RepEnTools: an automated repeat enrichment analysis package for ChIP-seq data reveals hUHRF1 Tandem-Tudor domain enrichment in young repeats.

BACKGROUND: Repeat elements (REs) play important roles for cell function in health and disease. However, RE enrichment analysis in short-read high-throughput sequencing (HTS) data, such as ChIP-seq, is a challenging task. RESULTS: Here, we present RepEnTools, a software package for genome-wide RE enrichment analysis of ChIP-seq and similar chromatin pulldown experiments. Our analysis package bundles together various software with carefully chosen and validated settings to provide a complete solution for RE analysis, starting from raw input files to tabular and graphical outputs. RepEnTools implementations are easily accessible even with minimal IT skills (Galaxy/UNIX). To demonstrate the performance of RepEnTools, we analysed chromatin pulldown data by the human UHRF1 TTD protein domain and discovered enrichment of TTD binding on young primate and hominid specific polymorphic repeats (SVA, L1PA1/L1HS) overlapping known enhancers and decorated with H3K4me1-K9me2/3 modifications. We corroborated these new bioinformatic findings with experimental data by qPCR assays using newly developed primate and hominid specific qPCR assays which complement similar research tools. Finally, we analysed mouse UHRF1 ChIP-seq data with RepEnTools and showed that the endogenous mUHRF1 protein colocalizes with H3K4me1-H3K9me3 on promoters of REs which were silenced by UHRF1. These new data suggest a functional role for UHRF1 in silencing of REs that is mediated by TTD binding to the H3K4me1-K9me3 double mark and conserved in two mammalian species. CONCLUSIONS: RepEnTools improves the previously available programmes for RE enrichment analysis in chromatin pulldown studies by leveraging new tools, enhancing accessibility and adding some key functions. RepEnTools can analyse RE enrichment rapidly, efficiently, and accurately, providing the community with an up-to-date, reliable and accessible tool for this important type of analysis.

CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice.

CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 µg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding.

Comparative Genomics: Insights on the Pathogenicity and Lifestyle of Rhizoctonia solani.

Proper management of agricultural disease is important to ensure sustainable food security. Staple food crops like rice, wheat, cereals, and other cash crops hold great export value for countries. Ensuring proper supply is critical; hence any biotic or abiotic factors contributing to the shortfall in yield of these crops should be alleviated. Rhizoctonia solani is a major biotic factor that results in yield losses in many agriculturally important crops. This paper focuses on genome informatics of our Malaysian Draft R. solani AG1-IA, and the comparative genomics (inter- and intra- AG) with four AGs including China AG1-IA (AG1-IA_KB317705.1), AG1-IB, AG3, and AG8. The genomic content of repeat elements, transposable elements (TEs), syntenic genomic blocks, functions of protein-coding genes as well as core orthologous genic information that underlies R. solani's pathogenicity strategy were investigated. Our analyses show that all studied AGs have low content and varying profiles of TEs. All AGs were dominant for Class I TE, much like other basidiomycete pathogens. All AGs demonstrate dominance in Glycoside Hydrolase protein-coding gene assignments suggesting its importance in infiltration and infection of host. Our profiling also provides a basis for further investigation on lack of correlation observed between number of pathogenicity and enzyme-related genes with host range. Despite being grouped within the same AG with China AG1-IA, our Draft AG1-IA exhibits differences in terms of protein-coding gene proportions and classifications. This implies that strains from similar AG do not necessarily have to retain similar proportions and classification of TE but must have the necessary arsenal to enable successful infiltration and colonization of host. In a larger perspective, all the studied AGs essentially share core genes that are generally involved in adhesion, penetration, and host colonization. However, the different infiltration strategies will depend on the level of host resilience where this is clearly exhibited by the gene sets encoded for the process of infiltration, infection, and protection from host.

AluMine: alignment-free method for the discovery of polymorphic Alu element insertions.

BACKGROUND: Recently, alignment-free sequence analysis methods have gained popularity in the field of personal genomics. These methods are based on counting frequencies of short k-mer sequences, thus allowing faster and more robust analysis compared to traditional alignment-based methods. RESULTS: We have created a fast alignment-free method, AluMine, to analyze polymorphic insertions of Alu elements in the human genome. We tested the method on 2,241 individuals from the Estonian Genome Project and identified 28,962 potential polymorphic Alu element insertions. Each tested individual had on average 1,574 Alu element insertions that were different from those in the reference genome. In addition, we propose an alignment-free genotyping method that uses the frequency of insertion/deletion-specific 32-mer pairs to call the genotype directly from raw sequencing reads. Using this method, the concordance between the predicted and experimentally observed genotypes was 98.7%. The running time of the discovery pipeline is approximately 2 h per individual. The genotyping of potential polymorphic insertions takes between 0.4 and 4 h per individual, depending on the hardware configuration. CONCLUSIONS: AluMine provides tools that allow discovery of novel Alu element insertions and/or genotyping of known Alu element insertions from personal genomes within few hours.

