BACKGROUND
cattle are an important source of meat, milk, and other goods in many parts of the world.
selective breeding has been used in conjunction with other approaches to increase the productivity of cattle and has contributed to dramatic changes in traits of interest.
in dairy cattle, increases of  <dig>  kg of milk,  <dig> kg of fat, and  <dig> kg of protein per cow per lactation have resulted from improvements in genetics, nutrition, and management during the past  <dig> years  <cit> .
more than half of the increase in milk production in us holstein cows achieved in the past  <dig> years is due to improved genetics  <cit> .
similarly, beef cattle have produced more meat of better quality than their recent ancestors due to selective breeding  <cit> .
considerable effort is also now focused on reducing the cost of raising animals by improving the efficiency of feed utilization  <cit> .
substantial gains in traits of interest have been made through selection of individuals for breeding based on their phenotypes, or those of their close relatives  <cit> .
more recently, genomics technologies like snp genotyping have been used to select animals on the basis of their genetic makeup  <cit> .
both of these methods can be applied without knowledge of the mechanisms that link the dna variations to the traits.
however, in addition to providing biological insights, identification of the specific dna differences associated with these traits can be used to develop more accurate tools for genomic selection as well as non-breeding approaches for modifying traits.
a large catalogue of genetic variation, especially snps, exists for cattle in publicly accessible databases, thanks largely to the bovine hapmap project  <cit> , the bovine genome project  <cit> , and large-scale snp discovery studies  <cit> .
nonetheless there is much genetic variation that remains to be discovered.
indeed, recent whole genome resequencing has revealed many novel snps  <cit> , and a recent comparative genomic hybridization study has identified numerous candidate cnvs  <cit> .
continued characterization of genetic variation, particularly in breeds that have not been thoroughly scrutinized, will be an important step towards deciphering the molecular mechanisms underlying trait variation.
in this work we describe the whole-genome resequencing of two individuals from distinct cattle breeds for the purpose of identifying dna differences.
one of the sequenced animals, goldwyn, is a bull from the holstein breed.
holstein cattle originated from the friesian breed in europe and were likely first imported to north america in  <dig>  <cit> .
they are known for their black-and-white markings and high milk production, and are the main source of dairy products in north america.
goldwyn was produced by the semex alliance  in  <dig>  and became one of the top dairy sires in the world by virtue of his daughters' impressive characteristics.
his semen, which currently sells for about $ <dig> per straw, has been used to sire over  <dig>  cows.
the second animal is a black angus bull.
the black angus breed originated in scotland and was imported to north america in the late 1800s, where it is now the most popular beef breed.
thus the two animals characterized in this study are from distinct populations shaped by selection for distinct traits.
in the case of the holstein breed, selection has been especially intense as has been the rate of performance gains.
we expect that these separate selection regimes have resulted in some different variants being favoured or fixed in each breed.
in our analyses of the holstein and black angus sequences we used assembly version btau <dig>   <cit>  as a reference sequence.
the btau <dig>  assembly was built using sequence from a hereford cow and her sire  <cit> .
the hereford breed originated in great britain in the 1700s and currently is a popular breed for beef production in many parts of the world.
a detailed snp-based comparison of the holstein, black angus, and hereford breeds shows that each is genetically distinct  <cit> .
snps were identified in this work as differences between the newly obtained genome sequences and the reference hereford sequence, whereas potential cnvs were detected as regions of unequal read depth between the two resequenced animals.
detailed annotation of the results and downstream validation suggest the presence of many novel genetic variants, with several of the variants affecting evolutionarily conserved protein regions.
the cnvs described in this work are enriched for immune system genes and genes that may contribute to lactation capacity.
most of the cnvs were detected as regions with higher abundance in the holstein bull.
the source and significance of this excess of cnv gains is not clear.
