BACKGROUND
centromeres are genomic sites of spindle attachment that are essential for ensuring proper chromosome segregation during cell division.
despite their recognized functional importance, centromeres are not well defined at a sequence level in most genomes  <cit> .
this has greatly limited efforts to understand in detail the nature and determinants of the synergistic relationship between genome sequence and epigenetics that is generally believed to underlie centromere identity and function  <cit> .
the relatively poor state of sequence assembly and annotation in centromeric regions is due to the presence and abundance of identical or near-identical satellite dna sequences that confound attempts to generate a reliable reference sequence  <cit> .
as a result, efforts to study the interaction of centromere proteins with the underlying genome sequence are largely incapable of distinguishing sequences that are ‘functional’ from those that are ‘non-functional’.
this remains a fundamental roadblock for sequence-based studies of centromere identity, variation and function in virtually all complex genomes.
robust genomic studies of centromeric sequences and their variation are not straightforward, as generating comprehensive and high-confidence inventories of satellite dna families requires substantial manual curation  <cit> .
this level of genomic resolution requires both long- and short-range sequence information capable of capturing sequence variation and spatial organization within a single satellite array, between satellites occupying distinct chromosomal domains, and within a given population  <cit> .
as a result, few detailed studies have been reported to date, largely limited to well characterized and intensely studied genomes  <cit> .
further, satellite-rich regions are known to turn over rapidly over short evolutionary periods, thus restricting comparative efforts to closely related species  <cit> .
to address the biological questions of centromere identity, evolution and function, therefore, there is a need to improve upon the current rate of sequence exploration in satellite-rich domains, thereby enabling detailed studies at the intersection of genomics and epigenetics.
the functional identification and annotation of centromeres depends on the availability of two comprehensive and complementary sequence datasets:  a reference sequence database that describes all centromeric sequence variation and its underlying organization, and  a functional sequence database that highlights the genomic features associated with centromere identity and function.
current attempts to construct such databases have focused on centromere-associated sequences, as functional sequences can be readily identified epigenetically through association with centromere-specific proteins, such as the histone h <dig> variant centromere protein a   <cit> .
this specialized component of centromeric nucleosomes is believed to be the fundamental epigenetic mark for defining kinetochore localization and is observed at discrete sites within satellite dna domains in many genomes  <cit> .
following this approach, inventories of sequences bound to cenp-a have been reported for several species to define, at least at the level of consensus sequences, the genomic content of centromeres in those genomes  <cit> .
while adequate for identifying particular classes of satellite dna associated with centromere function, chromatin immunoprecipitation sequencing  projects alone, however, provide only a broad view of centromere sequence organization that is largely incapable of distinguishing between sequences on different chromosomes and/or between closely related sequences, only a subset of which may be actually involved in centromere function.
as an alternative and complementary approach, efforts to work from the bottom up to generate high-resolution genomic libraries of centromeric sequences have been taxed by the level and precision of experimental effort needed to derive linear sequence predictions through long spans of near-identical repeats in these regions.
these limitations and difficulties notwithstanding, the value of paired genomic and epigenetic centromere datasets has been amply demonstrated in studies from human, plant, and drosophila genomes that have been valuable for defining current models of centromere specification, identity and function  <cit> .
to extend this to many other genomes that are less completely studied, however, will require new approaches to facilitate development and analysis of centromere datasets.
to address this gap in current knowledge, we present a strategy to produce a reference sequence database for satellite domains of less-characterized genomes in order to promote broader comparative studies on function and centromere sequence organization.
rather than characterizing satellite domains by extending linear maps from the assembled euchromatic chromosome arms  <cit> , we apply a computational approach to generate a preassembled satellite sequence database, resulting in a comprehensive list of satellite domain features as well as adjacent non-satellite sequences.
when paired with an equivalent epigenetic dataset of cenp-a-associated sequences, this enables one to functionally annotate satellite and non-satellite sequence variation, as well as describe the short- and long-range sequence organization associated with active centromeres.
to implement this strategy, we focus on the dog genome as an example, as it offers a high-quality whole-genome sanger sequences  and assembly  <cit> .
further, unlike the situation for many other high-quality genomes, at least some information about satellite dnas is available  <cit> .
these potential advantages are balanced, however, by limitations of linear assembly across canine satellite dna arrays and the enrichment of segmental duplications found at centric transitions  <cit> .
thus, the current understanding of centromeric sequence organization in the dog genome is summarized only by a small number of satellite family consensus sequences and marginal representation directly adjacent to centromere gaps  <cit> .