Maintenance of grafting-induced epigenetic variations in the asexual progeny of Brassica oleracea and B. juncea chimera.

Grafting-induced variations have been observed in many plant species, but the heritability of variation in progeny is not well understood. In our study, adventitious shoots from the C cell lineage of shoot apical meristem (SAM) grafting chimera TCC (where the origin of the outmost, middle and innermost cell layers, respectively, of SAM is designated by 'T' for tuber mustard and 'C' for red cabbage) were induced and identified as r-CCC (r = regenerated). To investigate the maintenance of grafting variations during cell propagation and regeneration, different generations of asexual progeny (r-CCCn, n = generation) were established through successive regeneration of axillary shoots from r-CCC. The fourth generation of r-CCC (r-CCC4) was selected to perform whole genome bisulfite sequencing for comparative analysis of hetero-grafting-induced global methylation changes relative to r-s-CCC4 (s = self-grafting). Increased CHH methylation levels and proportions were observed in r-CCC4, with substantial changes occurring in the repeat elements. Small RNA sequencing revealed 1135 specific small interfering RNA (siRNA) tags that were typically expressed in r-CCC, r-CCC2 and r-CCC4. Notably, 65% of these specific siRNAs were associated with repeat elements, termed RE siRNAs. Subsequent analysis revealed that the CHH methylation of RE siRNA-overlapping regions was mainly hypermethylation in r-CCC4, indicating that they were responsible for directing and maintaining grafting-induced CHH methylation. Moreover, the expression of 13 differentially methylated genes (DMGs) correlated with the phenotypic variation, showing differential expression levels between r-CCC4 and r-s-CCC4. These DMGs were predominantly CG hypermethylated, their methylation modifications corresponded to the transcription of relative methyltransferase.

DNA methylation and socioeconomic status in a Mexican-American birth cohort.

Background: Maternal social environmental stressors during pregnancy are associated with adverse birth and child developmental outcomes, and epigenetics has been proposed as a possible mechanism for such relationships. Methods: In a Mexican-American birth cohort of 241 maternal-infant pairs, cord blood samples were measured for repeat element DNA methylation (LINE-1 and Alu). Linear mixed effects regression was used to model associations between indicators of the social environment (low household income and education, neighborhood-level characteristics) and repeat element methylation. Results from a dietary questionnaire were also used to assess the interaction between maternal diet quality and the social environment on markers of repeat element DNA methylation. Results: After adjusting for confounders, living in the most impoverished neighborhoods was associated with higher cord blood LINE-1 methylation (ß = 0.78, 95%CI 0.06, 1.50, p = 0.03). No other neighborhood-, household-, or individual-level socioeconomic indicators were significantly associated with repeat element methylation. We observed a statistical trend showing that positive association between neighborhood poverty and LINE-1 methylation was strongest in cord blood of infants whose mothers reported better diet quality during pregnancy (pinteraction = 0.12). Conclusion: Our findings indicate a small yet unexpected positive association between neighborhood-level poverty during pregnancy and methylation of repetitive element DNA in infant cord blood and that this association is possibly modified by diet quality during pregnancy. However, our null findings for other adverse SES indicators do not provide strong evidence for an adverse association between early-life socioeconomic environment and repeat element DNA methylation in infants.

Development and validation of a real-time PCR assay for the detection of Toxoplasma gondii DNA in animal and meat samples.

We report a rapid and reliable method for the detection of Toxoplasma gondii in meat and animal tissues based on real-time polymerase chain reaction (PCR). Samples were collected from cattle, small ruminants, horses, and pigs raised or imported into Sicily, Italy. All DNA preparations were assayed by real-time PCR tests targeted to a 98-bp long fragment in the AF 529-bp repeat element and to the B1 gene using specific primers. Diagnostic sensitivity (100%), diagnostic specificity (100%), limit of detection (0.01 pg), efficiency (92-109%), and precision (mean coefficient of variation = 0.60%), repeatability (100%), reproducibility (100%), and robustness were evaluated using 240 DNA extracted samples (120 positives and 120 negative as per the OIE nested PCR method) from different matrices. Positive results were confirmed by the repetition of both real-time and nested PCR assays. Our study demonstrates the viability of a reliable, rapid, and specific real-time PCR on a large scale to monitor contamination with Toxoplasma cysts in meat and animal specimens. This validated method can be used for postmortem detection in domestic and wild animals and for food safety purposes.