RESULTS
genome sequencing, snp detection and snp validation
genomic dna from a black angus bull and a holstein bull were sequenced using the solid system and a combination of fragment and mate pair libraries .
the resulting reads were mapped to a reference bovine genome assembly  and yielded approximately 22-fold and 19-fold coverage for the two animals, respectively .
putative snps were detected by comparing the aligned reads to the reference assembly.
more than  <dig>  million and  <dig>  million snps were identified for the holstein and black angus genomes, respectively.
three libraries were constructed for each animal and sequenced using two or more instrument runs.
the numbers of reads obtained for fragment libraries is given in the f <dig> reads column.
mate-paired libraries yielded two read types .
megabases of coverage was calculated based on the numbers and lengths of reads that were successfully mapped to the reference.
fold coverage was calculated by dividing the megabases of coverage by the combined length of the reference chromosomes used for mapping .
to estimate the rate at which snps were missed by sequencing , the snp list for the holstein animal was compared to the genotypes obtained using an array-based genotyping assay.
of  <dig>  homozygous array calls that were different from the reference ,  <dig>   were identified as snps by sequencing, and  <dig>  of the  <dig>   snps showed concordant genotypes .
based on these results we calculated the false negative rate for homozygous snp detection as * <dig> = 10%.
of the  <dig>  heterozygous array calls,  <dig>   were called as sequencing snps, and  <dig>   of the  <dig>  snps had concordant genotypes.
from these results, we calculated the false negative rate for heterozygous snp detection as * <dig> = 21%.
examination of the discordant heterozygous calls reveals that the vast majority  represent cases where the sequencing indicated the presence of just one of the alleles assayed on the array.
the sequenced holstein animal was genotyped so that the ability of the sequencing to identify detectable snps  could be quantified .
the "sequencing calls" column gives the number of detectable array snps that were identified as snps through sequencing, regardless of whether the sequencing genotype matched the array genotype.
the "concordant" column gives the number of sequencing calls with a sequencing genotype that matched the array genotype.
to estimate the rate at which snps were called when no snp was actually present, a custom genotyping assay was designed and applied.
a group of  <dig> animals was genotyped using  <dig> and  <dig> snps selected from the holstein and black angus snp lists, respectively.
of the  <dig> holstein snps that were genotyped,  <dig>  were found to be true snps .
of the  <dig> black angus snps that were genotyped,  <dig>  were demonstrated to be true snps .
to estimate the false positive rate of snp discovery, a subset of the snps discovered by sequencing was genotyped in  <dig> animals.
snp annotation
the results of snp annotation using ngs-snp  <cit>  suggest that the holstein and black angus snps belong to a diverse range of functional classes.
most of the snps are located between genes or within introns .
a comparison of the snps identified in this work with those described in dbsnp build  <dig>  <cit>  indicates that 81% of the snps detected in the holstein animal, and 81% of the snps detected in the black angus animal, are novel.
subsets of the annotated snps are available in additional file  <dig> and additional file  <dig>
asnp is located 1- <dig> bases into an exon or 3- <dig> bases into an intron.
bsnp is located in the first two or the last two bases of an intron.
the predicted functional consequences of snps identified by sequencing of the holstein and black angus genomes.
values in parentheses are the percentage of snps that are in the functional class, out of the total snps in the column.
in humans and other animals, numerous phenotypes, both mendelian and quantitative, have been linked to nonsynonymous snps  <cit> .
one approach that is used to highlight potentially functionally important nonsynonymous snps involves comparing a protein sequence to its orthologues  <cit> .
to better characterize the large number of nonsynonymous snps identified in this work, we measured the severity of the corresponding amino acid changes by examining orthologous protein sequences in ensembl  <cit> .
the results were quantified for each nonsynonymous snp as an "alignment score change"  value.
in short, a negative value arises when the non-reference allele changes the protein so that it no longer resembles its orthologues, and a positive value arises when the non-reference allele changes the protein to make it more similar to its orthologues.
the majority of the nonsynonymous snps we identified involve minor changes from an evolutionary standpoint  .
there is a trend exhibited by the nonsynonymous snps in which those with lower asc values are less frequently detected as homozygous snps  and also less frequently shared between the two animals .
this trend can be explained by the non-conservative alleles being less prevalent in the population on average than those that yield a protein that resembles its orthologues.
cnv identification and validation
putative cnvs were detected by identifying regions of the btau <dig>  reference sequence with significantly different coverage between the holstein and black angus mapped read sets  <cit> .
in total,  <dig> cnvs were identified on the  <dig> autosomes analysed, involving approximately  <dig>  mbp of the reference assembly used for mapping .
the average and median cnv sizes are  <dig>  bp and  <dig>  bp, respectively, and the cnvs range in size from  <dig>  bp to  <dig>  bp.
the cnvs are not evenly distributed along the reference autosomes, with some chromosomes lacking cnvs and others having numerous such regions .
