Replication Slippage Research Articles

Novel gene rearrangements in mitochondrial genomes of four families of praying mantises (Insecta, Mantodea) and phylogenetic relationships of Mantodea

The mitochondrial genome (mitogenome) plays an important role in phylogenetic studies of many species. The mitogenomes of many praying mantis groups have been well-studied, but mitogenomes of special mimic praying mantises, especially Acanthopoidea and Galinthiadoidea species, are still sorely lacking in the NCBI database. The present study analyzes five mitogenomes from four species of Acanthopoidea (Angela sp., Callibia diana, Coptopteryx sp., Raptrix fusca) and one of Galinthiadoidea (Galinthias amoena) that were sequenced by the primer-walking method. Among Angela sp. and Coptopteryx sp., three gene rearrangements were detected in ND3-A-R-N-S-E-F and COX1-L2-COX2 gene regions, two of which were novel. In addition, individual tandem repeats were found in control regions of four mitogenomes (Angela sp., C. diana, Coptopteryx sp., G. amoena). For those, plausible explanations were derived from the tandem duplication-random loss (TDRL) model and the slipped-strand mispairing model. One potential motif was found in Acanthopidae that was seen as a synapomorphy. Several conserved block sequences (CBSs) were detected within Acanthopoidea that paved the way for the design of specific primers. Via BI and ML analysis, based on four datasets (PCG12, PCG12R, PCG123, PCG123R), the merged phylogenetic tree within Mantodea was reconstructed. This showed that the monophyly of Acanthopoidea was supported and that the PCG12R dataset was the most suitable for reconstructing the phylogenetic tree within Mantodea.

Slipped-strand mispairing within a polycytidine tract in transcriptional regulator mga leads to M protein phase variation and Mga length polymorphism in Group A Streptococcus.

The M protein, a major virulence factor of Group A Streptococcus (GAS), is regulated by the multigene regulator Mga. An unexplained phenomena frequently occurring with in vitro genetic manipulation or culturing of M1T1 GAS strains is the loss of M protein production. This study was aimed at elucidating the basis for the loss of M protein production. The majority of M protein-negative (M-) variants had one C deletion at a tract of 8 cytidines starting at base 1,571 of the M1 mga gene, which is designated as c.1571C[8]. The C deletion led to a c.1571C[7] mga variant that has an open reading frame shift and encodes a Mga-M protein fusion protein. Transformation with a plasmid containing wild-type mga restored the production of the M protein in the c.1571C[7] mga variant. Isolates producing M protein (M+) were recovered following growth of the c.1571C[7] M protein-negative variant subcutaneously in mice. The majority of the recovered isolates with reestablished M protein production had reverted back from c.1571C[7] to c.1571C[8] tract and some M+ isolates lost another C in the c.1571C[7] tract, leading to a c.1571C[6] variant that encodes a functional Mga with 13 extra amino acid residues at the C-terminus compared with wild-type Mga. The nonfunctional c.1571C[7] and functional c.1571C[6] variants are present in M1, M12, M14, and M23 strains in NCBI genome databases, and a G-to-A nonsense mutation at base 1,657 of M12 c.1574C[7] mga leads to a functional c.1574C[7]/1657A mga variant and is common in clinical M12 isolates. The numbers of the C repeats in this polycytidine tract and the polymorphism at base 1,657 lead to polymorphism in the size of Mga among clinical isolates. These findings demonstrate the slipped-strand mispairing within the c.1574C[8] tract of mga as a reversible switch controlling M protein production phase variation in multiple GAS common M types.

The Mechanism of Disease of Tri-Nucleotide Repeat Expansion Diseases (P9-11.002)

