BACKGROUND
tandemly repeated dna makes up a significant portion of the human genome.
although historically relegated as "junk dna", tandem repeats have taken on a new importance with the realization that their tandem organization provides potentially unique functional characteristics.
tandemly repeated dna is organized as multiple copies of a homologous dna sequence of a certain size  that are arranged in a head to tail pattern to form tandem arrays, and thus represent a distinct type of sequence organization shared by all sequenced genomes.
centromeres from fission yeast to humans contain tandem repeats that are critically important for establishing heterochromatin formation and proper chromosome segregation .
furthermore, tandem repeats have been shown to play a role in paramutation in maize  <cit>  and fwa gene regulation in arabidopsis  <cit> .
overall, many of these functions appear to involve rna interference- mediated chromatin modifications  <cit> .
tandem dna in the human genome shows a wide range of repeat sizes and organization, ranging from microsatellites of a few base pairs to megasatellites of up to several kb  <cit> .
microsatellites and variable number of tandem repeats  can be highly polymorphic and are important for use as genetic markers.
contraction of a  <dig>  kb polymorphic tandem repeat array on chromosome band 4q <dig> is associated with facioscapulohumeral muscular dystrophy   <cit> .
higher copy number of the salivary amylase  gene correlates with protein level in populations with a high starch diet  <cit> .
the duf <dig> protein domain, which is highly expressed in brain, was observed to be amplified specifically in the human lineage and to be undergoing positive selection  <cit> .
and the large macrosatellite array dxz <dig> appears to have a unique function in the process of x chromosome inactivation  <cit> .
thus, tandem repeats play important functional and evolutionary roles in genome biology.
centromeres of human chromosomes contain the largest tandem dna family in the human genome called alpha satellite dna, which has been extensively studied and has emerged as a paradigm for understanding the genomic organization of tandem dna  <cit> .
its fundamental repeat unit consist of  <dig> bp monomers, which are found in large highly homologous arrays of up to several million bp at the centromeres of all human chromosomes.
these tandem arrays are composed of either diverged monomers with no detectable higher-order structure, or as chromosome-specific higher order repeat units  characterized by distinct repeating linear arrangements of an integral set of  <dig> bp monomers  <cit> .
this hor structure correlates with centromere function  <cit> .
the assembled dna sequence of most human chromosomes ends abruptly in large gaps in centromeres and heterochromatic regions, often in arrays of diverged monomeric alpha satellite or other tandem repeated "satellite" dna.
to date, only chromosome  <dig> and the x chromosome end in the higher-order repeat units known to be at these centromeres  <cit> .
furthermore, the large regions of classical heterochromatin such as the those on the long arm of chromosomes  <dig>   <dig> and  <dig>  and the short arms of the acrocentric chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig> are poorly covered by assembled sequence.
these regions are rich in tandemly repeated dna families, which makes their sequence and assembly difficult.
similarly, the human y chromosome is rich in repetitive dna sequences, and required special efforts to obtain a detailed sequence  <cit> .
the following report represents the first overall genome-wide assessment of the number, position and organization of tandem repeats in the sequenced human genome, and therefore represents an important resource for further characterization and overall understanding of genomic organization.
an overall genome-wide view of the representation of major tandem repeat families found in the current version of the sequenced human genome  is important for the study of copy number variation , because many of these arrays will be highly variable in copy number between individuals.
therefore, bioinformatic analysis was performed in this report using both existing database software and additional searches to extend the databases.
the largest most prominent arrays ≥  <dig> kb in size currently found in the assembled genome are examined, including large arrays of "simple satellite" sequences such as gaatgn and vntrs, which reveal unexpected higher-order organization.
<dig> additional large arrays that are beyond the detection limit of current databases have been identified, which include multicopy gene families and large novel satellite dna families which display distinct tandem arrangements.
RESULTS
classical satellite repetitive dna in the human genome
we performed a bioinformatics analysis of the tandemly repeated dna using the output from tandem repeats finder  run against hg <dig>  <cit> , which reports  <dig>  arrays containing tandem repeats ranging in size from  <dig> to  <dig> bp  <cit> .
figure  <dig> shows these arrays > <dig> bp plotted by repeat unit size vs. array size .
in order to examine the largest most prominent tandem repeats in the human genome assembly, the  <dig> arrays larger than  <dig> kb were further classified into repeat class .
<dig>  of these large tandem arrays represented pericentromeric alpha satellite dna, which showed repeat unit sizes of ~ <dig> bp and multiples , found in arrays as large as  <dig> kb.
despite this prominence in the trf dataset, the majority of alpha satellite dna remains unassembled and is represented by megabased sized centromeric gaps  <cit> .
the trf output  includes some redundancies where the same array is reported more than once as multiples of a basic monomeric repeat unit e.g.
<dig> bp repeat units can also be reported as arrays of  <dig>   <dig>   <dig> bp repeat units, etc.
we therefore compiled all tandem arrays > <dig> kb found in the human genome sequence  , with all redundancies from trf removed.
we cross referenced these arrays with the "simple, low complexity, and satellite" repeat masker tracks  <cit>  from the ucsc genome browser and their associated entries in the repbase data base of repetitive elements  <cit> .
table  <dig> also includes several large arrays derived by combining multiple smaller  overlapping arrays found by trf but not highlighted in figure  <dig>  table  <dig> represents the most prominent arrays of satellite sequences currently found in the human genome sequence.
assembly- gdis- gap at distal end of array, gprox-gap at proximal end of array, gint- gap internal to array.
inv- inversion within array.
sd- array is associated with a surrounding segmental duplication .
intra- intrachromosomal duplication of related arrays on same chromosome inter- interchromosomal duplication of related arrays on different chromosomes.