here we describe an initial canine reference satellite domain database, utilizing both previously assembled and unassembled sequences, providing genome-wide descriptions of satellite families and annotation of all sequences physically linked to centromeric domains.
to annotate the database, we then extracted a library of informative satellite domain sequence features that include polymorphisms and junctions with interspersed repeats found within or adjacent to satellite arrays.
finally, to relate the genomic dataset to centromere function, we then developed a complementary dataset of cenp-a-associated sequences in the dog genome and determined the census of sequence features that occupy functional centromeres.
this combined genomic, epigenetic and functional approach reveals domains of satellite sequences that are not only distinguishable functionally and spatially, but also by sequence.
this approach should be generally applicable to any sequenced genome, with hopes of expanding our understanding of the genomic and functional definition of centromeres in complex genomes.
RESULTS
our approach utilizes all sequence reads from the dog wgs project, including those that are reported in the canine genome assembly  <cit>  and those that are missing from the assembly and fall within centromere “gaps”.
the strategy consists of three phases, outlined in figure 1: creating a database of sequence reads in and adjacent to the centromere “gaps” in the assembly, including satellite dnas known to localize to centromeric regions ; characterizing unique variants in that database to create a library of informative sequence features ; and developing a database of functional centromere sequences and sequence features associated with cenp-a .
a comprehensive centromeric sequence database
sequences in centromeric regions of complex genomes are generally of two types: those showing very limited variation, which remain unassembled and largely uncharacterized within centromeric “gaps” in the chromosome assemblies; and those adjacent to the gaps that have sufficient variation to allow standard assembly.
in the current dog reference genome sequence ,  <dig> , <dig> sequence reads  are currently unassigned to specific chromosomes and thus are candidates for sequences that map to the centromeric gaps.
consistent with what has been found in other complex genomes, a significant proportion of these unassembled and unassigned sequences consist of tandemly repeated satellite dnas .
notably, two centromeric satellite families, carnivore satellite  <dig>  and/or satellite  <dig> canis familiaris  – shown previously to hybridize to primary constrictions of dog chromosomes  <cit>  – are significantly enriched in the unmapped canfam <dig>  scaffolds and are also enriched in regions of the genome assembly directly adjacent to the centromere gaps.
these two centromere-associated satellite families account for reads containing  <dig>  mb and  <dig>  mb  and thus comprise a large proportion of the sequence content of canine centromere gaps .
because of their prior association with centromeric regions  <cit> , we used these two satellite families to nucleate the centromeric sequence database.
by surveying for satellite dnas on all assembled and unassembled sequences , we established that the dog genome contains, in addition to carsat <dig> and sat1cf, only nine satellite families estimated to account for more than 100 kb .
notably, members of five of these satellite families can be networked to carsat  <dig> and/or sat1cf by paired-read frequency and by proximity to centromere gaps and unmapped scaffolds in canfam <dig>  .
overall, the network of sequences that are anchored to the centromeric regions by their read linkages to carsat <dig> and sat1cf includes some  <dig>  mb  and  <dig>  reads, together accounting for  <dig> % of all unassigned sequences in canfam <dig> .
therefore, this approach yields a database  that contains a significant number of previously unassembled and uncharacterized sequences, suitable for exploring satellite family sequence variation and for identifying potential non-satellite sequences that might also be localized to centromeric regions in this genome.
while the vast majority of sequences in this database correspond to members of the seven satellite families , the  <dig>  reads also include members of other repetitive dna families, as well as non-repetitive dna .
about 7% of sequences in the database correspond to transposable elements embedded within reads otherwise consisting of satellite dna .
most of the transposable element families are underrepresented in canine satellite regions relative to the rest of the genome , as expected given the nature of tandemly repeated satellite dnas and their modes of homogenization.
however, at least some transposable element families appear enriched in reads containing specific satellite dnas.
for example, cferv1a, a canine-specific ltr family  <cit> , was found to be  <dig> -fold enriched in reads containing carsat <dig> sequence .
similarly, specific line subfamilies  were enriched  <dig> - and  <dig> -fold respectively in the sat2cf satellite family.
this enrichment notwithstanding, it should be emphasized that these embedded transposable elements represent only a small proportion of the overall content of satellite dnas in the centromeric read database, and it is unclear what role, if any, they might play in the maintenance or evolution of these regions of the genome.
nonetheless, as demonstrated for other genomes  <cit> , the elements described here should be useful as genomic landmarks within satellite domains.
we also uncovered small amounts of non-repetitive sequences within the centromeric satellite read database.
