Tandem Repeat Copy Number Research Articles

RegCloser: a robust regression approach to closing genome gaps

BackgroundClosing gaps in draft genomes leads to more complete and continuous genome assemblies. The ubiquitous genomic repeats are challenges to the existing gap-closing methods, based on either the k-mer representation by the de Bruijn graph or the overlap-layout-consensus paradigm. Besides, chimeric reads will cause erroneous k-mers in the former and false overlaps of reads in the latter.ResultsWe propose a novel local assembly approach to gap closing, called RegCloser. It represents read coordinates and their overlaps respectively by parameters and observations in a linear regression model. The optimal overlap is searched only in the restricted range consistent with insert sizes. Under this linear regression framework, the local DNA assembly becomes a robust parameter estimation problem. We solved the problem by a customized robust regression procedure that resists the influence of false overlaps by optimizing a convex global Huber loss function. The global optimum is obtained by iteratively solving the sparse system of linear equations. On both simulated and real datasets, RegCloser outperformed other popular methods in accurately resolving the copy number of tandem repeats, and achieved superior completeness and contiguity. Applying RegCloser to a plateau zokor draft genome that had been improved by long reads further increased contig N50 to 3-fold long. We also tested the robust regression approach on layout generation of long reads.ConclusionsRegCloser is a competitive gap-closing tool. The software is available at https://github.com/csh3/RegCloser. The robust regression approach has a prospect to be incorporated into the layout module of long read assemblers.

Genetic diversity of transmission-blocking vaccine candidate antigens Pvs25 and Pvs28 in Plasmodium vivax isolates from China

BackgroundTransmission-blocking vaccines (TBVs) target the sexual stages of malaria parasites to reduce or interrupt the transmission cycle in human and mosquito populations. The genetic diversity of TBVs candidate antigens, Pvs25 and Pvs28, in Plasmodium vivax could provide evidence for the development of TBVs.MethodsDry blood spots from P. vivax patients were collected from Dandong, Suining, Hainan, Nyingchi, Tengchong, and Yingjiang in China. The pvs25 and pvs28 genes were amplified and sequenced. The genetic diversity of pvs25 and pvs28 were analyzed using DNASTAR, MEGA6, and DnaSP 5.0 programs.ResultsA total of 377 samples were collected, among which 324 and 272 samples were successfully amplified in the pvs25 and pvs28 genes, respectively. Eight haplotypes were identified in Pvs25, for which the predominant mutation was I130T with 100% prevalence. A variety of 22 haplotypes in Pvs28 were identified. The number of GSGGE/D repeats of Pvs28 was a range of 4–8, among which, high (7–8) and low (4–5) copy numbers of tandem repeats were found in haplotypes H2 and H17, respectively. The nucleotide diversity of pvs28 (π = 0.00305 ± 0.00061) was slightly higher than that of pvs25 (π = 0.00146 ± 0.00007), thus they were not significantly different (P > 0.05). The Tajima's D value of pvs25 was positive whereas pvs28 was negative, which indicated that both genes were affected by natural selection.ConclusionThe genetic diversity of pvs25 and pvs28 genes in China was relatively limited, which provided valuable information for TBVs design and optimization.

Whole-genome sequencing: identification of additional pathogenic variation across the genome.

This scientific commentary refers to ‘Genome sequencing identifies rare tandem repeat expansions and copy number variants in Lennox–Gastaut syndrome’, by Qaiser et al. (https://doi.org/10.1093/braincomms/fcab207).

Genome sequencing identifies rare tandem repeat expansions and copy number variants in Lennox-Gastaut syndrome.