–  related arrays are grouped by number, either intra or interchromosomal, or both, as indicated.
the most abundant tandem repeats after alpha satellite dna are satellites ii and iii .
satellite iii is composed primarily of the pentameric sequence gaatgn , identified by repeat masker as a simple satellite.
this family forms a prominent family in the trf output , with many large arrays at  <dig> bp and multiples thereof including some repeat units as large as  <dig> bp, forming arrays of up to ~ <dig> kb.
satellite ii is based on highly diverged arrays of gaatg, of which trf finds  <dig> bp or  <dig> bp repeat units and approximate multiples which are identified by repeat masker as hsatii, based on comparison to a  <dig> bp consensus sequence listed in repbase  <cit> .
as expected, prominent arrays of these "classical" satellites are found in the pericentromeric regions of many chromosomes .
these repeats have been cytologically located to the heterochromatic blocks on chromosomes  <dig>   <dig> and  <dig>  <cit> .
however, in the current assembly these poorly sequenced and assembled regions are riddled with gaps and do not reflect the large amounts of satellite dna found there.
an ~ <dig> kb array of hsatii is seen on the proximal end of the long arm of chromosome  <dig>  directly abuting the heterochromatic gap, and several arrays of hsatii are found in the chromosome  <dig> random fragments.
on chromosome  <dig>  only a small ~ <dig>  kb array of satellite dna containing both gaatg and gagtg is present flanking the hererochromatin of chromosome  <dig>  consistent with satellite iii group ii which has been previously described there  <cit> .
an additional prominent ~ <dig> kb array of gaatg is found at the distal edge of the y long arm and is presumably representative of the large heterochromatic block on the y chromosome.
additional simple sequence repeats found in arrays larger than  <dig> kb include  <dig>   <dig> and  <dig> bp repeats identified by trf consisting of diverged ccttgn repeats as identified by repeat masker, found on xp <dig> .
<dig>   <dig>   <dig> and  <dig> bp repeats consisting of diverged cagcn as identified by repeat masker were found in arrays on chromosomes 2p <dig>  and 22q <dig> .
to round out the classical satellite dna is the centromeric repeats , based on a  <dig> bp repeat, which are found in several large arrays on the centromeric q arm of chromosomes  <dig> and  <dig>  as well as smaller arrays on chromosome  <dig>  another abundant type of satellite dna are the three families of gamma satellite dna , a highly diverged relatively gc rich ~ <dig> bp tandem repeat.
large arrays of gsat listed in repbase as a  <dig> bp repeat  <cit> , are found at the centromere of chromosome  <dig> .
gsatx, listed in repbase as a  <dig> bp repeat  <cit> , are found at xp <dig> and 8q <dig>  gsatii, listed in repbase as a  <dig> bp repeat, is found in a > <dig> kb array in 12p <dig>  sometimes mixed with gsatx repeats.
of these gamma satellites, only the gsatii arrays are found by trf in arrays > <dig> kb.
an additional satellite repeat is hsat <dig>  based on a  <dig> bp repeat  <cit>  found in prominent arrays abutting the centromere at chromosome xp, 16p and 19q, mixed with alpha satellite dna, and in a  <dig> kb array just proximal to the 5srna repeats in chromosome 1q <dig> .
also apparent from the trf output are  <dig> large arrays that consist of locus specific tandem repeats, ranging in repeat size from  <dig> bp up to  <dig> bp.
the largest array is the  <dig>  kb array  consisting of dyz <dig>  a tandem  <dig> bp repeat first reported by  <cit>  in the euchromatic portion of the y chromosome .
the inclusion of an array of this size in the finished human genome sequence likely reflects the heroic efforts made in sequencing the y chromosome.
the remaining arrays, which represent classical variable number of tandem repeat  arrays, are as large as  <dig> kb, and do not match known repeat classes as described by repeat masker.
in several cases, trf shows redundancy in these vntr repeats, e.g.
the  <dig> bp vntr from chromosome band 13q <dig> is also found as a  <dig> bp repeat at the same location .
higher-order structure in large arrays of satellite dna
self-similarity dot plot analysis was performed for all large > <dig> kb tandem arrays  in order to reveal any additional long range repeat unit structure .
at low stringency , all tandem arrays showed a very dense pattern indicative of the homology between repeat units across the arrays, which was not seen for standard complex dna at the boundaries of the tandem arrays .
this analysis revealed the presence of large scale inversions of the orientation of the repeat units in some arrays .
as "stringency" is increased , this dot plot analysis reveals distinct domains of repeat units with higher similarity within the larger array, shown for the large gsatii array on chromosome  <dig> .
remarkably, some "simple sequence" arrays are characterized by highly homologous higher-order repeats  encompassing part or all of the arrays, as indicated by vertical or horizontal lines that are strongly visible on high stringency dot-plots .
the first ~ <dig> kb of the large hsatii array on chromosome  <dig> shows hors of ~ <dig>  kb, with a reversal of orientation .
the distal ~ <dig> kb of the large array of gaatg satellite in the pericentromeric region of the y chromosome also contains  <dig> bp hors .
and the ~ <dig> kb array of gaatg seen in yq <dig> also shows a distinct  <dig> bp across the entire sequenced array .
this  <dig> bp hor is consistent with previously reported periodicity of restriction enzyme digests of human dna  <cit> .
several of the vntr arrays also demonstrated hor structure, such as the  <dig> kb array of a  <dig> bp repeat in the pseudoautosomal xp <dig> , which shows irregular hors of  <dig>  and  <dig>  kb in a complex arrangement .
these distinct sequence domains and higher-order repeat structures in these "simple sequence" satellite dna families were only revealed by examination of the large amount of contiguous sequence now available from the human genome.
large tandem arrays in the human genome
the trf analysis is limited to repeats less than  <dig> kb, and so alternative methods were used to identify tandem repeats larger that  <dig> kb from the human genome .
this revealed a set of  <dig> tandem repeat arrays  .
these represented unique regions which were usually highly visible on the ucsc genome browser as repeating patterns in the repeat masker track and in dot plot analyses as characteristic dense patterns of horizontal lines .
the vast majority of these were described as regions that showed copy number variation  according to the databases included in the ucsc genome browser , although these regions were not distinguished from other cnvs as containing tandem arrays.
some repeats that were less than  <dig> kb were included in table  <dig> and shown in figure  <dig> because they represented more complex repeats than those listed in table  <dig>  these included three arrays of the nbpf genes organized in ~ <dig>  kb repeats , and the largest exon of the hornerin  gene characterized by distinct ~ <dig>  kb repeating units .
arrays of 5srna genes from chromosomes  <dig>   <dig> and x are included .
and several arrays were included because each repeat contains interspersed transposable elements, including a ~ <dig>  kb repeat characterized by a mer5a <dig> element .