within the unmapped centromeric contigs in the canfam <dig>  genome assembly,  <dig>  mb correspond to contiguous sequences ≥100 bp that remain unmasked by the repeatmasker or satellite family libraries.
using available annotation, we identified  <dig>  kb of highly conserved sequences as reported by phastcons predictions  <cit> .
we also found  <dig> unmapped, centromere-linked contigs that contain sequences homologous to provisional, overlapping protein-coding and non-coding genes in organisms other than dog, taken from the reference sequences collection  .
many of these sequences, as expected  <cit> , correspond to segmental duplications in the dog genome.
together, these results indicate that analysis of satellite dna families, once annotated in a genomic context, can provide information on the sequence content and description of previously unmapped regions of the genome.
a sequence feature library for satellite domains
to explore sequence variation within the centromeric read database and as a prerequisite for searching for specific sequence features that distinguish functional centromeres from related  sequences, we reformatted the entire canine unmapped database and the remaining unassigned “gap” reads into a library of k-mers  .
while enrichment can be detected over a range of k-mer values, we have used k-mers 50 bp in length for all analyses described here, as this length maximizes the sequence-based information within our enrichment search while limiting edge effects observed when mapping exact matches in our short-read chip-seq database .
the library of 50-mers represents the frequency of all sequence signatures found within the reference database and allows one to annotate high- and low-frequency events, insertions and/or deletions within the highly repetitive sequences that dominate centromeric regions  <cit> .
we excluded 50-mers that also mapped to identical sequences in the canfam <dig>  genome assembly itself, leaving ~ <dig>  million different 50-mers that are specific to the unassembled gap regions of the genome .
by normalizing to single-copy 50-mer depth estimates , we could use this library to predict the abundance of individual sites within satellite domains, thus defining satellite sequence polymorphisms, transposable element insertions, and boundaries between satellite and non-satellite sequences within these regions.
identifying sequence features of functional centromeres
in the third phase of the strategy, we generated a library of sequences associated with canine cenp-a-containing nucleosomes, providing a functional context for the satellite dna domain database .
to identify all sequences associated with cenp-a in canine cells, we performed cenp-a chip-seq, generating  <dig>  million 72 bp illumina sequence reads  .
using these sequences, we surveyed the complete canine wgs database to identify sites of enrichment throughout both the current chromosome reference assembly, as well as the unmapped regions of the dog genome.
based on non-repetitive  alignments to canfam <dig>  using standard chip-seq mapping  and enrichment detection software , we found no evidence for significant cenp-a enrichment outside of the centromeric regions   <cit> .
next, to identify cenp-a-associated sequences within the unmapped gap regions, we compared the chip-seq reads to the unmapped 50-mer-based sequence feature set from phase ii.
from this analysis, we identified  <dig>  wgs reads that align with cenp-a chip-seq reads; notably,  <dig> % of these wgs reads are associated with the carsat <dig> and/or carsat <dig> satellite families , indicating significant enrichment .
carsat <dig> and carsat  <dig> are related satellite families, of lengths 738 bp and 1466 bp, respectively .
smaller enrichment sets were identified for several other satellite families; however, in sharp contrast to the carsat1/ <dig> datasets, these reads constitute <2% of the total number of reads that define each respective satellite family and thus are of uncertain significance .
to further subdivide the carsat1/ <dig> sequences, we focused on the 50-mers that are found in the chip-seq database  and used these to distinguish specific sequence features of cenp-a-associated versus non-associated copies of carsat1/ <dig> satellite repeats.
by this analysis, 60% of carsat1/ <dig> reads contain a minimum threshold of continuous bases of 50-mers that were >2-fold enriched , while 40% of reads contained no such enriched sequences.
this supports the hypothesis that only particular carsat1/ <dig> sequences are interacting with cenp-a, suggesting the presence of definable subtypes within the overall satellite domains at canine centromeres.
the majority of enriched 50-mers appear to be multi-copy, high-frequency satellite sequences in the carsat1/ <dig> arrays, thereby providing evidence for functional cenp-a domains that are predominantly found associated with near-identical satellite repeat units .
although not all multi-copy sites within the arrays are enriched, it is clear that high-frequency 50-mers  are most likely to be associated with cenp-a.
this may indicate that cenp-a is associated preferentially with specific, highly conserved positions within the majority of monomer units at the centromere, reflecting the sequence homogenization that is a common feature of satellite arrays  <cit> .
alternatively, the carsat1/ <dig> satellite families might be divided into distinct monomer types that are associated or not associated with cenp-a, suggesting the existence of different subtypes or subfamilies around the genome, similar to what is observed, for example, in primate alpha satellite  <cit> .