Epilepsies are a group of common neurological disorders with a substantial genetic basis. Despite this, the molecular diagnosis of epilepsies remains challenging due to its heterogeneity. Studies utilizing whole-genome sequencing may provide additional insights into genetic causes of epilepsies of unknown aetiology. Whole-genome sequencing was used to evaluate a cohort of adults with unexplained developmental and epileptic encephalopathies (n = 30), for whom prior genetic tests, including whole-exome sequencing in some cases, were negative or inconclusive. Rare single nucleotide variants, insertions/deletions, copy number variants and tandem repeat expansions were analysed. Seven pathogenic or likely pathogenic single nucleotide variants, and two pathogenic deleterious copy number variants were identified in nine patients (32.1% of the cohort). One of the copy number variants, identified in a patient with Lennox–Gastaut syndrome, was too small to be detected by chromosomal microarray techniques. We also identified two tandem repeat expansions with clinical implications in two other patients with Lennox–Gastaut syndrome: a CGG repeat expansion in the 5′untranslated region of DIP2B, and a CTG expansion in ATXN8OS (previously implicated in spinocerebellar ataxia type 8). Three patients had KCNA2 pathogenic variants. One of them died of sudden unexpected death in epilepsy. The other two patients had, in addition to a KCNA2 variant, a second de novo variant impacting potential epilepsy-relevant genes (KCNIP4 and UBR5). Overall, whole-genome sequencing provided a genetic explanation in 32.1% of the total cohort. This is also the first report of coding and non-coding tandem repeat expansions identified in patients with Lennox–Gastaut syndrome. This study demonstrates that using whole-genome sequencing, the examination of multiple types of rare genetic variation, including those found in the non-coding region of the genome, can help resolve unexplained epilepsies.

Multiple locus variable number tandem repeat analysis of Acinetobacter baumannii strains isolated from human clinical and animal meat samples

The current research was done to study the prevalence rate and molecular typing of Acinetobacter baumannii strains isolated from human and animal samples. One-hundred and seventy-four animal meat and 128 human clinical samples were collected and subjected to bacterial culture. A. baumannii isolates were confirmed using the Loop-mediated isothermal amplification. Approved strains were subjected to molecular typing using the multiple-locus variable number tandem repeat analysis method. Forty-four out of 174 (25.28%) raw meat and 64 out of 128 (50%) human samples were positive for A. baumannii strains. Ovine meat (39.28%) and urine (56.06%) samples had the highest prevalence of A. baumannii strains. Eighteen human isolates were located in eight separate profiles, whereas 18 animal isolates were located in six separate profiles. The highest similarities were found between human-based A. baumannii isolates nos 6, 7 and 18 with isolates nos 5, 11, 13 and 15 (85.6% similarity). The highest similarities were found between animal-based A. baumannii isolates nos 10, 11 and 17 (99.8% similarity). From a total of 10 studied variable copy numbers of tandem repeats (VNTR) loci, 0845, 0826 and 3406 were detected in all animal-based A. baumannii isolates. Moreover, 3406 VNTR loci was only detected in all 18 human-based A. baumannii isolates. A. baumannii isolate no 17 (harbored all 10 VNTR loci) and A. baumannii isolates nos 6, 7 and 18 (harbored 9 VNTR loci) were the most pathogenic human and animal-based strains. Multiple-locus variable number tandem repeat analysis was considered as an accurate and practical method for molecular typing of A. baumannii strains.

Pervasive cis effects of variation in copy number of large tandem repeats on local DNA methylation and gene expression

Variable number tandem repeats (VNTRs) are composed of large tandemly repeated motifs, many of which are highly polymorphic in copy number. However, because of their large size and repetitive nature, they remain poorly studied. To investigate the regulatory potential of VNTRs, we used read-depth data from Illumina whole-genome sequencing to perform association analysis between copy number of ∼70,000 VNTRs (motif size ≥ 10bp) with both gene expression (404 samples in 48 tissues) and DNA methylation (235 samples in peripheral blood), identifying thousands of VNTRs that are associated with local gene expression (eVNTRs) and DNA methylation levels (mVNTRs). Using an independent cohort, we validated 73%-80% of signals observed in the two discovery cohorts, while allelic analysis of VNTR length and CpG methylation in 30 Oxford Nanopore genomes gave additional support for mVNTR loci, thus providing robust evidence to support that these represent genuine associations. Further, conditional analysis indicated that many eVNTRs and mVNTRs act as QTLs independently of other local variation. We also observed strong enrichments of eVNTRs and mVNTRs for regulatory features such as enhancers and promoters. Using the Human Genome Diversity Panel, we define sets of VNTRs that show highly divergent copy numbers among human populations and show that these are enriched for regulatory effects and preferentially associate with genes that have been linked with human phenotypes through GWASs. Our study provides strong evidence supporting functional variation at thousands of VNTRs and defines candidate sets of VNTRs, copy number variation of which potentially plays a role in numerous human phenotypes.