the percentage of chromosomal length containing cnvs was less than 1% in all cases and ranged from  <dig> % to  <dig> % .
all the cnvs found in this work are described in additional file  <dig>
the distribution and size characteristics of cnvs detected through comparison of the holstein and black angus read sets mapped to the btau <dig>  reference assembly.
five genic cnvs and five non-genic cnvs  were randomly selected for evaluation by quantitative real-time pcr .
the cnvs identified as gains in the holstein animal  were quantified in  <dig> holstein animals and those identified as gains in the black angus animal  were quantified in  <dig> black angus animals.
all ten of the tested regions exhibited copy number differences .
five genic and five non-genic cnvs were selected for validation by qpcr.
entrez gene names are given for genic cnvs.
the log <dig> ratios and p-values obtained from the cnv-seq software are shown.
positive log <dig> ratios indicate higher read depth in the holstein animal.
cnv annotation and gene ontology analysis
to identify potential functional roles associated with the putative cnvs, genes completely or partially overlapping with these cnvs were retrieved from ensembl  <cit> .
a total of  <dig> genes were identified, involving  <dig> of the cnvs.
gene ontology  enrichment analysis  <cit>  indicates that genes related to "response to stimulus" , "immune system process" , and "growth"  are over-represented in the set of cnvs identified in this work .
the term "locomotion"  is enriched among the cnvs with higher copy number in black angus, while among the holstein cnv gains the terms "reproduction" , "reproductive process" , "membrane-enclosed lumen" , and "enzyme regulator activity"  are enriched .
go ids from the three go ontologies  enriched among the cnv gains in black angus , holstein , or both animals .
p-values are provided, when applicable, for the subset of cnvs detected as gains in the black angus animal  and the subset detected as gains in the holstein animal .
discussion
snp list characteristics
the proportions of novel snps identified in this work  are very close to the proportion of novel snps identified through the sequencing of a fleckvieh bull genome   <cit> .
these values suggest that a large number of dna variants remain to be identified in cattle.
the false negative rate of 10% and 21% for homozygous and heterozygous snps in the holstein bull indicates that our work does not provide a comprehensive list of the snps in the animals we sequenced, and that further sequencing or modified analysis procedures may be helpful for gaining a more complete picture of the genomes of these animals.
the low false-positive rates for both lists indicate that the vast majority of the snps we report are true snps.
the snps from both animals are available from dbsnp  <cit>  .
snps of potential functional significance
snp annotation aims to provide some indication as to which snps may be functionally relevant.
among the nonsynonymous snps we identified, those that cause a dramatic protein sequence change from the standpoint of an alignment-scoring matrix  may be of particular interest.
the snps that create stop codons can also be imagined to have important effects.
this class is enriched for heterozygous snps .
the annotation tool we used for this work  provides the names of protein features that overlap with snp-altered residues , as well as descriptions of protein function, gene names and identifiers, go information, and known phenotypes in cattle or in humans linked to the snp-affected gene or its human orthologue.
this information, particularly in conjunction with qtl mapping or genome-wide association results, should be useful for future work aimed at better understanding the genetic mechanisms underlying phenotypic differences in cattle.
comparison of cnvs to those identified in previous work
previous studies examining cnvs in cattle have employed array comparative genomic hybridization   <cit>  or snp arrays  <cit> .
next-generation sequencing has been used previously for cnv detection in humans  <cit> .
the sequencing approach can overcome the sensitivity limits of acgh and snp arrays, and can more precisely identify cnv boundaries  <cit> .
a substantial number of the cnvs from this work  are concordant with the cnvs previously identified in cattle using acgh  <cit> .
this concordance with the acgh findings, in conjunction with the pcr validation results, lends further support to the cnvs described in this study.
differences observed between the cnvs described here and those detected using acgh can be attributed to the particular breeds investigated and to differences between the technologies used.
the cnvs we detected by read-depth analysis are on average much smaller than those identified by acgh   <cit> .
in human studies, the use of sequencing also led to the identification of much shorter cnvs compared to acgh  <cit> .
the approach we used to detect cnvs can artificially break a single cnv into multiple cnvs.
for example, if read depth happens to drop in one or both of the animals in the middle of a cnv then two cnvs may be reported because the middle region does not meet the criteria for reporting.
gene ontology analysis of cnvs
gene ontology enrichment analysis indicates that genes related to "response to stimulus," "immune system process," and "growth" are over-represented in the set of cnvs identified in this work .
