. the first haplotype deletion/insertion is in the region corresponding to umd _chr,591–, and this region encoded an atpase and an aminophospholipid transporter. the second case is corresponding to umd chr,042–, there is a ~27 kb missing sequence in umd chr at this region, and the other part of this region encoded a solute carrier organic anion transporter fig. btom optical map comprehensively aligned to umd. optical contigs extensively span across the umd sequence assembly revealing a minimal number of gaps in the optical map construction of chromosome-wide optical maps and their comparison to umd and btau the optical contigs generated by iterative and de novo map assembly were merged through assembly of their consensus maps into final optical contigs. they were then ordered and oriented, through alignment against a bamhi in silico restriction map constructed from the umd sequence build. chromosome-wide optical maps, btom _chr1- and btom _chrx, were constructed with 500 kb gaps inserted between any two of optical map contigs anchored on the umd sequence assembly. this workflow constructed chromosome-wide optical maps that were aligned to both btau and umd sequences using local alignment. a series of contiguous restriction fragments that align between btom and the in silico maps of a sequence build is called a “map segment.” tabulations describing these aligned map segments are listed in table  in total, map segments present a total of aligned sequence segment mass of.08 mb, with an average size of  mb, or ~86 % of umd are covered by optical maps. map coverage of umd ranges from to 95 %. 50/ chromosome ends,  mb in total, within umd, are extended by optical maps. for btau, map segments align to btom, with a total mass of.74 mb and an aligned map segment size averaging  mb. the optical map coverage of btau by optical maps is less than that tabulated for umd and it ranges from ~ to 82 % for all bovine chromosomes. lastly, 55/ of the btau chromosome ends are extended by optical maps with a mass totalling  mb.fig. genome-wide alignment of btom to umd and btau sequence assemblies. tracks show btom alignments to umd and btau for each chromosome. red highlights restriction fragments aligned to both in silico maps of umd and btau; cyan highlights alignment of btom to umd, or btau; and white or black highlights no alignments to either sequence build. inset shows a zoomed view of chr detailing a large-scale discordance with the optical map: an inverted sequence assembly within btau. also note transposed sequence assemblies, flagged by black lines running between separate chromosomes fig. examples of optical map coverage within telomeric, or sub-telomeric regions. a: btom _chr aligned to chr of both umd and btau. lines show bamhi restriction sites with red highlighting those btom _chr track restriction fragments aligned to both umd _chr and btau _chr white highlights unaligned btom _chr restriction fragments and extend 517 kb past both the ummd _chr and btau _chr sequence. b: btom _chr aligned to chr of both umd and btau. the white region on btom _chr is unaligned to both sequence builds and extends ~625 kb past umd _chr22 discordance calling between optical maps and sequence assemblies discordances between btom and the umd were called based on the alignments between the consensus maps that were trimmed and stripped off from the last cycle of iterative assembly and then manually curated. complex discordances required directed alignment and assembly steps, complemented by additional curation, for their complete characterization. there are, in total, discordances called between btom and the umd based on only confident alignments and these discordances are tabulated as six categories: large segments of inverted/translocated sequence, complex-events/misassembly/inversion, ins-insertion/missing sequence, del-deletion/extra sequence included, ec-extra restriction site, and mc-missing restriction site.table statistics for the six categories of discordances between sequence builds and btom fig. circos plot of discordances found between btom and umd. the tracks a, b, c, d, e, and f represents the six categories of discordances: insertion, implying missing sequence; deletions implying extra sequence; compound multiple events including insertion/deletion/inversion etc.; missing restriction sites; extra restriction sites; and large inversions or translocations fig. examples of large-scale map vs. sequence discordances. a: gps viewer shows the btom _chr optical map alignments to umd _chr and btau _chr revealing 759 kb translocated region and a  kb inversion. color scheme is the same as fig.  with cyan highlighting btom _chr portions aligning to umd _chr or btu _chr b: ~1 mb region of chromosome 17: the in silico maps of umd _chr perfectly align to the btom _chr but btau _chr17/btom _chr alignment reveals two misassembled regions in the btau sequence build: an inversion and extraneous sequence. restriction fragments are color keyed as: gold; red; cyan; and purple. c: ~500 kb region on chromosome 19: alignment of btom _chr and btau _chr suggest that the btau _chr sequence was probably misplaced and should be inverted and placed nearby. btom _chr and umd _chr alignment suggests that sequence here also presents misassemblies similar to btau _chr19 similarly, btau discordances were tabulated as just described for umd, but relied on the same optical consensus maps created from umd. these efforts identified discordances in the btau sequence assembly. such tabulations include large segments of inverted/translocated, complex-complex events/misassembly/inversion, ins-insertion/missing sequences, del-deletion/extraneous sequence, ec-extra restriction sites, and mc-missing restriction sites. discussion and  a whole genome optical map, btom, of the b. taurus hereford breed, l dominette was constructed using the same animal employed for whole genome shotgun sequencing, which was also the daughter of the hereford bull l domino used for the construction of the previously analyzed bac library. the optical map spans.30 mb across the b. taurus genome and comprises optical contigs, which provide accurate size estimations for bamhi restriction fragments. alignments between btom and in silico restriction maps of umd and btau revealed numerous discordances at