DNA replication, transcription, and H3K56 acetylation regulate copy number and stability at tandem repeats

Tandem repeats are inherently unstable and exhibit extensive copy number polymorphisms. Despite mounting evidence for their adaptive potential, the mechanisms associated with regulation of the stability and copy number of tandem repeats remain largely unclear. To study copy number variation at tandem repeats, we used two well-studied repetitive arrays in the budding yeast genome, the ribosomal DNA (rDNA) locus, and the copper-inducible CUP1 gene array. We developed powerful, highly sensitive, and quantitative assays to measure repeat instability and copy number and used them in multiple high-throughput genetic screens to define pathways involved in regulating copy number variation. These screens revealed that rDNA stability and copy number are regulated by DNA replication, transcription, and histone acetylation. Through parallel studies of both arrays, we demonstrate that instability can be induced by DNA replication stress and transcription. Importantly, while changes in stability in response to stress are observed within a few cell divisions, a change in steady state repeat copy number requires selection over time. Further, H3K56 acetylation is required for regulating transcription and transcription-induced instability at the CUP1 array, and restricts transcription-induced amplification. Our work suggests that the modulation of replication and transcription is a direct, reversible strategy to alter stability at tandem repeats in response to environmental stimuli, which provides cells rapid adaptability through copy number variation. Additionally, histone acetylation may function to promote the normal adaptive program in response to transcriptional stress. Given the omnipresence of DNA replication, transcription, and chromatin marks like histone acetylation, the fundamental mechanisms we have uncovered significantly advance our understanding of the plasticity of tandem repeats more generally.

Genome-wide survey of tandem repeats by nanopore sequencing shows that disease-associated repeats are more polymorphic in the general population

BackgroundTandem repeats are highly mutable and contribute to the development of human disease by a variety of mechanisms. It is difficult to predict which tandem repeats may cause a disease. One hypothesis is that changeable tandem repeats are the source of genetic diseases, because disease-causing repeats are polymorphic in healthy individuals. However, it is not clear whether disease-causing repeats are more polymorphic than other repeats.MethodsWe performed a genome-wide survey of the millions of human tandem repeats using publicly available long read genome sequencing data from 21 humans. We measured tandem repeat copy number changes using tandem-genotypes. Length variation of known disease-associated repeats was compared to other repeat loci.ResultsWe found that known Mendelian disease-causing or disease-associated repeats, especially CAG and 5′UTR GGC repeats, are relatively long and polymorphic in the general population. We also show that repeat lengths of two disease-causing tandem repeats, in ATXN3 and GLS, are correlated with near-by GWAS SNP genotypes.ConclusionsWe provide a catalog of polymorphic tandem repeats across a variety of repeat unit lengths and sequences, from long read sequencing data. This method especially if used in genome wide association study, may indicate possible new candidates of pathogenic or biologically important tandem repeats in human genomes.

Physiological Changes in Barley mlo-11 Powdery Mildew Resistance Conditioned by Tandem Repeat Copy Number.

Wild barley accessions have evolved broad-spectrum defence against barley powdery mildew through recessive mlo mutations. However, the mlo defence response is associated with deleterious phenotypes with a cost to yield and fertility, with implications for natural fitness and agricultural productivity. This research elucidates the mechanism behind a novel mlo allele, designated mlo-11(cnv2), which has a milder phenotype compared to standard mlo-11. Bisulphite sequencing and histone ChIP-seq analyses using near-isogenic lines showed pronounced repression of the Mlo promoter in standard mlo-11 compared to mlo-11(cnv2), with repression governed by 24 nt heterochromatic small interfering RNAs. The mlo-11(cnv2) allele appears to largely reduce the physiological effects of mlo while still endorsing a high level of powdery mildew resistance. RNA sequencing showed that this is achieved through only partly restricted expression of Mlo, allowing adequate temporal induction of defence genes during infection and expression close to wild-type Mlo levels in the absence of infection. The two mlo-11 alleles showed copy number proportionate oxidase and peroxidase expression levels during infection, but lower amino acid and aromatic compound biosynthesis compared to the null allele mlo-5. Examination of highly expressed genes revealed a common WRKY W-box binding motif (consensus ACCCGGGACTAAAGG) and a transcription factor more highly expressed in mlo-11 resistance. In conclusion, mlo-11(cnv2) appears to significantly mitigate the trade-off between mlo defence and normal gene expression.

DNA Hypermethylation and Unstable Repeat Diseases: A Paradigm of Transcriptional Silencing to Decipher the Basis of Pathogenic Mechanisms.