"response to stimulus" is defined as a change in state or activity of a cell or an organism  as a result of a stimulus  <cit> .
"immune system response" is defined as any process involved in the development or functioning of the immune system: i.e., an organismal system for calibrated responses to potential internal or invasive threats  <cit> .
genes related to immunity and sensory response have been previously identified as being overrepresented in cnvs in cattle  <cit>  and in humans  <cit> .
it has been suggested that the increased dosage of genes related to infection response and sensing the environment offer a survivability benefit  <cit> .
"growth" is defined as the increase in size or mass of an entire organism or a part of an organism  <cit> .
perhaps enrichment of this term reflects the selection applied to these breeds, but as noted below none of these cnvs have been specifically associated with traits in cattle to our knowledge.
each cnv is detected in this work as a gain of sequence dosage in one animal relative to the other.
some go terms are enriched among the cnv gains in one animal but not among the cnv gains in the other.
the go term "locomotion" is enriched among the cnvs with higher copy number in black angus, while among the cnv gains in holstein the terms "reproduction", "reproductive process", "membrane-enclosed lumen", and "enzyme regulator activity" are enriched.
the term "locomotion" is defined as self-propelled movement of a cell or organism from one location to another and includes genes such as myotubularin related protein  <dig>  enrichment of this term could be imagined to reflect selection for lean muscle mass in beef cattle, while the enrichment of reproduction-related genes  in the holstein animal is consistent with the selection applied to the holstein breed.
at this point however there is no evidence linking the cnvs we detected to increased gene activity or to phenotypic differences.
a gene of interest overlapping with cnvs
several cnvs were found to overlap with genes that potentially influence beef or dairy traits of interest, such as milk production, health, or meat quality.
for example, cnvs overlapping with a pla2g2d gene were identified .
pla2g2d genes are thought to play roles in lipid metabolism, fat deposition, gonadotropin-releasing hormone signalling, and mapk signalling  <cit> .
the region shown in figure  <dig> also underlies qtl for body weight and carcass weight in beef cattle  <cit> .
one of the five cnvs located in the pla2g2d region was quantified using qpcr .
among the black angus animals tested by qpcr, copy number differences were observed relative to the holstein calibrator .
future work will be needed to establish whether these cnv differences are associated with phenotypic variation.
abundance of cnv gains in holstein
strong selection has led to impressive performance gains in the holstein breed, particularly in the past  <dig> years.
it is not possible to assess from our data whether the apparent abundance of cnv gains in the single holstein animal we examined is related to selection.
in other species, natural selection is thought to have favoured the expansion of cnvs that influence certain traits, such as immunity in the case of mice and humans  <cit> .
further research examining more individuals may allow us to discern whether artificial selection has had a role in shaping cnvs in cattle.
CONCLUSIONS
whole genome resequencing of a black angus bull and a holstein bull identified  <dig>  and  <dig>  million snps respectively, through comparisons with the hereford reference sequence.
numerous cnvs were also found through an analysis of read depth differences.
downstream validation suggests a low false-positive rate for snp and cnv detection, but that many snps were likely missed by sequencing.
more work is needed to investigate the source and significance of the higher proportion of cnv gains in the holstein animal.
the deeply annotated snps and cnvs identified in this resequencing study can serve as useful genetic tools, and as candidates in searches for phenotype-altering dna differences.