in phases i and ii of this strategy, we defined various non-satellite sequences embedded within satellite domains .
to determine if any of these sequences are enriched for cenp-a, we focused on 50-mers that provide junction information between satellite repeats and these non-satellite sequences.
notably, only transposable element junctions embedded in carsat1/ <dig> arrays appear to be enriched among cenp-a-associated 50-mers .
most of the detected cenp-a enrichment was observed at transposable element sequence junctions predicted  to be single-copy within the domain; only line elements in the carsat <dig> array appear to have an enrichment signature associated with homogenized repeat units .
however, we caution against any functional inference, since these enrichment patterns could simply reflect the presence of cenp-a over a region of the carsat <dig> array that happens to contain amplified line sequence in the particular canine genome tested here.
when we mapped cenp-a-enriched 50-mers, we also found enrichment over 323 kb of centromere-linked contigs identified in phase i. the majority of these sequences appear both by alignment and 50-mer frequency to be multi-copy.
it is notable, however, that we find overlap with sites of conserved sequence elements identified within the centromeric read database.
while these may correspond to segmental duplications in the region  <cit> , their significance remains to be determined.
investigating the unassembled sequences for functional centromere sequence variation and organization
to further study sequence organization of centromere sequence features, we investigated enrichment patterns in the wgs reads comprising the reference satellite read database .
focusing on the most abundant carsat <dig> family, we divided the carsat <dig> read database into those that contain cenp-a-associated k-mers  and those that lack entirely any association with cenp-a .
notably, the full-length cenp-a carsat <dig> monomers are phylogenetically distinct from cenp-a monomers .
therefore, cenp-a appears to be associated largely with a distinct subset of satellite sequences, suggesting that the functional component of the array can be delineated by local sequence variation.
to extend this observation, we clustered reads and focused on sequence patterns that distinguish the functionally distinct subtypes.
we reformatted carsat <dig> reads to evaluate sequence variation within overlapping 200 bp windows .
this revealed six major monomer types, three associated with cenp-a and three not .
different regions within these monomer types showed different patterns, with significant sequence differences between the left and right halves of the monomer  .
interestingly, these sequence groups could be readily distinguished by phylogenetic analysis .
while the above analysis distinguishes subfamilies of carsat <dig> monomers by sequence and functional attributes, it does not reveal how those subfamilies are organized in a genomic context.
to address this and to study the long-range, regional organization of cenp-a-enriched sequences, we next investigated paired-read frequencies between the predicted clusters.
high levels of ‘self-pairing’ between clusters of cenp-a monomers or cenp-a monomers provides evidence for homogenized functional satellite domains, suggesting that these reads are not only similar in sequence but are also spatially close to one another in the genome, with only limited spatial proximity between the cenp-a and cenp-a domains .
as expected, there is also evidence for a limited number of ‘intermediate’ monomers – cenp-a monomers that mate-pair with cenp-a monomers.
overall, these data are consistent with models that indicate that cenp-a-associated centromere sequences are clustered within centromeric domains in both human and other genomes  <cit> .
CONCLUSIONS
we have presented here a generally applicable strategy, outlined in figure  <dig>  to construct a comprehensive database of all sequences that occupy centromeric satellite dna domains in less characterized genomes with localized centromeres.
this strategy provides a comprehensive description of satellite sequence variation and organization, revealing in addition both embedded transposable element insertion sites and adjacent non-satellite sequences that are often missing from current genome assemblies.
global satellite sequence inventories -- defined both by wgs read characterization and by k-mer libraries of satellite sequence signature features – provide a platform to explore sequence variation within these domains, which has been masked previously by collapsed assembly efforts.
the ability to characterize the relative abundance and frequency of each genomic feature associated with centromere regions should promote studies to expand functionally annotated mapping efforts in these domains, as well as provide resources for exploring sequence evolution.
comprehensive databases of this sort could serve as a “reference database” for centromere regions in complex genomes, replacing the featureless gaps that exist now and providing features of satellite sequence and organization that can be used to explore trends in genome biology and function.
the results here provide an initial sequence definition of canine centromeres, while presenting a complete genomic reference database to further studies aimed to address centromere plasticity.
it remains to be tested, for example, how stable cenp-a sequence enrichment patterns are within the context of the same cell type, among different cell types of the same individual, or among different individuals or related species.
such questions can now be addressed using not only the broad classification of functional repeat subfamilies, but also extending such analyses to additional centromere sequence features, including precise single-copy junctions and rare sequence polymorphisms within these centromeric domains.