Unstable repeat disorders comprise a variable group of incurable human neurological and neuromuscular diseases caused by an increase in the copy number of tandem repeats located in various regions of their resident genes. It has become clear that dense DNA methylation in hyperexpanded non-coding repeats induces transcriptional silencing and, subsequently, insufficient protein synthesis. However, the ramifications of this paradigm reveal a far more profound role in disease pathogenesis. This review will summarize the significant progress made in a subset of non-coding repeat diseases demonstrating the role of dense landscapes of 5-methylcytosine (5mC) as a common disease modifier. However, the emerging findings suggest context-dependent models of 5mC-mediated silencing with distinct effects of excessive DNA methylation. An in-depth understanding of the molecular mechanisms underlying this peculiar group of human diseases constitutes a prerequisite that could help to discover novel pathogenic repeat loci, as well as to determine potential therapeutic targets. In this regard, we report on a brief description of advanced strategies in DNA methylation profiling for the identification of unstable Guanine-Cytosine (GC)-rich regions and on promising examples of molecular targeted therapies for Fragile X disease (FXS) and Friedrich ataxia (FRDA) that could pave the way for the application of this technique in other hypermethylated expansion disorders.

Using high-resolution annotation of insect mitochondrial DNA to decipher tandem repeats in the control region

ABSTRACTIn this study, we used a small RNA sequencing (sRNA-seq) based method to annotate the mitochondrial genome of the insect Erthesina fullo Thunberg at 1 bp resolution. The high-resolution annotations cover both entire strands of the mitochondrial genome without any gaps or overlaps. Most of the new annotations were consistent with the previous annotations which had been obtained using PacBio full-length transcripts. Two important findings were that animals transcribe both entire strands of mitochondrial genomes and the tandem repeats in the control region of the E. fullo mitochondrial genome contains the repeated Transcription Initiation Sites (TISs) of the heavy strand. In addition, we found that the copy numbers of tandem repeats showed a great diversity within an individual, suggesting that mitochondrial DNA recombination occurs in an individual. In conclusion, the sRNA-seq based method uses 5′ and 3′ end small RNAs to annotate nuclear non-coding and mitochondrial genes at 1 bp resolution, and can be used to identify new steady RNAs, particularly long non-coding RNAs (lncRNAs). The high-resolution annotations of mitochondrial genomes can also be used to study the molecular phylogenetics and evolution of animals or to investigate mitochondrial gene transcription, RNA processing, RNA maturation and several other related topics. The complete mitochondrial genome sequence of E. fullo with the new annotations using the sRNA-seq based method is available at the NCBI GenBank database under the accession number MK374364. We publish our theories, methods, the high quality sRNA-seq and RNA-seq data (SRA: SRP174926) for extensive use.

Physical location of tandem repeats in the wheat genome and application for chromosome identification

A general distribution of tandem repeats (TRs) in the wheat genome was predicted and a new web page combined with fluorescence in situ hybridization experiments, and the newly developed Oligo probes will improve the resolution for wheat chromosome identification. Comprehensive sequence analysis of tandem repeats (TR) in the wheat reference genome permits discovery and application of TRs for chromosome identification. Genome-wide localization of TRs was identified in the reference sequences of Chinese Spring using Tandem Repeat Finder (TRF). A database of repeats unit size, array number, and physical coverage length of TRs in the wheat genome was built. The distribution of TRs occupied 3-5% of the wheat chromosomes, with non-random dispersal across the A, B, and D genomes. Three classes of TRs surrounding the predicted genes were compared. An optimized computer-assisted website page B2DSC was constructed for the general distribution and chromosomally enriched zones of TR sequences to be displayed graphically. The physical distribution of predicted TRs in the wheat genome by B2DSC matched well with the corresponding hybridization signals obtained with fluorescence in situ hybridization (FISH). We developed 20 oligonucleotide probes representing 20-60bp lengths of high copy number of TRs and verified by FISH. An integrated physical map of TR-Oligo probes for wheat chromosome identification was constructed. Our results suggest that the combination of both molecular cytogenetics and genomic research will significantly benefit wheat breeding through chromosome manipulation and engineering.

GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing

BackgroundTandem repeats comprise significant proportion of the human genome including coding and regulatory regions. They are highly prone to repeat number variation and nucleotide mutation due to their repetitive and unstable nature, making them a major source of genomic variation between individuals. Despite recent advances in high throughput sequencing, analysis of tandem repeats in the context of complex diseases is still hindered by technical limitations. We report a novel targeted sequencing approach, which allows simultaneous analysis of hundreds of repeats. We developed a Bayesian algorithm, namely – GtTR - which combines information from a reference long-read dataset with a short read counting approach to genotype tandem repeats at population scale. PCR sizing analysis was used for validation.ResultsWe used a PacBio long-read sequenced sample to generate a reference tandem repeat genotype dataset with on average 13% absolute deviation from PCR sizing results. Using this reference dataset GtTR generated estimates of VNTR copy number with accuracy within 95% high posterior density (HPD) intervals of 68 and 83% for capture sequence data and 200X WGS data respectively, improving to 87 and 94% with use of a PCR reference. We show that the genotype resolution increases as a function of depth, such that the median 95% HPD interval lies within 25, 14, 12 and 8% of the its midpoint copy number value for 30X, 200X WGS, 395X and 800X capture sequence data respectively. We validated nine targets by PCR sizing analysis and genotype estimates from sequencing results correlated well with PCR results.ConclusionsThe novel genotyping approach described here presents a new cost-effective method to explore previously unrecognized class of repeat variation in GWAS studies of complex diseases at the population level. Further improvements in accuracy can be obtained by improving accuracy of the reference dataset.

Assembly and annotation of a draft genome sequence for Glycine latifolia, a perennial wild relative of soybean.

Glycine latifolia (Benth.) Newell & Hymowitz (2n=40), one of the 27 wild perennial relatives of soybean, possesses genetic diversity and agronomically favorable traits that are lacking in soybean. Here, we report the 939-Mb draft genome assembly of G.latifolia (PI 559298) using exclusively linked-reads sequenced from a single Chromium library. We organized scaffolds into 20 chromosome-scale pseudomolecules utilizing two genetic maps and the Glycine max (L.) Merr. genome sequence. High copy numbers of putative 91-bp centromere-specific tandem repeats were observed in consecutive blocks within predicted pericentromeric regions on several pseudomolecules. No 92-bp putative centromeric repeats, which are abundant in G.max, were detected in G.latifolia or Glycine tomentella. Annotation of the assembled genome and subsequent filtering yielded a high confidence gene set of 54475 protein-coding loci. In comparative analysis with five legume species, genes related to defense responses were significantly overrepresented in Glycine-specific orthologous gene families. A total of 304 putative nucleotide-binding site (NBS)-leucine-rich-repeat (LRR) genes were identified in this genome assembly. Different from other legume species, we observed a scarcity of TIR-NBS-LRR genes in G.latifolia. The G.latifolia genome was also predicted to contain genes encoding 367 LRR-receptor-like kinases, a family of proteins involved in basal defense responses and responses to abiotic stress. The genome sequence and annotation of G.latifolia provides a valuable source of alternative alleles and novel genes to facilitate soybean improvement. This study also highlights the efficacy and cost-effectiveness of the application of Chromium linked-reads in diploid plant genome de novo assembly.

STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods

The growing interest in liquid biopsies for cancer research and cell-based non-invasive prenatal testing (NIPT) invigorates the need for improved single cell analysis. In these applications, target cells are extremely rare and fragile in peripheral circulation, which makes the genetic analysis very challenging. To overcome these challenges, cell stabilization and unbiased whole genome amplification are required. This study investigates the performance of four WGA methods on single or a limited number of cells after 24 hour of Streck Cell-Free DNA BCT preservation. The suitability of the DNA, amplified with Ampli1, DOPlify, PicoPLEX and REPLI-g, was assessed for both short tandem repeat (STR) profiling and copy number variant (CNV) analysis after shallow whole genome massively parallel sequencing (MPS). Results demonstrate that Ampli1, DOPlify and PicoPLEX perform well for both applications, with some differences between the methods. Samples amplified with REPLI-g did not result in suitable STR or CNV profiles, indicating that this WGA method is not able to generate high quality DNA after Streck Cell-Free DNA BCT stabilization of the cells.

Establishment and molecular characterisation of seven novel soft-tissue sarcoma cell lines.