The RIDL hypothesis: transposable elements as functional domains of long noncoding RNAs.

Our genome contains tens of thousands of long noncoding RNAs (lncRNAs), many of which are likely to have genetic regulatory functions. It has been proposed that lncRNA are organized into combinations of discrete functional domains, but the nature of these and their identification remain elusive. One class of sequence elements that is enriched in lncRNA is represented by transposable elements (TEs), repetitive mobile genetic sequences that have contributed widely to genome evolution through a process termed exaptation. Here, we link these two concepts by proposing that exonic TEs act as RNA domains that are essential for lncRNA function. We term such elements Repeat Insertion Domains of LncRNAs (RIDLs). A growing number of RIDLs have been experimentally defined, where TE-derived fragments of lncRNA act as RNA-, DNA-, and protein-binding domains. We propose that these reflect a more general phenomenon of exaptation during lncRNA evolution, where inserted TE sequences are repurposed as recognition sites for both protein and nucleic acids. We discuss a series of genomic screens that may be used in the future to systematically discover RIDLs. The RIDL hypothesis has the potential to explain how functional evolution can keep pace with the rapid gene evolution observed in lncRNA. More practically, TE maps may in the future be used to predict lncRNA function.

Major chromosomal breakpoint intervals in breast cancer co-localize with differentially methylated regions.

Solid tumors exhibit chromosomal rearrangements resulting in gain or loss of multiple chromosomal loci (copy number variation, or CNV), and translocations that occasionally result in the creation of novel chimeric genes. In the case of breast cancer, although most individual tumors each have unique CNV landscape, the breakpoints, as measured over large datasets, appear to be non-randomly distributed in the genome. Breakpoints show a significant regional concentration at genomic loci spanning perhaps several megabases. The proximal cause of these breakpoint concentrations is a subject of speculation, but is, as yet, largely unknown. To shed light on this issue, we have performed a bio-statistical analysis on our previously published data for a set of 119 breast tumors and normal controls (Wiedswang et al., 2003), where each sample has both high-resolution CNV and methylation data. The method examined the distribution of closeness of breakpoint regions with differentially methylated regions (DMR), coupled with additional genomic parameters, such as repeat elements and designated "fragile sites" in the reference genome. Through this analysis, we have identified a set of 93 regional loci called breakpoint enriched DMR (BEDMRs) characterized by altered DNA methylation in cancer compared to normal cells that are associated with frequent breakpoint concentrations within a distance of 1 Mb. BEDMR loci are further associated with local hypomethylation (66%), concentrations of the Alu SINE repeats within 3 Mb (35% of the cases), and tend to occur near a number of cancer related genes such as the protocadherins, AKT1, DUB3, GAB2. Furthermore, BEDMRs seem to deregulate members of the histone gene family and chromatin remodeling factors, e.g., JMJD1B, which might affect the chromatin structure and disrupt coordinate signaling and repair. From this analysis we propose that preference for chromosomal breakpoints is related to genome structure coupled with alterations in DNA methylation and hence, chromatin structure, associated with tumorigenesis.

REPEATOME: A Database for Repeat Element Comparative Analysis in Human and Chimpanzee

An increasing number of primate genomes are being sequenced. A direct comparison of repeat elements in human genes and their corresponding chimpanzee orthologs will not only give information on their evolution, but also shed light on the major evolutionary events that shaped our species. We have developed REPEATOME to enable visualization and subsequent comparisons of human and chimpanzee repeat elements. REPEATOME (http://www.repeatome.org/) provides easy access to a complete repeat element map of the human genome, as well as repeat element-associated information. It provides a convenient and effective way to access the repeat elements within or spanning the functional regions in human and chimpanzee genome sequences. REPEATOME includes information to compare repeat elements and gene structures of human genes and their counterparts in chimpanzee. This database can be accessed using comparative search options such as intersection, union, and difference to find lineage-specific or common repeat elements. REPEATOME allows researchers to perform visualization and comparative analysis of repeat elements in human and chimpanzee.

RepWeb: A Web-Based Search Tool for Repeat-Related Literatures

Repetitive sequences such as SINE, LINE, and LTR elements form a major part of eukaryotic genomes. A literature search tool that summarizes the information contained within repeat elements would provide biologists in the field of genomics with a useful tool for analyzing genomic sequence features. We developed a java program designed to make literature access easier by using two search engines simultaneously. RepWeb is a web-based search system that provides a user friendly interface for searching the reference data and journals for information related to repeat elements by using the search engines, Google Scholar and PubMed, simultaneously. It provides an interface that displays the repeat element- related biological information, and includes useful functions such as the production of a repeat tree, clickable links to PubMed and Google Scholar, exporting, and sorting a field into date, author, journal and title.