<dig> repeat families were found in multiple arrays, either located on the same chromosome  or on different chromosomes , or both .
chromosome band 8p <dig>  has the highest concentration of tandem arrays , and additional regions with multiple tandem repeats includes chromosome 19q and the y chromosome, both of which are known to be enriched in tandem repeats  <cit> .
several of these arrays appear in poorly assembled regions, characterized by sequence gaps within the array e.g.
the gor <dig> repeats in 8q <dig>  , or directly abutting them on the proximal or distal side e.g.
the nbpf gene family found in several arrays in 1q <dig>    <cit> .
furthermore, several arrays may have assembly errors, suggested by the fact that overlap of assembled bac clones is found precisely at the end of the array or at a change in orientation of the repeat units e.g.
the array at 5p <dig> .
these gaps and poor assembly likely indicate significant variation in these regions.
in order to further investigate this variation in a repeat array, the gor <dig> repeats in 8p <dig>  were examined, which are represented in the genome by ~ <dig> copies of a  <dig> kb repeat that is found on either side of an  <dig> kb gap .
pfg analysis was performed to assess the size and copy number variation in this repeat, using the pmei restriction enzyme, which does not cut within the repeat unit and thus releases the whole array in a single large fragment .
a total of  <dig> arrays in two pedigrees were analyzed.
these arrays were polymorphic and spanned a region from  <dig> kb to  <dig>  mbp, representing ~50– <dig> repeat units, but were stably transmitted through up to three generations.
to our knowledge, this is the largest array of tandemly repeated dna in the genome after the centromeric alpha satellite arrays.
the large tandem repeat arrays in table  <dig> fell into several categories depending on the organization of genes, if any, within them.
<dig> tandem repeats contain a gene in most or every repeat unit, such that variation in the copy number of the tandem array would change the gene copy number , e.g.
the ct <dig> gene found in  <dig> precise tandem copies   <cit> .
the dub <dig> gene is represented in the genome by  <dig> precise tandem copies , which was previously found in meiotically unstable arrays from  <dig> to  <dig> copies  <cit> .
additional repeat arrays are wholly contained within a gene, such that each repeat unit represents an exon or protein domain , such as the dmbt gene  <cit>  , the nbpf genes   <cit> , and the lpa gene  <cit>  .
some additional arrays may contain one or a few genes , and several are contained within an intron of a gene .
other arrays represent large classes of megasatellite dna which do not contain genes, but are characterized by repeating patterns of interspersed transposable elements  .
figure 2k shows an example of a  <dig>  kb repeat which contains a mer <dig> dna transposon, several ltr retrotransposons, and many sine and line elements, found in two large arrays on chromosome 19q <dig>  figure 2l shows ~ <dig> kb repeat units from band 4p <dig> that contains portions of lines, ltrs and dna transposons, as well as ~ <dig>  kb blocks of the  <dig> bp acro satellite dna identified by repeat masker.
the presence of horizontal and vertical lines on the dot-plot indicates that there is an inversion within this array.
fish probes made from the non-transposon regions between the acro satellite blocks  hybridize strongly to the short arms of all acrocentric chromosomes as well as chromosome 4p and 3p , indicating that it is this entire  <dig> kb repeat that is amplified on the acrocentric chromosomes.
other large arrays include the sst family of  <dig>  kb repeats on chromosomes 4q <dig>  and 19q <dig>  <cit> , which are listed in repbase as  <dig> bp repeats, with additional diverged arrays on chromosomes 7p <dig> , 17p <dig>  and 20p <dig> .
a  <dig> bp fish probe made to the region between the  <dig> bp "sst" repeats from chromosome  <dig> hybridizes specifically to the arrays on chromosomes  <dig> and  <dig> .
a large polymorphic  <dig> kb repeat  has been described on the x chromosome in an array of 50– <dig> copies, which does not contain any repeat masker identified tes  <cit> .
another striking array at band 2q <dig>  consists of  <dig> kb of diverged  <dig> bp repeats that are identified by repeat masker as a dense block of highly diverged fragments of the dna transposon mer <dig> .
a large tandem dna family consists of amplified ltr retrotransposons
one unique megasatellite family was observed which consists entirely of the malr class of ltr retrotransposons.
this family is organized into ~ <dig>  kb monomeric repeat units containing fragments of both msta and the- <dig> ltrs and internal open reading frames  in both orientations , which have undergone expansion into a tandem array.
these "ltr arrays" are found on  <dig> different chromosomes in  <dig> arrays, covering ~ <dig> kb of dna , and account for a significant portion of the msta ltr transposons in the genome.
these arrays appear as solid blocks of ltr retrotransposons in the ucsc genome browser , with  <dig> distinct arrays on chromosome  <dig>  and the three largest arrays in the pericentromeric regions of chromosomes 13q, 18p and 21q .
fish analysis using probes designed to conserved regions of these ltr array repeats reveals the  <dig> arrays, including small arrays seen in the genome browser with less than  <dig> repeat units  on chromosomes 9q <dig> , 3q <dig>  and 2q <dig> as well as additional arrays on chromosomes  <dig>   <dig>   <dig>  and  <dig> .
thus, this family is the largest most widespread megasatellite family yet described, and represents a novel arrangement and expansion of the abundant malr class of ltr retrotransposons.
fish analysis of  <dig> unrelated individuals showed no variation in the presence of ltr arrays on non-acrocentric chromosomes, but revealed significant variation in the presence and number of ltr arrays on the acrocentric chromosomes .
a centromeric array was seen on all  <dig> chromosome 13s examined,  <dig> of which also had an array on the short arm.
a centromeric array was also seen on all  <dig> chromosome 21s examined,  <dig> of which also has a short arm array,  <dig> of which had a centromeric and a slightly more distal second array, and  <dig> of which had all three arrays.
seven chromosomes  <dig> had only a short arm array,  <dig> had only a centromeric array, while  <dig> had both and  <dig> had no detectable array.
six of the chromosome 15s examined had only a short arm array, and  <dig> had no detectable array.
thus, the acrocentric chromosomes show up to three arrays resolvable by fish  and a large amount of variability in the population.
the presence of ltr arrays at the centromeres of chromosomes  <dig> and  <dig> in the genomic sequence are consistent with these results.
these ltr arrays are in general embedded in large interrelated segmental duplications, which on chromosomes  <dig>   <dig> and  <dig> are complex highly related mosaics several hundred kb in size .
the large ltr arrays from chromosomes  <dig>   <dig> and  <dig> are each characterized by higher order repeat  structures consisting of  <dig> ~ <dig>  kb monomeric repeats , with distinct patterns of msta fragments repeated once per hor .