Background:Soft-tissue sarcomas (STS) are a diverse group of malignancies that remain a diagnostic and therapeutic challenge. Relatively few reliable cell lines currently exist. Rapidly developing technology for genomic profiling with emerging insights into candidate functional (driver) aberrations raises the need for more models for in vitro functional validation of molecular targets.Methods:Primary cell culture was performed on STS tumours utilising a differential attachment approach. Cell lines were characterised by morphology, immunocytochemistry, proliferation assays, short tandem repeat (STR) and microarray-based genomic copy number profiling.Results:Of 47 STS cases of various subtypes, half formed adherent monolayers. Seven formed self-immortalised cell lines, including three undifferentiated pleomorphic sarcomas, two dedifferentiated liposarcomas (one of which had received radiotherapy), a leiomyosarcoma and a myxofibrosarcoma. Two morphologically distinct yet genetically identical variants were established in separate cultures for the latter two tumours. All cell lines demonstrated genomic and phenotypic features that not only confirm their malignant characteristics but also confirm retention of DNA copy number aberrations present in their parent tumours that likely include drivers.Conclusions:These primary cell lines are much-needed additions to the number of reliable cell lines of STS with complex genomics available for initial functional validation of candidate molecular targets.

The mitogenomes of the pouched lamprey (Geotria australis) and least brook lamprey (Lampetra aepyptera) with phylogenetic considerations

We report the mitogenomes of the pouched lamprey (Geotria australis) and least brook lamprey (Lampetra aepyptera) in the families Geotriidae and Petromyzontidae, respectively. Both of the mitogenomes contain the 37 typical vertebrate genes. Their gene order and contents are identical to those of previously described lamprey mitogenomes. The mitogenome of G. australis (17 080 bp) is the largest among the 10 reported lamprey mitogenomes, owed to two long noncoding regions. The mitogenome of L. aepyptera is 77 bp longer (16 236 bp) than that of the congeneric European river lamprey L. fluviatilis, a size difference mostly due to different copy numbers of tandem repeats in the noncoding regions. The phylogenetic analysis supports that the pouched lamprey (Geotriidae) diverged earlier from the common ancestor of lampreys than the Petromyzonids, and the placement of the least brook lamprey in the genus Lampetra.

Comprehensive Survey of Genetic Diversity in Chloroplast Genomes and 45S nrDNAs within Panax ginseng Species.

We report complete sequences of chloroplast (cp) genome and 45S nuclear ribosomal DNA (45S nrDNA) for 11 Panax ginseng cultivars. We have obtained complete sequences of cp and 45S nrDNA, the representative barcoding target sequences for cytoplasm and nuclear genome, respectively, based on low coverage NGS sequence of each cultivar. The cp genomes sizes ranged from 156,241 to 156,425 bp and the major size variation was derived from differences in copy number of tandem repeats in the ycf1 gene and in the intergenic regions of rps16-trnUUG and rpl32-trnUAG. The complete 45S nrDNA unit sequences were 11,091 bp, representing a consensus single transcriptional unit with an intergenic spacer region. Comparative analysis of these sequences as well as those previously reported for three Chinese accessions identified very rare but unique polymorphism in the cp genome within P. ginseng cultivars. There were 12 intra-species polymorphisms (six SNPs and six InDels) among 14 cultivars. We also identified five SNPs from 45S nrDNA of 11 Korean ginseng cultivars. From the 17 unique informative polymorphic sites, we developed six reliable markers for analysis of ginseng diversity and cultivar authentication.

Digital Genotyping of Macrosatellites and Multicopy Genes Reveals Novel Biological Functions Associated with Copy Number Variation of Large Tandem Repeats

Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in modulating both genomic and phenotypic diversity.

Variation of Intragenic Tandem Repeat Tract of tolA Modulates Escherichia coli Stress Tolerance

In recent work we discovered that the intragenic tandem repeat (TR) region of the tolA gene is highly variable among different Escherichia coli strains. The aim of this study was therefore to investigate the biological function and dynamics of TR variation in E. coli tolA. The biological impact of TR variation was examined by comparing the ability of a set of synthetic tolA variants with in frame repeat copies varying from 2 to 39 to rescue the altered susceptibility of an E. coli ΔtolA mutant to deoxycholic acid, sodium dodecyl sulfate, hyperosmolarity, and infection with filamentous bacteriophage. Interestingly, although each of the TolA variants was able to at least partly rescue the ΔtolA mutant, the extent was clearly dependent on both the repeat number and the type of stress imposed, indicating the existence of opposing selective forces with regard to the optimal TR copy number. Subsequently, TR dynamics in a clonal population were assayed, and we could demonstrate that TR contractions are RecA dependent and enhanced in a DNA repair deficient uvrD background, and can occur at a frequency of 6.9×10−5.