 centromeres are genomic sites of spindle attachment that are essential for ensuring proper chromosome segregation during cell division. despite their recognized functional importance, centromeres are not well defined at a sequence level in most genomes. this has greatly limited efforts to understand in detail the nature and determinants of the synergistic relationship between genome sequence and epigenetics that is generally believed to underlie centromere identity and function. the relatively poor state of sequence assembly and annotation in centromeric regions is due to the presence and abundance of identical or near-identical satellite dna sequences that confound attempts to generate a reliable reference sequence. as a result, efforts to study the interaction of centromere proteins with the underlying genome sequence are largely incapable of distinguishing sequences that are ‘functional’ from those that are ‘non-functional’. this remains a fundamental roadblock for sequence-based studies of centromere identity, variation and function in virtually all complex genomes. robust genomic studies of centromeric sequences and their variation are not straightforward, as generating comprehensive and high-confidence inventories of satellite dna families requires substantial manual curation. this level of genomic resolution requires both long- and short-range sequence information capable of capturing sequence variation and spatial organization within a single satellite array, between satellites occupying distinct chromosomal domains, and within a given population. as a result, few detailed studies have been reported to date, largely limited to well characterized and intensely studied genomes. further, satellite-rich regions are known to turn over rapidly over short evolutionary periods, thus restricting comparative efforts to closely related species. to address the biological questions of centromere identity, evolution and function, therefore, there is a need to improve upon the current rate of sequence exploration in satellite-rich domains, thereby enabling detailed studies at the intersection of genomics and epigenetics. the functional identification and annotation of centromeres depends on the availability of two comprehensive and complementary sequence datasets: a reference sequence database that describes all centromeric sequence variation and its underlying organization, and a functional sequence database that highlights the genomic features associated with centromere identity and function. current attempts to construct such databases have focused on centromere-associated sequences, as functional sequences can be readily identified epigenetically through association with centromere-specific proteins, such as the histone h variant centromere protein a. this specialized component of centromeric nucleosomes is believed to be the fundamental epigenetic mark for defining kinetochore localization and is observed at discrete sites within satellite dna domains in many genomes. following this approach, inventories of sequences bound to cenp-a have been reported for several species to define, at least at the level of consensus sequences, the genomic content of centromeres in those genomes. while adequate for identifying particular classes of satellite dna associated with centromere function, chromatin immunoprecipitation sequencing projects alone, however, provide only a broad view of centromere sequence organization that is largely incapable of distinguishing between sequences on different chromosomes and/or between closely related sequences, only a subset of which may be actually involved in centromere function. as an alternative and complementary approach, efforts to work from the bottom up to generate high-resolution genomic libraries of centromeric sequences have been taxed by the level and precision of experimental effort needed to derive linear sequence predictions through long spans of near-identical repeats in these regions. these limitations and difficulties notwithstanding, the value of paired genomic and epigenetic centromere datasets has been amply demonstrated in studies from human, plant, and drosophila genomes that have been valuable for defining current models of centromere specification, identity and function. to extend this to many other genomes that are less completely studied, however, will require new approaches to facilitate development and analysis of centromere datasets. to address this gap in current knowledge, we present a strategy to produce a reference sequence database for satellite domains of less-characterized genomes in order to promote broader comparative studies on function and centromere sequence organization. rather than characterizing satellite domains by extending linear maps from the assembled euchromatic chromosome arms, we apply a computational approach to generate a preassembled satellite sequence database, resulting in a comprehensive list of satellite domain features as well as adjacent non-satellite sequences. when paired with an equivalent epigenetic dataset of cenp-a-associated sequences, this enables one to functionally annotate satellite and non-satellite sequence variation, as well as describe the short- and long-range sequence organization associated with active centromeres. to implement this strategy, we focus on the dog genome as an example, as it offers a high-quality whole-genome sanger sequences and assembly. further, unlike the situation for many other high-quality genomes, at least some information about satellite dnas is available. these potential advantages are balanced, however, by limitations of linear assembly across canine satellite dna arrays and the enrichment of segmental duplications found at centric transitions. thus, the current understanding of centromeric sequence organization in the dog genome is summarized only by a small number of satellite family consensus sequences and marginal representation directly adjacent to centromere gaps. here we describe an initial canine reference satellite domain database, utilizing both previously assembled and unassembled sequences, providing genome-wide descriptions of satellite families and annotation of all sequences physically linked to centromeric domains. to annotate the database, we then extracted a library of informative satellite domain sequence features that include polymorphisms and junctions with interspersed repeats found within or adjacent to satellite arrays. finally, to relate the genomic dataset to centromere function, we then developed a complementary dataset of cenp-a-associated sequences in the dog genome and determined the census of sequence features that occupy functional centromeres. this combined genomic, epigenetic and functional approach reveals domains of satellite sequences that are not only distinguishable functionally and spatially, but also by sequence. this approach should be generally applicable to any sequenced genome, with hopes of expanding our understanding of the genomic and functional definition of centromeres in complex genomes.  