<h3>Objective:</h3> To clarify the mechanism of disease of tri-nucleotide repeat (<b>TNR</b>) expansion diseases such as Huntington’s Disease, Myotonic Dystrophy and Friedreich’s Ataxia. <h3>Background:</h3> The current explanation for TNR expansion diseases is DNA slippage during DNA duplication; however, it does not explain why risk of disease starts with 30–40 TNRs or 90–120 repetitive nucleotides and occurs in non-dividing neurons. An alternative explanation is that (1) TNRs are mutation-prone; (2) a constant environmental factor induces constant DNA damage and mutations independent of DNA duplication; and (3) existing DNA repair mechanisms are prone to fail starting at 90–120 repetitive nucleotides. <h3>Design/Methods:</h3> TNR sequences were evaluated for the presence of mutation-prone DNA: GC-rich sequence and somatic hypermutation (<b>SHM</b>) hotspots. Environmental factors able to cause constant DNA damage independent of geographic location or time were investigated. Existing DNA repair mechanisms were assessed for likelihood of failure at or above a TNR sequence of 90–120 repetitive nucleotides. <h3>Results:</h3> All TNR sequences consist of GC-rich DNA or SHM hotspots. Among environmental factors only oxidative stress from minimum ionizing particle (<b>MIP</b>) radiation has the energy spectrum, constant presence, and geographic distribution required to cause double-strand DNA breaks in unwound, single-strand DNA during transcription. Homologous recombination repair is a repair mechanism for double-strand DNA breaks that is present in terminally differentiated neurons at time of active transcription; it requires the immediate presence of template duplex DNA to initiate repair synthesis. With increasing TNRs, the size of the D-loop template (average length 200 nucleotides) may be larger (often) or smaller (rarely) than the actual number of damaged repeats, explaining expansion or contraction of TNRs in successive generations. <h3>Conclusions:</h3> The combination of mutation-prone GC-rich DNA or SHM hotspots, MIP radiation-related oxidative stress as constant source of mutations, and a DNA repair mechanism that fails with long stretches of TNRs explains the etiology of TNR expansion diseases. <b>Disclosure:</b> The institution of Prof. De Groen has received research support from Pfizer. The institution of Prof. De Groen has received research support from NIH. Prof. De Groen has received intellectual property interests from a discovery or technology relating to health care. Prof. De Groen has received personal compensation in the range of $500-$4,999 for serving as a Invited speaker at annual conference x2 with AAFPE. Prof. De Groen has received personal compensation in the range of $0-$499 for serving as a None with None.

Tandem Repeat Generation and Novel Isothermal Amplification Based on Nonspecific Tailing and Replication Slippage.

Isothermal amplification is considered to be one of the most promising tools for point-of-care testing molecular diagnosis. However, its clinical application is severely hindered by nonspecific amplification. Thus, it is important to investigate the exact mechanism of nonspecific amplification and develop a high-specific isothermal amplification assay. Four sets of primer pairs were incubated with Bst DNA polymerase to produce nonspecific amplification. Gel electrophoresis, DNA sequencing, and sequence function analysis were used to investigate the mechanism of nonspecific product generation, which was discovered to be nonspecific tailing and replication slippage mediated tandem repeats generation (NT&RS). Using this knowledge, a novel isothermal amplification technology, bridging primer assisted slippage isothermal amplification (BASIS), was developed. During NT&RS, the Bst DNA polymerase triggers nonspecific tailing on the 3'-ends of DNAs, thereby producing sticky-end DNAs over time. The hybridization and extension between these sticky DNAs generate repetitive DNAs, which can trigger self-extension via replication slippage, thereby leading to nonspecific tandem repeats (TRs) generation and nonspecific amplification. Based on the NT&RS, we developed the BASIS assay. The BASIS is carried out by using a well-designed bridging primer, which can form hybrids with primer-based amplicons, thereby generating specific repetitive DNA and triggering specific amplification. The BASIS can detect 10 copies of target DNA, resist interfering DNA disruption, and provide genotyping ability, thereby offering 100% accuracy for type 16 human papillomavirus detection. We discovered the mechanism for Bst-mediated nonspecific TRs generation and developed a novel isothermal amplification assay (BASIS), which can detect nucleic acids with high sensitivity and specificity.

The nucleotide landscape of polyXY regions

PolyXY regions are compositionally biased regions composed of two different amino acids. They are classified according to the arrangement of the two amino acid types ‘X′ and ‘Y′ into direpeats (composed of alternating amino acids, e.g. ‘XYXYXY’), joined (composed of two consecutive stretches of each amino acid, e.g. ‘XXXYYY’) and shuffled (other arrangements, e.g., ‘XYXXYY’). They have been characterized at the amino acid level in all domains of life, and are described as often found within intrinsically disordered regions. Since DNA replication slippage has been proposed as a driver of repeat variation, and given that some polyXY have a repetitive nature, we hypothesized that characterizing the nucleotide coding of various types of polyXY could give hints about their origin and evolution. To test this, we obtained all polyXY regions in the human transcriptome, categorized them, and studied their coding nucleotide sequences. We observed that polyXY exacerbates the codon biases, and that the similarity between the X and Y codons is higher than in the background proteome. Our results support a general mechanism of emergence and evolution of polyXY from single-codon polyX. PolyXY are revealed as hotspots for replication slippage, particularly those composed of repeats: joined and direpeat polyXY. Inter-conversion to shuffled polyXY disrupts nucleotide repeats and restricts further evolution by replication slippage, a mechanism that we previously observed in polyX. Our results shed light on polyXY composition and should simplify the determination of their functions.

