Barrier activity is a general property of tDNAs .

<p>A. (Upper) Representation of <i>S. pombe</i> centromere 1 (cen1). The central core (black) is surrounded by inverted repeats including the inner repeat (imr1; red) and outer repeats (otr1-; light blue, navy and purple). Black vertical lines within imr represent tDNAs in cen1. (Lower) Map of cen1 tDNA^Ala (green) and surrounding sequence. The <i>ura4⁺</i>reporter gene (ORF,yellow arrow; surrounding sequence, black line) was inserted into imr1 of cen1. PCR primers are shown as black arrows. B. Illustrations to the left of the graph as above. tDNA alanine (green), isoleucine (gray) and glutamine (light blue) are represented as colored triangles in imr1. Real time RT-PCR analysis of centromeric <i>ura4⁺</i> transcription was normalized to endogenous <i>ura4⁺</i>transcription and compared among indicated strains. Error bars represent the standard error of the mean (SEM).</p

Types of barriers in fission yeast.

<p>A. Boundaries of the distinct chromatin domains (dark blue, heterochromatin; pink, euchromatin) are fluid and established through counteracting processes dependent upon the local concentrations of activators and repressors. B. Inverted Repeat barriers are transcribed by RNA pol II (orange arrow) and associated with active chromatin modifications. C. B-box barriers associate with TFIIIC (red star) and coalesce at the nuclear periphery. D. cen tDNA barriers distinguish pericentromeric heterochromatin from centromeric chromatin and require association of both TFIIIC and RNA Pol III (green circle). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001099#s3" target="_blank">discussion</a> for details.</p

Barrier activity requires the RNA polymerase III complex.

<p>A. Chromatin IP analysis of cen1 for enrichment of Sfc6 (TFIIIC) and Rpc130 (Pol III). The X-axis represents 1.5 kb of cen1 imr including tDNA^Ala (green) and nearby tDNA^Glu (white). Centromere specific primers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001099#s4" target="_blank">Materials and Methods</a>. Error bars represent SEM. B . Real time RT-PCR analysis of centromeric <i>ura4⁺</i> transcription normalized to endogenous <i>ura4⁺</i> transcription in strains containing wild-type or mutant barriers (Left). Indicated strains were analyzed by chromatin IP for Sfc6 (Middle, dark gray) and Rpc130 (Right, light gray) enrichment. Error bars represent SEM.</p

Barrier activity is independent of tDNA orientation

a<p>Strains contained the ura4⁺ reporter gene at centromere 1 in both wild type and clr4⁻ backgrounds.</p>b<p>Transcriptional orientation of tDNA^Ala: N, native (antisense strand); R, reverse (sense strand).</p>c<p>Transcriptional orientation of the ura4⁺ reporter gene: F, forward (sense strand); R, Reverse (antisense strand).</p>d<p>ura4⁺ transcript levels were measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001099#pone-0001099-g001" target="_blank">Figure 1</a>.</p>e<p>standard error of the mean</p

Strains used in this study

<p>Strains used in this study</p

SD3_HSat3A6ArraySizeEstimates

SD3_HSat3A6ArraySizeEstimates.txt: This file lists the 396 male samples for whom we estimated HSat3A6 array sizes. The first column lists the Sample ID, the second column lists the sample population (abbreviated according to the conventions used by the 1000 Genomes Project), the third and fourth columns specify the Y haplogroup assignment of each sample (from personal correspondence with Chris Tyler-Smith), and the fifth column lists our HSat3A6 array size estimate in bp

Genomic Characterization of Large Heterochromatic Gaps in the Human Genome Assembly

<div><p>The largest gaps in the human genome assembly correspond to multi-megabase heterochromatic regions composed primarily of two related families of tandem repeats, Human Satellites 2 and 3 (HSat2,3). The abundance of repetitive DNA in these regions challenges standard mapping and assembly algorithms, and as a result, the sequence composition and potential biological functions of these regions remain largely unexplored. Furthermore, existing genomic tools designed to predict consensus-based descriptions of repeat families cannot be readily applied to complex satellite repeats such as HSat2,3, which lack a consistent repeat unit reference sequence. Here we present an alignment-free method to characterize complex satellites using whole-genome shotgun read datasets. Utilizing this approach, we classify HSat2,3 sequences into fourteen subfamilies and predict their chromosomal distributions, resulting in a comprehensive satellite reference database to further enable genomic studies of heterochromatic regions. We also identify 1.3 Mb of non-repetitive sequence interspersed with HSat2,3 across 17 unmapped assembly scaffolds, including eight annotated gene predictions. Finally, we apply our satellite reference database to high-throughput sequence data from 396 males to estimate array size variation of the predominant HSat3 array on the Y chromosome, confirming that satellite array sizes can vary between individuals over an order of magnitude (7 to 98 Mb) and further demonstrating that array sizes are distributed differently within distinct Y haplogroups. In summary, we present a novel framework for generating initial reference databases for unassembled genomic regions enriched with complex satellite DNA, and we further demonstrate the utility of these reference databases for studying patterns of sequence variation within human populations.</p></div

SD2_SubfamilySpecific24mers

SD2_SubfamilySpecific24mers.txt: This file lists all subfamily-specific 24-mers (column 1) along with their subfamily assignments (column 2). A subfamily-specific 24-mer is defined as a 24-mer that occurs on >1% of reads within a given HSat2,3 subfamily and on no more than 0.1% of reads in any other subfamily (and on no non-HSat2,3 reads). Column 3 lists the proportion of reads containing that 24-mer in that subfamily, and column 4 lists the proportion of High Quality (phred>20 for all bases) 24-bp windows matching that 24-mer in that subfamily

Recursive identification of highly connected subgraphs identifies fourteen HSat2,3 subfamilies.

<p>This tree illustrates the iterative binary divisions of the complete HSat2,3 dataset into subfamilies. Each plot is a PCA projection (on principal components 1 and 2) of the normalized 5-mer frequency vectors for all reads in that subgraph. Each point corresponds to one read, colored red or blue by its cluster assignment. Final cluster projections are colored black. The box at the upper right illustrates the concept of self-mate-pair frequencies within the first subgraph division. Arrows below each subgraph are labeled with the self-mate-pair frequency of each daughter cluster, and they are colored to match their cluster of origin in the parent subgraph.</p

Unmapped scaffold uniquely mapped to HSat2-rich region on chromosome 1.

<p>This unmapped scaffold (HuRef SCAF_1103279187792) defines an inter-satellite region in the large centromeric/heterochromatin gap on chromosome 1 (<b>a</b>), which is located between alpha satellite (centromeric region) and Human Satellites 2,3 (heterochromatic gap) (<b>b</b>). It contains roughly 60kb of non-RepeatMasked sequence (<b>c</b>), most of which represents ancient segmental duplications to the pericentromeric regions of chromosomes 1, 2, 7, 10, 16, and 20. Also shown are the positions of annotated gene predictions and HSat2-containing reads used in the assembly of this scaffold (<b>d</b>).</p