our approach utilizes all sequence reads from the dog wgs project, including those that are reported in the canine genome assembly and those that are missing from the assembly and fall within centromere “gaps”. the strategy consists of three phases, outlined in figure 1: creating a database of sequence reads in and adjacent to the centromere “gaps” in the assembly, including satellite dnas known to localize to centromeric regions; characterizing unique variants in that database to create a library of informative sequence features; and developing a database of functional centromere sequences and sequence features associated with cenp-a. a comprehensive centromeric sequence database sequences in centromeric regions of complex genomes are generally of two types: those showing very limited variation, which remain unassembled and largely uncharacterized within centromeric “gaps” in the chromosome assemblies; and those adjacent to the gaps that have sufficient variation to allow standard assembly. in the current dog reference genome sequence,, sequence reads are currently unassigned to specific chromosomes and thus are candidates for sequences that map to the centromeric gaps. consistent with what has been found in other complex genomes, a significant proportion of these unassembled and unassigned sequences consist of tandemly repeated satellite dnas. notably, two centromeric satellite families, carnivore satellite and/or satellite canis familiaris – shown previously to hybridize to primary constrictions of dog chromosomes – are significantly enriched in the unmapped canfam scaffolds and are also enriched in regions of the genome assembly directly adjacent to the centromere gaps. these two centromere-associated satellite families account for reads containing  mb and  mb and thus comprise a large proportion of the sequence content of canine centromere gaps. because of their prior association with centromeric regions, we used these two satellite families to nucleate the centromeric sequence database. by surveying for satellite dnas on all assembled and unassembled sequences, we established that the dog genome contains, in addition to carsat and sat1cf, only nine satellite families estimated to account for more than 100 kb. notably, members of five of these satellite families can be networked to carsat and/or sat1cf by paired-read frequency and by proximity to centromere gaps and unmapped scaffolds in canfam. overall, the network of sequences that are anchored to the centromeric regions by their read linkages to carsat and sat1cf includes some  mb and reads, together accounting for % of all unassigned sequences in canfam. therefore, this approach yields a database that contains a significant number of previously unassembled and uncharacterized sequences, suitable for exploring satellite family sequence variation and for identifying potential non-satellite sequences that might also be localized to centromeric regions in this genome. while the vast majority of sequences in this database correspond to members of the seven satellite families, the reads also include members of other repetitive dna families, as well as non-repetitive dna. about 7% of sequences in the database correspond to transposable elements embedded within reads otherwise consisting of satellite dna. most of the transposable element families are underrepresented in canine satellite regions relative to the rest of the genome, as expected given the nature of tandemly repeated satellite dnas and their modes of homogenization. however, at least some transposable element families appear enriched in reads containing specific satellite dnas. for example, cferv1a, a canine-specific ltr family, was found to be -fold enriched in reads containing carsat sequence. similarly, specific line subfamilies were enriched - and -fold respectively in the sat2cf satellite family. this enrichment notwithstanding, it should be emphasized that these embedded transposable elements represent only a small proportion of the overall content of satellite dnas in the centromeric read database, and it is unclear what role, if any, they might play in the maintenance or evolution of these regions of the genome. nonetheless, as demonstrated for other genomes, the elements described here should be useful as genomic landmarks within satellite domains. we also uncovered small amounts of non-repetitive sequences within the centromeric satellite read database. within the unmapped centromeric contigs in the canfam genome assembly,  mb correspond to contiguous sequences ≥100 bp that remain unmasked by the repeatmasker or satellite family libraries. using available annotation, we identified  kb of highly conserved sequences as reported by phastcons predictions. we also found unmapped, centromere-linked contigs that contain sequences homologous to provisional, overlapping protein-coding and non-coding genes in organisms other than dog, taken from the reference sequences collection. many of these sequences, as expected, correspond to segmental duplications in the dog genome. together, these results indicate that analysis of satellite dna families, once annotated in a genomic context, can provide information on the sequence content and description of previously unmapped regions of the genome. a sequence feature library for satellite domains to explore sequence variation within the