Numerical investigation into the effects of corrosion on the shear performance of pretensioned bridge girders with cast-in-place slabs

Corrosion and shear cracking are frequently observed near supports of pretensioned bridge girders in coastal climates, so non-linear finite element analysis was used to study the effect of corrosion on shear performance in a real case study. Varying degrees of corrosion and various locations (top strands, bottom strands, vertical stirrups and girder-slab interface) were considered. The analyses included construction phases, concrete creep and shrinkage, and the effects of corrosion on the properties of prestressing and reinforcing steel, concrete, and the bond between concrete and reinforcement. The study shows that high (20%) corrosion in the bottom layer of strands can modify the failure mode from concrete crushing in the web to strand slippage or crushing of concrete in the support zone with limited cracking. Although severe strand corrosion significantly compromises girder capacity and ductility, failure occurs only when there is overloading. The predicted failure mode was also sensitive to material parameters, in particular the corrosion-induced crack widths used for modelling the reduced concrete strength. Nevertheless, some similarities were noticed between observed and predicted cracking occurrence. For moderate corrosion (10%), girder capacity was limited by strand fracture, but extensive flexure and shear cracking would appear before failure. 20% corrosion in the vertical stirrups in the web seems to have potentially smaller effect on the shear capacity than 20% corrosion in the strands in support, while corrosion in the top strands or stirrups in the girder-slab interface did not affect the girder capacity.

32. Clin-MSI: A machine learning-based approach to MSI detection in endometrial cancers

Microsatellite instability (MSI) is a hallmark of cancers with defective DNA mismatch repair (MMR) including Lynch Syndrome and POLE defects, wherein these repeats are prone to strand slippage replication errors that fail to repair, creating insertion/deletion mutations. Clinically, PCR assays have been used historically to classify microsatellite status, although these are prone to errors based on subjective comparisons to control tissues and the vagaries of Sanger sequencing data. We recently developed a hybrid capture-based gene panel assay to survey Lynch Syndrome genes, POLE and 20 microsatellite sites commonly surveyed for MSI by PCR methods. Using data from this panel to study patients with known MSI status, we extracted NGS reads aligned to the 20 microsatellite sites and trained a gradient boosted decision tree model using xgBoost. Using this model, called Clin-MSI, we were able to classify novel endometrial tumor samples with >97% accuracy. As an analytical tool for use in clinical reporting, we utilized SHAP (SHapley Additive exPlanations), a software package that provides granular information to interpret each feature of the model's decision-making process. The combined NGS assay and Clin-MSI predictor were clinically validated in our CAP-CLIA laboratory and have been utilized to clinically characterize over 700 endometrial cancer samples to-date, as an NCI-funded initiative that tests patient samples submitted from across the state of Ohio. These results can provide important information for patients and providers regarding treatment options as well as the need for family-based germline testing.

Migrating bubble synthesis promotes mutagenesis through lesions in its template.

Break-induced replication (BIR) proceeds via a migrating D-loop for hundreds of kilobases and is highly mutagenic. Previous studies identified long single-stranded (ss) nascent DNA that accumulates during leading strand synthesis to be a target for DNA damage and a primary source of BIR-induced mutagenesis. Here, we describe a new important source of mutagenic ssDNA formed during BIR: the ssDNA template for leading strand BIR synthesis formed during D-loop migration. Specifically, we demonstrate that this D-loop bottom template strand (D-BTS) is susceptible to APOBEC3A (A3A)-induced DNA lesions leading to mutations associated with BIR. Also, we demonstrate that BIR-associated ssDNA promotes an additional type of genetic instability: replication slippage between microhomologies stimulated by inverted DNA repeats. Based on our results we propose that these events are stimulated by both known sources of ssDNA formed during BIR, nascent DNA formed by leading strand synthesis, and the D-BTS that we describe here. Together we report a new source of mutagenesis during BIR that may also be shared by other homologous recombination pathways driven by D-loop repair synthesis.