in particular, each hor on chromosomes  <dig>   <dig> and  <dig> is marked by insertion of another type of ltr transposon, a full length ltr6a .
these hors are readily visible as bold solid horizontal lines on dotplots .
the dispersal of these hors likely occurred due to the interchromosomal segmental duplications between chromosomes  <dig>  and  <dig>  however, the hors from each chromosomal array shows unique rearrangements .
on chromosome  <dig>  the third hor is missing a  <dig>  kb monomeric repeat unit, presumably due to unequal crossing over between hors .
the array on chromosome  <dig> has a similar crossover event, but the monomeric repeat was deleted from the second hor .
and the array from chromosome  <dig> has a  <dig> monomer deletion in the third hor.
thus, these unique hor rearrangements on each array suggest that unequal crossing over events have been ongoing since the dispersal of the arrays to each chromosome.
discussion and
CONCLUSIONS
tandemly repeated dna represents a unique class of dna in the human genome with unusual sequence organization in regular repeat units.
we have presented here genomic analysis of the largest tandem arrays in the human genome .
for example, analysis of the largest arrays of simple sequence "satellite" dna in the genome  revealed unexpected higher-order structure, including inversions, domains of homology, and extensive hor structures .
hors in "simple" sequence have been suggested by restriction enzyme periodicities seen as large bands on southern blots e.g.
<cit> , but the size and extent of these hors and the diverse sizes seen here for several different classes of satellites has not been previously described.
elucidation of such structures was only possible because of the large  contiguous sequences available from the human genome sequence.
the hor structure of human centromeric alpha satellite has been implicated as important in centromere function  <cit> .
by analogy, it is possible that the simple satellites that display hor and domain structures  may represent a subclass involved in chromosome function.
we have also examined arrays that contain larger repeats, generally greater than  <dig> kb, with some monomeric repeat units as large as  <dig> kb .
tandem dna represents an important source of copy number variation because the number of repeat units can vary by several fold at individual arrays, which for these large arrays can represent mbps of dna sequence.
we have shown, for example, that the size of a tandem array consisting of  <dig> kb repeats at chromosome 8q <dig>  can vary from  <dig> kb to  <dig> mbp, even though these were represented in the genome as 5– <dig> repeats spanning an " <dig> kb" gap.
many of the repeats listed in table  <dig> have been observed to be found in much larger polymorphic arrays than represented in the genome sequence, such as the dux <dig> genes which are associated with fshd  <cit> .
moreover, the majority of the tandem repeats are found in regions with detectable copy number variation, usually by performing acgh with bac arrays or sequence analysis of fosmids  <cit>  and found in databases for human cnvs.
however, these detection methods such as acgh do not clearly distinguish these large tandem arrays, which may vary by hundreds of copies, from other types of cnvs that are found in fewer copies, such as insertions and deletions.
ideally, analysis of the array size and copy number at each individual allele would be desirable, in order to distinguish between, for example, two medium size arrays versus one large and one small array, which may have significant phenotypic differences.
currently, pfg gel analysis as shown in figure  <dig> has been used to distinguish individual alleles of large tandem dna arrays.
chromosome band 8p <dig> contains the highest concentration of large tandem repeats, which occur in the two repetitive proximal and distal regions  which flank a large  <dig>  mbp polymorphic inversion  <cit> .
a  <dig> kb repeat found in  <dig> different arrays of up to  <dig> repeat units were seen in opposite orientations in repd, and homologous single copies of this repeat are seen in repp .
two additional arrays consisting of  <dig>  kb repeats are seen in both repp and repd, and share homology with the dub <dig> array on 4p <dig>  <cit> .
whether these tandem repeats contribute to, or are a consequence of, the common inversions and duplications seen in 8p <dig> is not known.
the large tandem repeats listed in table  <dig> have been categorized according to whether they contain genes or are contained within genes.
<dig> arrays contain repeated genes with one gene per repeat unit .
in these cases, variation in copy number of the tandem repeat would lead to different numbers of genes, which may effect dosage of the protein product and lead to phenotypic changes, such as observed for the amy <dig> gene  <cit> .
<dig> repeat arrays were wholly contained within genes , such that each repeat unit contained one or more exons representing protein domains  <cit> .
in these cases, a change in copy number may represent a change in the number of these repeated domains present in a protein product, which may have a strong effect on function and phenotype.
<dig> other arrays were more difficult to categorize .
some appeared to contain single genes , including small ncrna sequences.
others showed the repeat array contained entirely within an intron, which may represent a source of micrornas involved in gene regulation e.g.
see flj <dig> .
<dig> additional tandem arrays that did not contain genes were categorized as megasatellites because they are made up of repeat units that contain interspersed tes.
these regions presumably represent regions of the genome that accumulated tes in a normal fashion over time, and were subsequently amplified into large tandem repeats.
each is distinguished by striking repetitive patterns in the repeat masker tracks from the ucsc genome browser and distinct dot plots containing dense patterns of horizontal and/or vertical lines .
several arrays appear in the repeat masker tracks as large dense blocks of ltr or dna transposons.
for example, the large  <dig> kb array of  <dig> bp repeats in yq <dig>   <cit>  appears in the ltr track as a solid block of  <dig> copies of highly diverged fragments of ltr12b.
the  <dig> bp consensus of ltr12b listed in repbase has within it  <dig> tandem copies of an ~ <dig> bp repeat which is ~85% similar to the  <dig> bp repeat units seen in this array, and thus it seems possible that this  <dig> bp repeat is derived from a hugely expanded portion of a ltr12b sequence.
another large array on chromosome 2q <dig> consists of  <dig> bp repeats with ~80% homology to positions 128– <dig> of the dna transposon mer <dig>  the beginning of this array contains a full length mer <dig>  in the same orientation, and thus this array likely represents tandem expansion of the latter  <dig> bp of this ltr transposon into the large array.
the ltr arrays  also illustrate novel composition, organization, and evolutionary processes in a tandem repeat.
these arrays consist entirely of the malr class of ltr transposons, including both ltrs and internal open reading frames of the msta elements, organized into  <dig>  kb monomeric repeat units.
these have been dispersed to over  <dig> arrays on ~ <dig> chromosomes, including the acrocentric chromosomes with significant variation for the presence of arrays in both the short arm and pericentromeric regions .