 cattle are an important source of meat, milk, and other goods in many parts of the world. selective breeding has been used in conjunction with other approaches to increase the productivity of cattle and has contributed to dramatic changes in traits of interest. in dairy cattle, increases of kg of milk, kg of fat, and kg of protein per cow per lactation have resulted from improvements in genetics, nutrition, and management during the past years. more than half of the increase in milk production in us holstein cows achieved in the past years is due to improved genetics. similarly, beef cattle have produced more meat of better quality than their recent ancestors due to selective breeding. considerable effort is also now focused on reducing the cost of raising animals by improving the efficiency of feed utilization. substantial gains in traits of interest have been made through selection of individuals for breeding based on their phenotypes, or those of their close relatives. more recently, genomics technologies like snp genotyping have been used to select animals on the basis of their genetic makeup. both of these methods can be applied without knowledge of the mechanisms that link the dna variations to the traits. however, in addition to providing biological insights, identification of the specific dna differences associated with these traits can be used to develop more accurate tools for genomic selection as well as non-breeding approaches for modifying traits. a large catalogue of genetic variation, especially snps, exists for cattle in publicly accessible databases, thanks largely to the bovine hapmap project, the bovine genome project, and large-scale snp discovery studies. nonetheless there is much genetic variation that remains to be discovered. indeed, recent whole genome resequencing has revealed many novel snps, and a recent comparative genomic hybridization study has identified numerous candidate cnvs. continued characterization of genetic variation, particularly in breeds that have not been thoroughly scrutinized, will be an important step towards deciphering the molecular mechanisms underlying trait variation. in this work we describe the whole-genome resequencing of two individuals from distinct cattle breeds for the purpose of identifying dna differences. one of the sequenced animals, goldwyn, is a bull from the holstein breed. holstein cattle originated from the friesian breed in europe and were likely first imported to north america in. they are known for their black-and-white markings and high milk production, and are the main source of dairy products in north america. goldwyn was produced by the semex alliance in and became one of the top dairy sires in the world by virtue of his daughters' impressive characteristics. his semen, which currently sells for about $ per straw, has been used to sire over cows. the second animal is a black angus bull. the black angus breed originated in scotland and was imported to north america in the late 1800s, where it is now the most popular beef breed. thus the two animals characterized in this study are from distinct populations shaped by selection for distinct traits. in the case of the holstein breed, selection has been especially intense as has been the rate of performance gains. we expect that these separate selection regimes have resulted in some different variants being favoured or fixed in each breed. in our analyses of the holstein and black angus sequences we used assembly version btau as a reference sequence. the btau assembly was built using sequence from a hereford cow and her sire. the hereford breed originated in great britain in the 1700s and currently is a popular breed for beef production in many parts of the world. a detailed snp-based comparison of the holstein, black angus, and hereford breeds shows that each is genetically distinct. snps were identified in this work as differences between the newly obtained genome sequences and the reference hereford sequence, whereas potential cnvs were detected as regions of unequal read depth between the two resequenced animals. detailed annotation of the results and downstream validation suggest the presence of many novel genetic variants, with several of the variants affecting evolutionarily conserved protein regions. the cnvs described in this work are enriched for immune system genes and genes that may contribute to lactation capacity. most of the cnvs were detected as regions with higher abundance in the holstein bull. the source and significance of this excess of cnv gains is not clear.  genome sequencing, snp detection and snp validation genomic dna from a black angus bull and a holstein bull were sequenced using the solid system and a combination of fragment and mate pair libraries. the resulting reads were mapped to a reference bovine genome assembly and yielded approximately 22-fold and 19-fold coverage for the two animals, respectively. putative snps were detected by comparing the aligned reads to the reference assembly. more than million and million snps were identified for the holstein and black angus genomes, respectively. three libraries were constructed for each animal and sequenced using two or more instrument runs. the numbers of reads obtained for fragment libraries is given in the f reads column. mate-paired libraries yielded two read types. megabases of coverage was calculated based on the numbers and lengths of reads that were successfully mapped to the reference. fold coverage was calculated by dividing the megabases of coverage by the combined length of the reference chromosomes used for mapping. to estimate the rate at which snps were missed by sequencing, the snp list for the holstein animal was compared to the genotypes obtained using an array-based genotyping assay. of homozygous array calls that were different from the reference, were identified as snps by sequencing, and of the snps showed concordant genotypes. based on these results we calculated the false negative rate for homozygous snp detection as * = 10%. of the heterozygous array calls, were called as sequencing snps, and of the snps had concordant genotypes. from these results, we calculated the false negative rate for heterozygous snp detection as * = 21%. examination of the discordant heterozygous calls reveals that the vast majority represent cases where the sequencing indicated the presence of just one of the alleles assayed on the array. the sequenced holstein animal