Maximum depth sequencing reveals an ON/OFF replication slippage switch and apparent in vivo selection for bifidobacterial pilus expression

The human gut microbiome, of which the genus Bifidobacterium is a prevalent and abundant member, is thought to sustain and enhance human health. Several surface-exposed structures, including so-called sortase-dependent pili, represent important bifidobacterial gut colonization factors. Here we show that expression of two sortase-dependent pilus clusters of the prototype Bifidobacterium breve UCC2003 depends on replication slippage at an intragenic G-tract, equivalents of which are present in various members of the Bifidobacterium genus. The nature and extent of this slippage is modulated by the host environment. Involvement of such sortase-dependent pilus clusters in microbe-host interactions, including bacterial attachment to the gut epithelial cells, has been shown previously and is corroborated here for one case. Using a Maximum Depth Sequencing strategy aimed at excluding PCR and sequencing errors introduced by DNA polymerase reagents, specific G-tract sequences in B. breve UCC2003 reveal a range of G-tract lengths whose plasticity within the population is functionally utilized. Interestingly, replication slippage is shown to be modulated under in vivo conditions in a murine model. This in vivo modulation causes an enrichment of a G-tract length which appears to allow biosynthesis of these sortase-dependent pili. This work provides the first example of productive replication slippage influenced by in vivo conditions. It highlights the potential for microdiversity generation in “beneficial” gut commensals.

Partners in crime: Tbf1 and Vid22 promote expansions of long human telomeric repeats at an interstitial chromosome position in yeast.

In humans, telomeric repeats (TTAGGG)n are known to be present at internal chromosomal sites. These interstitial telomeric sequences (ITSs) are an important source of genomic instability, including repeat length polymorphism, but the molecular mechanisms responsible for this instability remain to be understood. Here, we studied the mechanisms responsible for expansions of human telomeric (Htel) repeats that were artificially inserted inside a yeast chromosome. We found that Htel repeats in an interstitial chromosome position are prone to expansions. The propensity of Htel repeats to expand depends on the presence of a complex of two yeast proteins: Tbf1 and Vid22. These two proteins are physically bound to an interstitial Htel repeat, and together they slow replication fork progression through it. We propose that slow progression of the replication fork through the protein complex formed by the Tbf1 and Vid22 partners at the Htel repeat cause DNA strand slippage, ultimately resulting in repeat expansions.

Evaluation of the Simple Sequence Repeats to Work as a Phase Variation with the Neisseria meningitidis Genome

Background: The most crucial mechanism of genetic variation in N. meningitidis is the slipped strand mispairing, this mechanism generates Phase variation using simple sequence repeat (SSR) and is commonly used by the N. meningitidis to escape the immune system despite its function in eradicating the pathogenic and commensal bacteria. Some of simple sequence repeats (SSRs) that located within the genome works as phase variation while other SSRs have no role in generating phase variation mechanisms. Therefore,&#x0D; Aim: the main goal of the current in silico study was to detect the probability of SSR to enroll with phase variation for the entire N. meningitidis genome.&#x0D; Methods: Different criteria were used to judge SSR as it works in phase variation and these criteria were taken from the current literature. These criteria involve the Z score value of the synonymous shuffling model and Markov model of SSR, the position of SSR in the gene or the promoter, instability and polymorphism of SSR in genomes of different strains, and the length of SSR.&#x0D; Results: The positive Z score value of SSR, SSR being variable in length among genomes of different strains and SSR location in 3 prime end of a gene or in the promoter indicates that the SSR generates phase variation in a particular gene.&#x0D; Conclusion: 67 out of 327 putative phase variable genes located on the N. meningitidis genome were determined to fit these criteria. We assume, therefore, that SSR in these genes may be connected with phase variation mechanism. We recommend that experimental evidence should be generated to confirm these findings.

Genomic instability is enriched in localized prostate cancers from men of African ancestry.