on chromosomes  <dig>   <dig> and  <dig>  the available dna sequence reveals distinct hor structure consisting of  <dig> monomeric repeat units, each of which contain an insertion of an ltr6a element .
the dispersal to multiple chromosomes may be related to the large segmental duplications in which these arrays are embedded.
however, unequal crossing over events have occurred within these hors since this dispersal to multiple chromosomes.
thus, we can putatively order a series of events that shaped these arrays, 1) the transposition of the malr elements and amplification into the  <dig>  kb monomeric repeat units, 2) insertion of the ltr6a element into one of the monomers, 3) expansion into hors containing  <dig> monomers 4) dispersal to chromosomes  <dig>   <dig> and  <dig> and 5) unequal crossing over in the hors on each chromosome.
in summary, we have compiled the collection of largest tandem arrays from the sequenced human genome.
we have demonstrated unexpected secondary higher-order structure in "simple sequence" dna.
we have added to current databases the list of tandem arrays with repeat units greater than  <dig> kb, including novel megasatellites that contain amplified patterns of interspersed transposable elements.
the tandem repeats described in this paper represent an important resource for understanding the genomic organization of the human genome.

 tandemly repeated dna makes up a significant portion of the human genome. although historically relegated as "junk dna", tandem repeats have taken on a new importance with the realization that their tandem organization provides potentially unique functional characteristics. tandemly repeated dna is organized as multiple copies of a homologous dna sequence of a certain size that are arranged in a head to tail pattern to form tandem arrays, and thus represent a distinct type of sequence organization shared by all sequenced genomes. centromeres from fission yeast to humans contain tandem repeats that are critically important for establishing heterochromatin formation and proper chromosome segregation. furthermore, tandem repeats have been shown to play a role in paramutation in maize and fwa gene regulation in arabidopsis. overall, many of these functions appear to involve rna interference- mediated chromatin modifications. tandem dna in the human genome shows a wide range of repeat sizes and organization, ranging from microsatellites of a few base pairs to megasatellites of up to several kb. microsatellites and variable number of tandem repeats can be highly polymorphic and are important for use as genetic markers. contraction of a kb polymorphic tandem repeat array on chromosome band 4q is associated with facioscapulohumeral muscular dystrophy. higher copy number of the salivary amylase gene correlates with protein level in populations with a high starch diet. the duf protein domain, which is highly expressed in brain, was observed to be amplified specifically in the human lineage and to be undergoing positive selection. and the large macrosatellite array dxz appears to have a unique function in the process of x chromosome inactivation. thus, tandem repeats play important functional and evolutionary roles in genome biology. centromeres of human chromosomes contain the largest tandem dna family in the human genome called alpha satellite dna, which has been extensively studied and has emerged as a paradigm for understanding the genomic organization of tandem dna. its fundamental repeat unit consist of bp monomers, which are found in large highly homologous arrays of up to several million bp at the centromeres of all human chromosomes. these tandem arrays are composed of either diverged monomers with no detectable higher-order structure, or as chromosome-specific higher order repeat units characterized by distinct repeating linear arrangements of an integral set of bp monomers. this hor structure correlates with centromere function. the assembled dna sequence of most human chromosomes ends abruptly in large gaps in centromeres and heterochromatic regions, often in arrays of diverged monomeric alpha satellite or other tandem repeated "satellite" dna. to date, only chromosome and the x chromosome end in the higher-order repeat units known to be at these centromeres. furthermore, the large regions of classical heterochromatin such as the those on the long arm of chromosomes and and the short arms of the acrocentric chromosomes and are poorly covered by assembled sequence. these regions are rich in tandemly repeated dna families, which makes their sequence and assembly difficult. similarly, the human y chromosome is rich in repetitive dna sequences, and required special efforts to obtain a detailed sequence. the following report represents the first overall genome-wide assessment of the number, position and organization of tandem repeats in the sequenced human genome, and therefore represents an important resource for further characterization and overall understanding of genomic organization. an overall genome-wide view of the representation of major tandem repeat families found in the current version of the sequenced human genome is important for the study of copy number variation, because many of these arrays will be highly variable in copy number between individuals. therefore, bioinformatic analysis was performed in this report using both existing database software and additional searches to extend the databases. the largest most prominent arrays ≥ kb in size currently found in the assembled genome are examined, including large arrays of "simple satellite" sequences such as gaatgn and vntrs, which reveal unexpected higher-order organization. additional large arrays that are beyond the detection limit of current databases have been identified, which include multicopy gene families and large novel satellite dna families which display distinct tandem arrangements.  classical satellite repetitive dna in the human genome we performed a bioinformatics analysis of the tandemly repeated dna using the output from tandem repeats finder run against hg, which reports arrays containing tandem repeats ranging in size from to bp. figure shows these arrays > bp plotted by repeat unit size vs. array size. in order to examine the largest most prominent tandem repeats in the human genome assembly, the arrays larger than kb were further classified into repeat class. of these large tandem arrays represented pericentromeric alpha satellite dna, which showed repeat unit sizes of ~ bp and multiples, found in arrays as large as kb. despite this prominence in the trf dataset, the majority of alpha satellite dna remains unassembled and is represented by megabased sized centromeric gaps. the trf output includes some redundancies where the same array is reported more than once as multiples of a basic monomeric repeat unit e.g. bp repeat units can also be reported as arrays of bp repeat units, etc. we therefore compiled all tandem arrays > kb found in the human genome sequence, with all redundancies from trf removed. we cross referenced these arrays with the "simple, low complexity, and satellite" repeat masker tracks from the ucsc genome browser and their associated entries in the repbase data base of repetitive elements. table also includes several large arrays derived by combining multiple smaller overlapping arrays found by trf but not highlighted in figure table represents the most prominent arrays of satellite sequences currently found in the human genome sequence. assembly- gdis- gap at distal end of array, gprox-gap at proximal end of array, gint- gap internal to array. inv- inversion within array. sd- array is associated with a surrounding segmental duplication. intra- intrachromosomal duplication of related arrays on same chromosome inter- interchromosomal duplication of related arrays on different chromosomes. – related arrays are grouped by number, either intra or interchromosomal, or both, as indicated. the most abundant tandem repeats after alpha satellite dna are satellites ii and iii. satellite iii is composed primarily of the pentameric sequence gaatgn, identified