was genotyped so that the ability of the sequencing to identify detectable snps could be quantified. the "sequencing calls" column gives the number of detectable array snps that were identified as snps through sequencing, regardless of whether the sequencing genotype matched the array genotype. the "concordant" column gives the number of sequencing calls with a sequencing genotype that matched the array genotype. to estimate the rate at which snps were called when no snp was actually present, a custom genotyping assay was designed and applied. a group of animals was genotyped using and snps selected from the holstein and black angus snp lists, respectively. of the holstein snps that were genotyped, were found to be true snps. of the black angus snps that were genotyped, were demonstrated to be true snps. to estimate the false positive rate of snp discovery, a subset of the snps discovered by sequencing was genotyped in animals. snp annotation the results of snp annotation using ngs-snp suggest that the holstein and black angus snps belong to a diverse range of functional classes. most of the snps are located between genes or within introns. a comparison of the snps identified in this work with those described in dbsnp build indicates that 81% of the snps detected in the holstein animal, and 81% of the snps detected in the black angus animal, are novel. subsets of the annotated snps are available in additional file and additional file asnp is located 1- bases into an exon or 3- bases into an intron. bsnp is located in the first two or the last two bases of an intron. the predicted functional consequences of snps identified by sequencing of the holstein and black angus genomes. values in parentheses are the percentage of snps that are in the functional class, out of the total snps in the column. in humans and other animals, numerous phenotypes, both mendelian and quantitative, have been linked to nonsynonymous snps. one approach that is used to highlight potentially functionally important nonsynonymous snps involves comparing a protein sequence to its orthologues. to better characterize the large number of nonsynonymous snps identified in this work, we measured the severity of the corresponding amino acid changes by examining orthologous protein sequences in ensembl. the results were quantified for each nonsynonymous snp as an "alignment score change" value. in short, a negative value arises when the non-reference allele changes the protein so that it no longer resembles its orthologues, and a positive value arises when the non-reference allele changes the protein to make it more similar to its orthologues. the majority of the nonsynonymous snps we identified involve minor changes from an evolutionary standpoint. there is a trend exhibited by the nonsynonymous snps in which those with lower asc values are less frequently detected as homozygous snps and also less frequently shared between the two animals. this trend can be explained by the non-conservative alleles being less prevalent in the population on average than those that yield a protein that resembles its orthologues. cnv identification and validation putative cnvs were detected by identifying regions of the btau reference sequence with significantly different coverage between the holstein and black angus mapped read sets. in total, cnvs were identified on the autosomes analysed, involving approximately mbp of the reference assembly used for mapping. the average and median cnv sizes are bp and bp, respectively, and the cnvs range in size from bp to bp. the cnvs are not evenly distributed along the reference autosomes, with some chromosomes lacking cnvs and others having numerous such regions. the percentage of chromosomal length containing cnvs was less than 1% in all cases and ranged from % to %. all the cnvs found in this work are described in additional file the distribution and size characteristics of cnvs detected through comparison of the holstein and black angus read sets mapped to the btau reference assembly. five genic cnvs and five non-genic cnvs were randomly selected for evaluation by quantitative real-time pcr. the cnvs identified as gains in the holstein animal were quantified in holstein animals and those identified as gains in the black angus animal were quantified in black angus animals. all ten of the tested regions exhibited copy number differences. five genic and five non-genic cnvs were selected for validation by qpcr. entrez gene names are given for genic cnvs. the log ratios and p-values obtained from the cnv-seq software are shown. positive log ratios indicate higher read depth in the holstein animal. cnv annotation and gene ontology analysis to identify potential functional roles associated with the putative cnvs, genes completely or partially overlapping with these cnvs were retrieved from ensembl. a total of genes were identified, involving of the cnvs. gene ontology enrichment analysis indicates that genes related to "response to stimulus", "immune system process", and "growth" are over-represented in the set of cnvs identified in this work. the term "locomotion" is enriched among the cnvs with higher copy number in black angus, while among the holstein cnv gains the terms "reproduction", "reproductive process", "membrane-enclosed lumen", and "enzyme regulator activity" are enriched. go ids from the three go ontologies enriched among the cnv gains in black angus, holstein, or both animals. p-values are provided, when applicable, for the subset of cnvs detected as gains in the black angus animal and the subset detected as gains in the holstein animal. discussion snp list characteristics the proportions of novel snps identified in this work are very close to the proportion of novel snps identified through the sequencing of a fleckvieh bull genome. these values suggest that a large number of dna variants remain to be identified in cattle. the false negative rate of 10% and 21% for homozygous and heterozygous snps in the holstein bull indicates that our work does not provide a comprehensive list of the snps in the animals we sequenced, and that further sequencing or modified analysis procedures may be helpful for gaining a more complete picture of the genomes of these animals. the low false-positive rates for both lists indicate that the vast majority of the snps we report are true snps. the snps from both animals are available from dbsnp. snps of potential functional significance snp annotation