centromeric read database and as a prerequisite for searching for specific sequence features that distinguish functional centromeres from related sequences, we reformatted the entire canine unmapped database and the remaining unassigned “gap” reads into a library of k-mers. while enrichment can be detected over a range of k-mer values, we have used k-mers 50 bp in length for all analyses described here, as this length maximizes the sequence-based information within our enrichment search while limiting edge effects observed when mapping exact matches in our short-read chip-seq database. the library of 50-mers represents the frequency of all sequence signatures found within the reference database and allows one to annotate high- and low-frequency events, insertions and/or deletions within the highly repetitive sequences that dominate centromeric regions. we excluded 50-mers that also mapped to identical sequences in the canfam genome assembly itself, leaving ~ million different 50-mers that are specific to the unassembled gap regions of the genome. by normalizing to single-copy 50-mer depth estimates, we could use this library to predict the abundance of individual sites within satellite domains, thus defining satellite sequence polymorphisms, transposable element insertions, and boundaries between satellite and non-satellite sequences within these regions. identifying sequence features of functional centromeres in the third phase of the strategy, we generated a library of sequences associated with canine cenp-a-containing nucleosomes, providing a functional context for the satellite dna domain database. to identify all sequences associated with cenp-a in canine cells, we performed cenp-a chip-seq, generating million 72 bp illumina sequence reads. using these sequences, we surveyed the complete canine wgs database to identify sites of enrichment throughout both the current chromosome reference assembly, as well as the unmapped regions of the dog genome. based on non-repetitive alignments to canfam using standard chip-seq mapping and enrichment detection software, we found no evidence for significant cenp-a enrichment outside of the centromeric regions. next, to identify cenp-a-associated sequences within the unmapped gap regions, we compared the chip-seq reads to the unmapped 50-mer-based sequence feature set from phase ii. from this analysis, we identified wgs reads that align with cenp-a chip-seq reads; notably, % of these wgs reads are associated with the carsat and/or carsat satellite families, indicating significant enrichment. carsat and carsat are related satellite families, of lengths 738 bp and 1466 bp, respectively. smaller enrichment sets were identified for several other satellite families; however, in sharp contrast to the carsat1/ datasets, these reads constitute <2% of the total number of reads that define each respective satellite family and thus are of uncertain significance. to further subdivide the carsat1/ sequences, we focused on the 50-mers that are found in the chip-seq database and used these to distinguish specific sequence features of cenp-a-associated versus non-associated copies of carsat1/ satellite repeats. by this analysis, 60% of carsat1/ reads contain a minimum threshold of continuous bases of 50-mers that were >2-fold enriched, while 40% of reads contained no such enriched sequences. this supports the hypothesis that only particular carsat1/ sequences are interacting with cenp-a, suggesting the presence of definable subtypes within the overall satellite domains at canine centromeres. the majority of enriched 50-mers appear to be multi-copy, high-frequency satellite sequences in the carsat1/ arrays, thereby providing evidence for functional cenp-a domains that are predominantly found associated with near-identical satellite repeat units. although not all multi-copy sites within the arrays are enriched, it is clear that high-frequency 50-mers are most likely to be associated with cenp-a. this may indicate that cenp-a is associated preferentially with specific, highly conserved positions within the majority of monomer units at the centromere, reflecting the sequence homogenization that is a common feature of satellite arrays. alternatively, the carsat1/ satellite families might be divided into distinct monomer types that are associated or not associated with cenp-a, suggesting the existence of different subtypes or subfamilies around the genome, similar to what is observed, for example, in primate alpha satellite. in phases i and ii of this strategy, we defined various non-satellite sequences embedded within satellite domains. to determine if any of these sequences are enriched for cenp-a, we focused on 50-mers that provide junction information between satellite repeats and these non-satellite sequences. notably, only transposable element junctions embedded in carsat1/ arrays appear to be enriched among cenp-a-associated 50-mers. most of the detected cenp-a enrichment was observed at transposable element sequence junctions predicted to be single-copy within the domain; only line elements in the carsat array appear to have an enrichment signature associated with homogenized repeat units. however, we caution against any functional inference, since these enrichment patterns could simply reflect the presence of cenp-a over a region of the carsat array that happens to contain amplified line sequence in the particular canine genome tested here. when we mapped cenp-a-enriched 50-mers, we also found enrichment over 323 kb of centromere-linked contigs identified in phase i. the majority of these sequences appear both by alignment and 50-mer frequency to be multi-copy. it is notable, however, that we find overlap with sites of conserved sequence elements identified within the centromeric read database. while these may correspond to segmental duplications in the region, their significance remains to be determined. investigating the unassembled sequences for functional centromere sequence variation and organization to further study sequence organization of centromere sequence features, we investigated enrichment patterns in the wgs reads comprising the reference satellite read database. focusing on the most