genomic length scales reaching from a restriction site to portions of a chromosomal arm. on average, there is a bamhi site every  kb, and such “marker” density is far greater than the bovine genetic map and the composite map which combines linkage and radiation hybrid resources. the size of the b. taurus genome, estimated by optical mapping, is similar to those estimates provided by the umd and btau genome sequence assemblies, but is ~17 % smaller than the  mb size estimated by bac fingerprinting. genome size differences may stem, in part, from genome analysis efforts that employed dna samples from three different cattle breeds: hereford, holstein and angus, whose separate bac ibraries were used for previous mapping efforts. our analysis, through alignments to btom, showed that btau presented far more discordances as compared to umd. overall, we found that btau presented almost double the number of discordances across most of its categories of sequence vs. map discrepancies. they include complex discordances as compared to umd, which more than doubled the amount of affected sequence. further comparisons of btau to umd also showed a doubled count of dels discordances and the amount of affected sequence. similarly, btau presented a doubled rate of called ins and inverted/translocated discordances. comparisons of btau and umd to btom also revealed large-scale difference between these sequence assemblies. such issues became most apparent through our analysis of optical map alignments at telomeric regions, or ends of chromosomes. table  shows that btau is missing more sequence at chromosome ends as compared to umd. also, the btau assembly of the x chromosome excluded ~60 mb of sequence relative to btom. therefore, our comparative analysis results of umd and btau based on alignments to btom are in line with previous reports, and affirm the ncbi’s current designation of umd sequence assembly as the “reference” assembly and the btau. assembly as the “alternate” assembly.fig. map alignments among btom chromosome x and umd and btau. gps reveals numerous and very large-scale discordances as a series of misassemblies. zoomed portion shows details of such misassembled regions there are numerous sequence gaps in the two b. taurus genome sequence assemblies. however, most of the sequence gaps are small, in that there are only sequence gaps ≥ 2 kb in umd and in btau. importantly, greater than 96 % of these gaps in umd and btau were successfully bridged by btom. accordingly, this analysis begs the question: are the discordances called in umd and btau, through alignments to btom, largely due to sequence gaps inserted into the two sequence assemblies? we explored this question by intersecting the sequence gap and discordance coordinates from both sequence assemblies, and identified within umd only 162/ discordances:, which intersected just 167/ sequence gaps. for btau we found /:, which intersected / sequence gaps. thus,  % of the large sequence gaps in umd contribute to only  % of the discordances called in umd, while  % of the large sequence gaps in btau are responsible for  % of the called discordances in this assembly. as such, this simple analysis further substantiates the superior quality of umd vs. btau, which in part, is reflected by the high rate of parsimoniously inserted sequence gaps. our systematic tabulation and curation of discordances found through comparison of btom vs. umd, or btau will greatly facilitate future improvements of b. taurus genome sequence assemblies in order to build a more accurate and unified version of the reference sequence. because btom was constructed from dna derived from the very same animal that was sequenced, this physical map provides direct comparisons to these other resources that are not affected by genotype differences manifested by other breeds, or even animals of the same breed. although there are many map resources available for the b. taurus genome, which include genetic linkage maps, radiation hybrid maps, bac physical maps, cytogenetic maps and comparative maps between cattle and human, the resolution of these maps can be modest. consider that the b. taurus composite map of integrated linkage/radiation hybrid maps and bac physical maps features the greatest number of markers, but with a density of only ~180 kb/marker. in comparison, btom boasts an average restriction site density of  kb, which fostered resolution of difficult-to-discern errors in sequence assembly. for example, fig.  shows a 79 kb region that was inverted and misplaced based on alignment to btom, which was also substantiated by new sequence data and pcr.fig. optical map reveals a 79 kb sequence assembly as being transposed and inverted in umd _chr alignment shows an unaligned 79 kb segment within the optical map and a corresponding gap in the sequence. same 79 kb segment within the umd build but apparently transposed to,754 bp and inverted. accordingly, there is a 79 kb gap in the optical map. illumina paired-end dominette l sequence data, aligned to umd corroborates sequence misjoining points at,000 bp and,500 bp. pcr experiments confirms that the 79 kb segment should be placed between and on chromosome. paired-end reads mapped to umd showing correct orientations with both ends mapped are named an intact pair. when only one end is mapped, or mapping shows wrong orientation, or revealing discordant distances between mapped read pairs, these events are termed broken pairs. reads of a broken pair that map to a unique location against the reference are colored green or red, according to whether they mapped in the forward, or reverse, orientation respectively during the course of writing this manuscript, a reviewer questioned the extent of map errors and possible biases that may be introduced through our selective use of umd as the reference genome for btom. although our previous publications report a high degree of accuracy and minimal biases stemming from the iterative assembly pipeline, we compared the iterative assembly of optical maps constructed from umd vs. btau using chromosomes and. using btau as the reference sequence, eight iterations and merging of optical contigs produced three optical map contigs for chromosome and a single optical map contig was derived for chromosome alignments show that these new maps are