aims to provide some indication as to which snps may be functionally relevant. among the nonsynonymous snps we identified, those that cause a dramatic protein sequence change from the standpoint of an alignment-scoring matrix may be of particular interest. the snps that create stop codons can also be imagined to have important effects. this class is enriched for heterozygous snps. the annotation tool we used for this work provides the names of protein features that overlap with snp-altered residues, as well as descriptions of protein function, gene names and identifiers, go information, and known phenotypes in cattle or in humans linked to the snp-affected gene or its human orthologue. this information, particularly in conjunction with qtl mapping or genome-wide association results, should be useful for future work aimed at better understanding the genetic mechanisms underlying phenotypic differences in cattle. comparison of cnvs to those identified in previous work previous studies examining cnvs in cattle have employed array comparative genomic hybridization or snp arrays. next-generation sequencing has been used previously for cnv detection in humans. the sequencing approach can overcome the sensitivity limits of acgh and snp arrays, and can more precisely identify cnv boundaries. a substantial number of the cnvs from this work are concordant with the cnvs previously identified in cattle using acgh. this concordance with the acgh findings, in conjunction with the pcr validation results, lends further support to the cnvs described in this study. differences observed between the cnvs described here and those detected using acgh can be attributed to the particular breeds investigated and to differences between the technologies used. the cnvs we detected by read-depth analysis are on average much smaller than those identified by acgh. in human studies, the use of sequencing also led to the identification of much shorter cnvs compared to acgh. the approach we used to detect cnvs can artificially break a single cnv into multiple cnvs. for example, if read depth happens to drop in one or both of the animals in the middle of a cnv then two cnvs may be reported because the middle region does not meet the criteria for reporting. gene ontology analysis of cnvs gene ontology enrichment analysis indicates that genes related to "response to stimulus," "immune system process," and "growth" are over-represented in the set of cnvs identified in this work. "response to stimulus" is defined as a change in state or activity of a cell or an organism as a result of a stimulus. "immune system response" is defined as any process involved in the development or functioning of the immune system: i.e., an organismal system for calibrated responses to potential internal or invasive threats. genes related to immunity and sensory response have been previously identified as being overrepresented in cnvs in cattle and in humans. it has been suggested that the increased dosage of genes related to infection response and sensing the environment offer a survivability benefit. "growth" is defined as the increase in size or mass of an entire organism or a part of an organism. perhaps enrichment of this term reflects the selection applied to these breeds, but as noted below none of these cnvs have been specifically associated with traits in cattle to our knowledge. each cnv is detected in this work as a gain of sequence dosage in one animal relative to the other. some go terms are enriched among the cnv gains in one animal but not among the cnv gains in the other. the go term "locomotion" is enriched among the cnvs with higher copy number in black angus, while among the cnv gains in holstein the terms "reproduction", "reproductive process", "membrane-enclosed lumen", and "enzyme regulator activity" are enriched. the term "locomotion" is defined as self-propelled movement of a cell or organism from one location to another and includes genes such as myotubularin related protein enrichment of this term could be imagined to reflect selection for lean muscle mass in beef cattle, while the enrichment of reproduction-related genes in the holstein animal is consistent with the selection applied to the holstein breed. at this point however there is no evidence linking the cnvs we detected to increased gene activity or to phenotypic differences. a gene of interest overlapping with cnvs several cnvs were found to overlap with genes that potentially influence beef or dairy traits of interest, such as milk production, health, or meat quality. for example, cnvs overlapping with a pla2g2d gene were identified. pla2g2d genes are thought to play roles in lipid metabolism, fat deposition, gonadotropin-releasing hormone signalling, and mapk signalling. the region shown in figure also underlies qtl for body weight and carcass weight in beef cattle. one of the five cnvs located in the pla2g2d region was quantified using qpcr. among the black angus animals tested by qpcr, copy number differences were observed relative to the holstein calibrator. future work will be needed to establish whether these cnv differences are associated with phenotypic variation. abundance of cnv gains in holstein strong selection has led to impressive performance gains in the holstein breed, particularly in the past years. it is not possible to assess from our data whether the apparent abundance of cnv gains in the single holstein animal we examined is related to selection. in other species, natural selection is thought to have favoured the expansion of cnvs that influence certain traits, such as immunity in the case of mice and humans. further research examining more individuals may allow us to discern whether artificial selection has had a role in shaping cnvs in cattle.  whole genome resequencing of a black angus bull and a holstein bull identified and million snps respectively, through comparisons with the hereford reference sequence. numerous cnvs were also found through an analysis of read depth differences. downstream validation suggests a low false-positive rate for snp and cnv detection, but that many snps were likely missed by sequencing. more work is needed to investigate the source and significance of the higher proportion of cnv gains in the holstein animal. the deeply annotated snps and cnvs identified in this resequencing study can serve as useful genetic tools, and as candidates in searches for phenotype-altering dna differences. 