by repeat masker as a simple satellite. this family forms a prominent family in the trf output, with many large arrays at bp and multiples thereof including some repeat units as large as bp, forming arrays of up to ~ kb. satellite ii is based on highly diverged arrays of gaatg, of which trf finds bp or bp repeat units and approximate multiples which are identified by repeat masker as hsatii, based on comparison to a bp consensus sequence listed in repbase. as expected, prominent arrays of these "classical" satellites are found in the pericentromeric regions of many chromosomes. these repeats have been cytologically located to the heterochromatic blocks on chromosomes and. however, in the current assembly these poorly sequenced and assembled regions are riddled with gaps and do not reflect the large amounts of satellite dna found there. an ~ kb array of hsatii is seen on the proximal end of the long arm of chromosome directly abuting the heterochromatic gap, and several arrays of hsatii are found in the chromosome random fragments. on chromosome only a small ~ kb array of satellite dna containing both gaatg and gagtg is present flanking the hererochromatin of chromosome consistent with satellite iii group ii which has been previously described there. an additional prominent ~ kb array of gaatg is found at the distal edge of the y long arm and is presumably representative of the large heterochromatic block on the y chromosome. additional simple sequence repeats found in arrays larger than kb include and bp repeats identified by trf consisting of diverged ccttgn repeats as identified by repeat masker, found on xp. and bp repeats consisting of diverged cagcn as identified by repeat masker were found in arrays on chromosomes 2p and 22q. to round out the classical satellite dna is the centromeric repeats, based on a bp repeat, which are found in several large arrays on the centromeric q arm of chromosomes and as well as smaller arrays on chromosome another abundant type of satellite dna are the three families of gamma satellite dna, a highly diverged relatively gc rich ~ bp tandem repeat. large arrays of gsat listed in repbase as a bp repeat, are found at the centromere of chromosome. gsatx, listed in repbase as a bp repeat, are found at xp and 8q gsatii, listed in repbase as a bp repeat, is found in a > kb array in 12p sometimes mixed with gsatx repeats. of these gamma satellites, only the gsatii arrays are found by trf in arrays > kb. an additional satellite repeat is hsat based on a bp repeat found in prominent arrays abutting the centromere at chromosome xp, 16p and 19q, mixed with alpha satellite dna, and in a kb array just proximal to the 5srna repeats in chromosome 1q. also apparent from the trf output are large arrays that consist of locus specific tandem repeats, ranging in repeat size from bp up to bp. the largest array is the kb array consisting of dyz a tandem bp repeat first reported by in the euchromatic portion of the y chromosome. the inclusion of an array of this size in the finished human genome sequence likely reflects the heroic efforts made in sequencing the y chromosome. the remaining arrays, which represent classical variable number of tandem repeat arrays, are as large as kb, and do not match known repeat classes as described by repeat masker. in several cases, trf shows redundancy in these vntr repeats, e.g. the bp vntr from chromosome band 13q is also found as a bp repeat at the same location. higher-order structure in large arrays of satellite dna self-similarity dot plot analysis was performed for all large > kb tandem arrays in order to reveal any additional long range repeat unit structure. at low stringency, all tandem arrays showed a very dense pattern indicative of the homology between repeat units across the arrays, which was not seen for standard complex dna at the boundaries of the tandem arrays. this analysis revealed the presence of large scale inversions of the orientation of the repeat units in some arrays. as "stringency" is increased, this dot plot analysis reveals distinct domains of repeat units with higher similarity within the larger array, shown for the large gsatii array on chromosome. remarkably, some "simple sequence" arrays are characterized by highly homologous higher-order repeats encompassing part or all of the arrays, as indicated by vertical or horizontal lines that are strongly visible on high stringency dot-plots. the first ~ kb of the large hsatii array on chromosome shows hors of ~ kb, with a reversal of orientation. the distal ~ kb of the large array of gaatg satellite in the pericentromeric region of the y chromosome also contains bp hors. and the ~ kb array of gaatg seen in yq also shows a distinct bp across the entire sequenced array. this bp hor is consistent with previously reported periodicity of restriction enzyme digests of human dna. several of the vntr arrays also demonstrated hor structure, such as the kb array of a bp repeat in the pseudoautosomal xp, which shows irregular hors of and kb in a complex arrangement. these distinct sequence domains and higher-order repeat structures in these "simple sequence" satellite dna families were only revealed by examination of the large amount of contiguous sequence now available from the human genome. large tandem arrays in the human genome the trf analysis is limited to repeats less than kb, and so alternative methods were used to identify tandem repeats larger that kb from the human genome. this revealed a set of tandem repeat arrays. these represented unique regions which were usually highly visible on the ucsc genome browser as repeating patterns in the repeat masker track and in dot plot analyses as characteristic dense patterns of horizontal lines. the vast majority of these were described as regions that showed copy number variation according to the databases included in the ucsc genome browser, although these regions were not distinguished from other cnvs as containing tandem arrays. some repeats that were less than kb were included in table and shown in figure because they represented more complex repeats than those listed in table these included three arrays of the nbpf genes organized in ~ kb repeats, and the largest exon of the hornerin gene characterized by distinct ~ kb repeating units. arrays of 5srna genes from chromosomes and x are included. and several arrays were included because each repeat contains interspersed transposable elements, including a ~ kb repeat characterized by a mer5a element. repeat families were found in multiple arrays, either located on the same chromosome or on different chromosomes, or both. chromosome band 8p has the highest concentration of tandem arrays, and additional regions with multiple tandem repeats includes chromosome 19q and the y chromosome, both of which are known to be enriched in tandem repeats. several of these arrays appear in poorly assembled regions, characterized by sequence gaps within the array e.g. the gor repeats in 8q, or directly abutting them on the proximal or distal side e.g. the nbpf gene family found in several arrays in 1q. furthermore, several arrays may have assembly errors, suggested by the fact that overlap of assembled bac clones is found precisely at the end of the array or at a change in orientation of the repeat units e.g. the array at 5p. these gaps and poor assembly likely indicate significant variation in these regions. in order to further investigate this variation in a repeat array, the gor repeats in 8p were examined, which are represented in the genome by ~ copies of a kb repeat that is found on either side of an kb gap. pfg analysis was performed to assess the size and copy number variation in this repeat, using the pmei restriction enzyme, which does not cut within the repeat unit and thus releases the whole array in a single large fragment. a total of arrays in two pedigrees were analyzed. these arrays were polymorphic and spanned a region from kb to mbp, representing ~50– repeat units, but were stably transmitted through up to three generations. to our knowledge, this is the largest array of tandemly repeated