abundant carsat family, we divided the carsat read database into those that contain cenp-a-associated k-mers and those that lack entirely any association with cenp-a. notably, the full-length cenp-a carsat monomers are phylogenetically distinct from cenp-a monomers. therefore, cenp-a appears to be associated largely with a distinct subset of satellite sequences, suggesting that the functional component of the array can be delineated by local sequence variation. to extend this observation, we clustered reads and focused on sequence patterns that distinguish the functionally distinct subtypes. we reformatted carsat reads to evaluate sequence variation within overlapping 200 bp windows. this revealed six major monomer types, three associated with cenp-a and three not. different regions within these monomer types showed different patterns, with significant sequence differences between the left and right halves of the monomer. interestingly, these sequence groups could be readily distinguished by phylogenetic analysis. while the above analysis distinguishes subfamilies of carsat monomers by sequence and functional attributes, it does not reveal how those subfamilies are organized in a genomic context. to address this and to study the long-range, regional organization of cenp-a-enriched sequences, we next investigated paired-read frequencies between the predicted clusters. high levels of ‘self-pairing’ between clusters of cenp-a monomers or cenp-a monomers provides evidence for homogenized functional satellite domains, suggesting that these reads are not only similar in sequence but are also spatially close to one another in the genome, with only limited spatial proximity between the cenp-a and cenp-a domains. as expected, there is also evidence for a limited number of ‘intermediate’ monomers – cenp-a monomers that mate-pair with cenp-a monomers. overall, these data are consistent with models that indicate that cenp-a-associated centromere sequences are clustered within centromeric domains in both human and other genomes.  we have presented here a generally applicable strategy, outlined in figure  to construct a comprehensive database of all sequences that occupy centromeric satellite dna domains in less characterized genomes with localized centromeres. this strategy provides a comprehensive description of satellite sequence variation and organization, revealing in addition both embedded transposable element insertion sites and adjacent non-satellite sequences that are often missing from current genome assemblies. global satellite sequence inventories -- defined both by wgs read characterization and by k-mer libraries of satellite sequence signature features – provide a platform to explore sequence variation within these domains, which has been masked previously by collapsed assembly efforts. the ability to characterize the relative abundance and frequency of each genomic feature associated with centromere regions should promote studies to expand functionally annotated mapping efforts in these domains, as well as provide resources for exploring sequence evolution. comprehensive databases of this sort could serve as a “reference database” for centromere regions in complex genomes, replacing the featureless gaps that exist now and providing features of satellite sequence and organization that can be used to explore trends in genome biology and function. the results here provide an initial sequence definition of canine centromeres, while presenting a complete genomic reference database to further studies aimed to address centromere plasticity. it remains to be tested, for example, how stable cenp-a sequence enrichment patterns are within the context of the same cell type, among different cell types of the same individual, or among different individuals or related species. such questions can now be addressed using not only the broad classification of functional repeat subfamilies, but also extending such analyses to additional centromere sequence features, including precise single-copy junctions and rare sequence polymorphisms within these centromeric domains. 
we have developed an approach to characterize both satellite and non-satellite centromeric sequences that are missing from current assemblies in complex genomes, using the dog genome as an example. these findings establish a genomic and epigenetic foundation for exploring the functional role of centromeric sequences in the previously sequenced dog genome and provide a model for similar studies within the context of less-characterized genomes. combining this genomic reference with an epigenetic dataset corresponding to sequences associated with the histone h variant centromere protein a, we identify active satellite sequence domains that appear to be both functionally and spatially distinct within the overall definition of satellite families. while the sequence basis for centromere identity remains a subject of considerable debate, one approach is to examine the genomic organization at these active sites that are correlated with epigenetic marks of centromere function.
 centromeres are sites of chromosomal spindle attachment during mitosis and meiosis. while the sequence basis for centromere identity remains a subject of considerable debate, one approach is to examine the genomic organization at these active sites that are correlated with epigenetic marks of centromere function.  we have developed an approach to characterize both satellite and non-satellite centromeric sequences that are missing from current assemblies in complex genomes, using the dog genome as an example. combining this genomic reference with an epigenetic dataset corresponding to sequences associated with the histone h variant centromere protein a, we identify active satellite sequence domains that appear to be both functionally and spatially distinct within the overall definition of satellite families.  these findings establish a genomic and epigenetic foundation for exploring the functional role of centromeric sequences in the previously sequenced dog genome and provide a model for similar studies within the context of less-characterized genomes. centromeresatellite dnascenp-acentromere protein acanis familiaris 