a comparison between the sequencing snps and genotyping results from the bovinehd high-density genotyping chip indicates a detection rate of 91% for homozygous snps and 81% for heterozygous snps. substantial genetic differences exist between the black angus and holstein animals sequenced in this work and the hereford reference sequence, and some of this variation is predicted to affect evolutionarily conserved amino acids or gene copy number. the genomes of a black angus bull and a holstein bull were sequenced to 22-fold and 19-fold coverage, respectively, using the abi solid system. characterizing the genetic variation in livestock species is an important step towards linking genes or genomic regions with phenotypes. the completion of the bovine genome sequence and recent advances in dna sequencing technology allow for in-depth characterization of the genetic variations present in cattle. here we describe the whole-genome resequencing of two bos taurus bulls from distinct breeds for the purpose of identifying and annotating novel forms of genetic variation in cattle. of the total snps found in holstein, black angus, and in both animals, 81%, 81%, and 75% respectively are novel. the majority of the cnvs were detected as regions with higher abundance in the holstein bull. the deeply annotated snps and cnvs identified in this resequencing study can serve as useful genetic tools, and as candidates in searches for phenotype-altering dna differences. the false positive rate is estimated to be about 2% for both the black angus and holstein snp sets, based on follow-up genotyping of and snps, respectively. the cnvs are enriched for immune system genes and include genes that may contribute to lactation capacity. comparisons of the sequences with the btau reference assembly yielded million single nucleotide polymorphisms, 24% of which were identified in both animals. comparisons of read depth between the two bulls along the reference assembly identified putative copy-number variations. ten randomly selected cnvs, five genic and five non-genic, were successfully validated using quantitative real-time pcr.
 one of the goals of livestock genomics research is to identify the genetic differences responsible for variation in phenotypic traits, particularly those of economic importance. characterizing the genetic variation in livestock species is an important step towards linking genes or genomic regions with phenotypes. the completion of the bovine genome sequence and recent advances in dna sequencing technology allow for in-depth characterization of the genetic variations present in cattle. here we describe the whole-genome resequencing of two bos taurus bulls from distinct breeds for the purpose of identifying and annotating novel forms of genetic variation in cattle.  the genomes of a black angus bull and a holstein bull were sequenced to 22-fold and 19-fold coverage, respectively, using the abi solid system. comparisons of the sequences with the btau reference assembly yielded million single nucleotide polymorphisms, 24% of which were identified in both animals. of the total snps found in holstein, black angus, and in both animals, 81%, 81%, and 75% respectively are novel. in-depth annotations of the data identified more than thousand distinct non-synonymous snps between the two datasets. alignments between the snp-altered proteins and orthologues from numerous species indicate that many of the snps alter well-conserved amino acids. several snps predicted to create or remove stop codons were also found. a comparison between the sequencing snps and genotyping results from the bovinehd high-density genotyping chip indicates a detection rate of 91% for homozygous snps and 81% for heterozygous snps. the false positive rate is estimated to be about 2% for both the black angus and holstein snp sets, based on follow-up genotyping of and snps, respectively. comparisons of read depth between the two bulls along the reference assembly identified putative copy-number variations. ten randomly selected cnvs, five genic and five non-genic, were successfully validated using quantitative real-time pcr. the cnvs are enriched for immune system genes and include genes that may contribute to lactation capacity. the majority of the cnvs were detected as regions with higher abundance in the holstein bull.  substantial genetic differences exist between the black angus and holstein animals sequenced in this work and the hereford reference sequence, and some of this variation is predicted to affect evolutionarily conserved amino acids or gene copy number. the deeply annotated snps and cnvs identified in this resequencing study can serve as useful genetic tools, and as candidates in searches for phenotype-altering dna differences. 