dna in the genome after the centromeric alpha satellite arrays. the large tandem repeat arrays in table fell into several categories depending on the organization of genes, if any, within them. tandem repeats contain a gene in most or every repeat unit, such that variation in the copy number of the tandem array would change the gene copy number, e.g. the ct gene found in precise tandem copies. the dub gene is represented in the genome by precise tandem copies, which was previously found in meiotically unstable arrays from to copies. additional repeat arrays are wholly contained within a gene, such that each repeat unit represents an exon or protein domain, such as the dmbt gene, the nbpf genes, and the lpa gene. some additional arrays may contain one or a few genes, and several are contained within an intron of a gene. other arrays represent large classes of megasatellite dna which do not contain genes, but are characterized by repeating patterns of interspersed transposable elements. figure 2k shows an example of a kb repeat which contains a mer dna transposon, several ltr retrotransposons, and many sine and line elements, found in two large arrays on chromosome 19q figure 2l shows ~ kb repeat units from band 4p that contains portions of lines, ltrs and dna transposons, as well as ~ kb blocks of the bp acro satellite dna identified by repeat masker. the presence of horizontal and vertical lines on the dot-plot indicates that there is an inversion within this array. fish probes made from the non-transposon regions between the acro satellite blocks hybridize strongly to the short arms of all acrocentric chromosomes as well as chromosome 4p and 3p, indicating that it is this entire kb repeat that is amplified on the acrocentric chromosomes. other large arrays include the sst family of kb repeats on chromosomes 4q and 19q, which are listed in repbase as bp repeats, with additional diverged arrays on chromosomes 7p, 17p and 20p. a bp fish probe made to the region between the bp "sst" repeats from chromosome hybridizes specifically to the arrays on chromosomes and. a large polymorphic kb repeat has been described on the x chromosome in an array of 50– copies, which does not contain any repeat masker identified tes. another striking array at band 2q consists of kb of diverged bp repeats that are identified by repeat masker as a dense block of highly diverged fragments of the dna transposon mer. a large tandem dna family consists of amplified ltr retrotransposons one unique megasatellite family was observed which consists entirely of the malr class of ltr retrotransposons. this family is organized into ~ kb monomeric repeat units containing fragments of both msta and the- ltrs and internal open reading frames in both orientations, which have undergone expansion into a tandem array. these "ltr arrays" are found on different chromosomes in arrays, covering ~ kb of dna, and account for a significant portion of the msta ltr transposons in the genome. these arrays appear as solid blocks of ltr retrotransposons in the ucsc genome browser, with distinct arrays on chromosome and the three largest arrays in the pericentromeric regions of chromosomes 13q, 18p and 21q. fish analysis using probes designed to conserved regions of these ltr array repeats reveals the arrays, including small arrays seen in the genome browser with less than repeat units on chromosomes 9q, 3q and 2q as well as additional arrays on chromosomes and. thus, this family is the largest most widespread megasatellite family yet described, and represents a novel arrangement and expansion of the abundant malr class of ltr retrotransposons. fish analysis of unrelated individuals showed no variation in the presence of ltr arrays on non-acrocentric chromosomes, but revealed significant variation in the presence and number of ltr arrays on the acrocentric chromosomes. a centromeric array was seen on all chromosome 13s examined, of which also had an array on the short arm. a centromeric array was also seen on all chromosome 21s examined, of which also has a short arm array, of which had a centromeric and a slightly more distal second array, and of which had all three arrays. seven chromosomes had only a short arm array, had only a centromeric array, while had both and had no detectable array. six of the chromosome 15s examined had only a short arm array, and had no detectable array. thus, the acrocentric chromosomes show up to three arrays resolvable by fish and a large amount of variability in the population. the presence of ltr arrays at the centromeres of chromosomes and in the genomic sequence are consistent with these results. these ltr arrays are in general embedded in large interrelated segmental duplications, which on chromosomes and are complex highly related mosaics several hundred kb in size. the large ltr arrays from chromosomes and are each characterized by higher order repeat structures consisting of ~ kb monomeric repeats, with distinct patterns of msta fragments repeated once per hor. in particular, each hor on chromosomes and is marked by insertion of another type of ltr transposon, a full length ltr6a. these hors are readily visible as bold solid horizontal lines on dotplots. the dispersal of these hors likely occurred due to the interchromosomal segmental duplications between chromosomes and however, the hors from each chromosomal array shows unique rearrangements. on chromosome the third hor is missing a kb monomeric repeat unit, presumably due to unequal crossing over between hors. the array on chromosome has a similar crossover event, but the monomeric repeat was deleted from the second hor. and the array from chromosome has a monomer deletion in the third hor. thus, these unique hor rearrangements on each array suggest that unequal crossing over events have been ongoing since the dispersal of the arrays to each chromosome. discussion and  tandemly repeated dna represents a unique class of dna in the human genome with unusual sequence organization in regular repeat units. we have presented here genomic analysis of the largest tandem arrays in the human genome. for example, analysis of the largest arrays of simple sequence "satellite" dna in the genome revealed unexpected higher-order structure, including inversions, domains of homology, and extensive hor structures. hors in "simple" sequence have been suggested by restriction enzyme periodicities seen as large bands on southern blots e.g., but the size and extent of these hors and the diverse sizes seen here for several different classes of satellites has not been previously described. elucidation of such structures was only possible because of the large contiguous sequences available from the human genome sequence. the hor structure of human centromeric alpha satellite has been implicated as important in centromere function. by analogy, it is possible that the simple satellites that display hor and domain structures may represent a subclass involved in chromosome function. we have also examined arrays that contain larger repeats, generally greater than kb, with some monomeric repeat units as large as kb. tandem dna represents an important source of copy number variation because the number of repeat units can vary by several fold at individual arrays, which for these large arrays can represent mbps of dna sequence. we have shown, for example, that the size of a tandem array consisting of kb repeats at chromosome 8q can vary from kb to mbp, even though these were represented in the genome as 5– repeats spanning an " kb" gap. many of the repeats listed in table have been observed to be found in much larger polymorphic arrays than represented in the genome sequence, such as the dux genes which are associated with fshd. moreover, the majority of the tandem repeats are found in regions with detectable copy number variation, usually by performing acgh with bac arrays or sequence analysis of fosmids and found in databases for human cnvs. however, these detection methods such as acgh do not clearly distinguish these large tandem arrays, which may vary by hundreds of copies, from other types