essentially identical to btom except for a few restriction site differences. we attribute these minor differences to heterozygosity, since our calling of discordances uses a single representation of the physical map created by optical mapping. however, over an entire chromosome multi-mb-scale differences are apparent. fig. 10b shows three optical map contigs aligned to btau, chromosome presenting a large gap between contig and contig, and no gap between contig and contig relative to btom; while a single contig spans the same chromosome using umd chromosome in comparison, chromosome shows a single optical map contig generated from the btau sequence as the starting reference, while two optical map contigs formed using the umd sequence for chromosome. the absence of the small contig from the btau derived optical contigs implies reduced coverage for chromosome as such, our analysis shows that btom bears minimal local biases stemming from the choice of sequence build used for iterative assembly, but the overall optical map coverage varies. fortunately, absent, or problematic genomic regions would then be covered, as required, by optical maps constructed by de novo techniques. consequently, the need for de novo assembly steps is minimized by judicious selection of a reference genome for iterative assembly of an optical map.fig. comparisons of optical maps assemblies seeded by umd, or btau.. black vertical lines demarcate restriction fragments, or map alignments.) a: alignments of btom _chrs against the in silico maps of umd --chrs and. b: iterative assembly results for chrs and using btau as the reference sequence. note large inversion, not present in umd, or optical maps, revealed within the btau chr sequence. c: alignment of the optical maps shown in and. inset shows  mb region; vertical lines locate bamhi restriction sites. pink fragment shows the gap between the two optical map contigs in btom chromosome 28 we conclude that btom will prove to be a valuable resource for advancing the state of current sequence assemblies, by serving as a largely independent physical scaffold, as shown in figs.  and but perhaps, more importantly, as a platform to support future comparative studies, focusing on structural variation amongst different cattle breeds, or within populations. lastly, errors always accompany any ambitious effort pointed at comprehensive analysis of entire genomes. accordingly, the true merits and accuracy of a new resource, such as btom, will be comprehensively assessed over time by individual researchers in the bovine community. 
alignments of umd and btau to btom reveal discordances commensurate with previous reports, and affirm the ncbi’s current designation of umd sequence assembly as the “reference assembly” and the btau as the “alternate assembly.” the cattle genome optical map, btom, when used as a comprehensive and largely independent guide, will greatly assist improvements to existing sequence builds, and later serve as an accurate physical scaffold for studies concerning the comparative genomics of cattle breeds. however, past comparisons between these assemblies revealed substantial differences. consequently, such discordances have engendered ambiguities when using reference sequence data, impacting genomic studies in cattle and motivating construction of a new optical map resource--btom --to guide comparisons and improvements to the current sequence builds. accordingly, our comprehensive comparisons of btom against the umd  and btau sequence builds tabulate large-to-immediate scale discordances requiring mediation. the optical map, btom, spanning the b. taurus genome was assembled from an optical map dataset consisting of, single molecule optical maps generated by the optical mapping system. currently, there are two cattle genome sequence assemblies from groups using dissimilar assembly algorithms, which were complemented by genetic and physical map resources. btom is a high-resolution physical map featuring an average restriction fragment size of  kb. the cattle genome was originally selected for sequencing due to its economic importance and unique biology as a model organism for understanding other ruminants, or mammals.
 the cattle genome was originally selected for sequencing due to its economic importance and unique biology as a model organism for understanding other ruminants, or mammals. currently, there are two cattle genome sequence assemblies from groups using dissimilar assembly algorithms, which were complemented by genetic and physical map resources. however, past comparisons between these assemblies revealed substantial differences. consequently, such discordances have engendered ambiguities when using reference sequence data, impacting genomic studies in cattle and motivating construction of a new optical map resource--btom --to guide comparisons and improvements to the current sequence builds. accordingly, our comprehensive comparisons of btom against the umd  and btau sequence builds tabulate large-to-immediate scale discordances requiring mediation.  the optical map, btom, spanning the b. taurus genome was assembled from an optical map dataset consisting of, single molecule optical maps generated by the optical mapping system. the bamhi map spans.30 mb and comprises optical contigs assembled by a combination of iterative and de novo assembly techniques. btom is a high-resolution physical map featuring an average restriction fragment size of  kb. comparisons of btom vs. umd, or btau, revealed that btau presented far more discordances vs. umd. overall, we found that btau presented almost double the number of discordances than umd across most of the categories of sequence vs. map discrepancies, which are: complex, dels, inss, its, ecs and mcs.  alignments of umd and btau to btom reveal discordances commensurate with previous reports, and affirm the ncbi’s current designation of umd sequence assembly as the “reference assembly” and the btau as the “alternate assembly.” the cattle genome optical map, btom, when used as a comprehensive and largely independent guide, will greatly assist improvements to existing sequence builds, and later serve as an accurate physical scaffold for studies concerning the comparative genomics of cattle breeds. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 