of cnvs that are found in fewer copies, such as insertions and deletions. ideally, analysis of the array size and copy number at each individual allele would be desirable, in order to distinguish between, for example, two medium size arrays versus one large and one small array, which may have significant phenotypic differences. currently, pfg gel analysis as shown in figure has been used to distinguish individual alleles of large tandem dna arrays. chromosome band 8p contains the highest concentration of large tandem repeats, which occur in the two repetitive proximal and distal regions which flank a large mbp polymorphic inversion. a kb repeat found in different arrays of up to repeat units were seen in opposite orientations in repd, and homologous single copies of this repeat are seen in repp. two additional arrays consisting of kb repeats are seen in both repp and repd, and share homology with the dub array on 4p. whether these tandem repeats contribute to, or are a consequence of, the common inversions and duplications seen in 8p is not known. the large tandem repeats listed in table have been categorized according to whether they contain genes or are contained within genes. arrays contain repeated genes with one gene per repeat unit. in these cases, variation in copy number of the tandem repeat would lead to different numbers of genes, which may effect dosage of the protein product and lead to phenotypic changes, such as observed for the amy gene. repeat arrays were wholly contained within genes, such that each repeat unit contained one or more exons representing protein domains. in these cases, a change in copy number may represent a change in the number of these repeated domains present in a protein product, which may have a strong effect on function and phenotype. other arrays were more difficult to categorize. some appeared to contain single genes, including small ncrna sequences. others showed the repeat array contained entirely within an intron, which may represent a source of micrornas involved in gene regulation e.g. see flj. additional tandem arrays that did not contain genes were categorized as megasatellites because they are made up of repeat units that contain interspersed tes. these regions presumably represent regions of the genome that accumulated tes in a normal fashion over time, and were subsequently amplified into large tandem repeats. each is distinguished by striking repetitive patterns in the repeat masker tracks from the ucsc genome browser and distinct dot plots containing dense patterns of horizontal and/or vertical lines. several arrays appear in the repeat masker tracks as large dense blocks of ltr or dna transposons. for example, the large kb array of bp repeats in yq appears in the ltr track as a solid block of copies of highly diverged fragments of ltr12b. the bp consensus of ltr12b listed in repbase has within it tandem copies of an ~ bp repeat which is ~85% similar to the bp repeat units seen in this array, and thus it seems possible that this bp repeat is derived from a hugely expanded portion of a ltr12b sequence. another large array on chromosome 2q consists of bp repeats with ~80% homology to positions 128– of the dna transposon mer the beginning of this array contains a full length mer in the same orientation, and thus this array likely represents tandem expansion of the latter bp of this ltr transposon into the large array. the ltr arrays also illustrate novel composition, organization, and evolutionary processes in a tandem repeat. these arrays consist entirely of the malr class of ltr transposons, including both ltrs and internal open reading frames of the msta elements, organized into kb monomeric repeat units. these have been dispersed to over arrays on ~ chromosomes, including the acrocentric chromosomes with significant variation for the presence of arrays in both the short arm and pericentromeric regions. on chromosomes and the available dna sequence reveals distinct hor structure consisting of monomeric repeat units, each of which contain an insertion of an ltr6a element. the dispersal to multiple chromosomes may be related to the large segmental duplications in which these arrays are embedded. however, unequal crossing over events have occurred within these hors since this dispersal to multiple chromosomes. thus, we can putatively order a series of events that shaped these arrays, 1) the transposition of the malr elements and amplification into the kb monomeric repeat units, 2) insertion of the ltr6a element into one of the monomers, 3) expansion into hors containing monomers 4) dispersal to chromosomes and and 5) unequal crossing over in the hors on each chromosome. in summary, we have compiled the collection of largest tandem arrays from the sequenced human genome. we have demonstrated unexpected secondary higher-order structure in "simple sequence" dna. we have added to current databases the list of tandem arrays with repeat units greater than kb, including novel megasatellites that contain amplified patterns of interspersed transposable elements. the tandem repeats described in this paper represent an important resource for understanding the genomic organization of the human genome. 
tandemly repeated dna represents a large portion of the human genome, and accounts for a significant amount of copy number variation. this study represents the most comprehensive genome wide analysis of large tandem repeats in the human genome, and will serve as an important resource towards understanding the organization and copy number variation of these complex dna families. several novel megasatellite tandem dna families were observed that are characterized by repeating patterns of interspersed transposable elements that have expanded presumably by unequal crossing over. using tandem repeats finder, tandem repeat arrays greater than kb in total size were identified, and classified into simple sequence e.g. alpha satellite dna, and locus specific vntr arrays. using additional methods, we further identified a total of additional arrays with tandem repeat units greater than kb, of which contained genes or repeated exons. here we present a genome wide analysis of the largest tandem repeats found in the human genome sequence. one of these families is found on different chromosomes in > arrays, and represents one of the largest most widespread megasatellite dna families. analysis of these large sequenced regions revealed that several "simple sequence" arrays actually showed complex domain and/or higher order repeat organization.
 tandemly repeated dna represents a large portion of the human genome, and accounts for a significant amount of copy number variation. here we present a genome wide analysis of the largest tandem repeats found in the human genome sequence.  using tandem repeats finder, tandem repeat arrays greater than kb in total size were identified, and classified into simple sequence e.g. gaatg, classical satellites e.g. alpha satellite dna, and locus specific vntr arrays. analysis of these large sequenced regions revealed that several "simple sequence" arrays actually showed complex domain and/or higher order repeat organization. using additional methods, we further identified a total of additional arrays with tandem repeat units greater than kb, of which contained genes or repeated exons. the overall size of an array of tandem kb repeats which spanned a gap on chromosome was found to be kb to mbp in size, representing one of the largest non-centromeric arrays characterized. several novel megasatellite tandem dna families were observed that are characterized by repeating patterns of interspersed transposable elements that have expanded presumably by unequal crossing over. one of these families is found on different chromosomes in > arrays, and represents one of the largest most widespread megasatellite dna families.  this study represents the most comprehensive genome wide analysis of large tandem repeats in the human genome, and will serve as an important resource towards understanding the organization and copy number variation of these complex dna families. 