270 Background: Whole genome sequencing (WGS) of prostate cancers (PC) from American men of African ancestry (AA) is limited despite AA men having twice the incidence of and mortality from PC as compared to their European ancestry (EA) men. Herein we describe analysis of AA PC WGS to identify genomic contributions to incidence and outcome disparities. Methods: WGS data from AA localized hormone naïve (HN) PCs (n = 23) from our institution were combined with publicly available WGS HNPC datasets (n = 5); quantitative predominant genome-wide ancestry was approximated via RFMix. A comparable EA cohort (n = 224) was similarly assembled. Ancestry groups were compared via regression models correcting for Gleason grade, PSA, and pathologic stage. Results: Analysis of known HNPC driver genes revealed lower frequency of PTEN (2/28 v 70/224, 7% v 31%, p = 0.006) and higher frequency of MYC (5/28 v 13/224, 18% v 5%, p = 0.014) and FOXA1 (7/28 v 24/224, 25% v 11%, p = 0.018) alterations in AA tumors relative to EA. An unbiased search for coding and noncoding drivers uncovered recurrent FOXA1 promoter (n = 8, p = 1.1e-8, RR = 3.92) and gene body protein-coding (n = 5, p = 1.2e-6, RR = 7.83) mutations, as targets of somatic selection and affecting nearly half of AA cases. Despite comparable tumor mutational burdens in each group, analysis of genome-wide mutational signatures revealed an AA-specific enrichment of SNVs in trinucleotide contexts associated with mismatch repair deficiency (SBS6, p = 1.68e-2, RR = 49.1). AA tumors also had significantly more small deletions (sig. ID2) relative to EA samples (p = 2.25e-31, RR = 9.47), implying replication slippage at lagging strands. Finally, AA PC genomes had consistently higher MSI scores relative to EA (median [IQR]: 4.03 [3.8-4.53] v 1.52 [1.21-1.85], p = 2.95e-44), yet lower than MSI-H colon cancers. These associations were independent of tissue preservation method or source, suggesting they reflect an ancestry-specific mutational process. Comparison of germline and somatic variants between AA and EA uncovered candidate DNA damage response genes (DDR) for further functional validation. Conclusions: Analysis of the largest AA cohort to date of WGS HNPC has revealed an ancestry-specific somatic mutational processes resulting in elevated rate of MMR-linked SNVs, replication-slippage associated small deletions, and MSI, relative to EA. The uniformity of these data suggest that AAs may harbor an inherited factor contributing to increased somatic genomic instability in the HNPC context. These results are compatible with published analyses demonstrating lower expression of DDR genes in AA versus EA PCs. Greater MSI and indels (via neoantigen formation) may explain the higher response proportions in AA PC patients treated with immune- and radiotherapies. Studies are ongoing to define mechanisms via associations between germline predisposition, somatic modification, and transcriptional outcome.

Long-Read Sequencing Reveals Genetic Adaptation of Bartonella Adhesin A Among Different Bartonella henselae Isolates.

Bartonella henselae is the causative agent of cat scratch disease and other clinical entities such as endocarditis and bacillary angiomatosis. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations. Human, feline, and laboratory adapted B. henselae isolates often display genomic and phenotypic differences that are related to the expression of outer membrane proteins, for example the Bartonella adhesin A (BadA). This modularly-structured trimeric autotransporter adhesin is a major virulence factor of B. henselae and is crucial for the initial binding to the host via the extracellular matrix proteins fibronectin and collagen. By using next-generation long-read sequencing we demonstrate a conserved genome among eight B. henselae isolates and identify a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes. Two of the eight tested B. henselae strains lack BadA expression because of frameshift mutations. We suggest that active recombination mechanisms, possibly via phase variation (i.e., slipped-strand mispairing and site-specific recombination) within the repetitive badA island facilitate reshuffling of homologous domain arrays. The resulting variations among the different BadA proteins might contribute to host immune evasion and enhance long-term and efficient colonisation in the differing host environments. Considering the role of BadA as a key virulence factor, it remains important to check consistently and regularly for BadA surface expression during experimental infection procedures.

Interaction between DNA mismatch repair proteins and triplet repeat DNA observed from single-molecule FRET measurements

DNA Mismatch Repair (MMR) is a conserved system that maintains genomic fidelity through correction of DNA replication errors. However, DNA MMR displays mutagenic actions that contribute to DNA triplet repeat expansions. Here we used single molecule FRET measurements to characterize MMR protein binding to DNA structures containing triplet repeats. DNA primary and secondary structures were demonstrated to affect strand slippage dynamics as well as DNA-protein binding kinetics in trinucleotide repeat DNA hairpins and related three-way junctions.

Comparative Genome Analysis Reveals Accumulation of Single-Nucleotide Repeats in Pathogenic Escherichia Lineages.

Homopolymeric tracts (HPTs) can lead to phase variation and DNA replication slippage, driving adaptation to environmental changes and evolution of genes and genomes. However, there is limited information on HPTs in Escherichia; therefore, we conducted a comprehensive cross-strain search for HPTs in Escherichia genomes. We determined the HPT genomic distribution and identified a pattern of high-frequency HPT localization in pathogenic Escherichia lineages. Notably, HPTs localized near transcriptional regulatory genes. Additionally, excessive repeats accumulated in toxin-coding genes. Moreover, the genomic localization of some HPTs might be derived from exogenous DNA, such as that of bacteriophages. Altogether, our findings may prove useful for understanding the role of HPTs in Escherichia genomes.

A Small Molecule Exploits Hidden Structural Features within the RNA Repeat Expansion That Causes c9ALS/FTD and Rescues Pathological Hallmarks.

The hexanucleotide repeat expansion GGGGCC [r(G4C2)exp] within intron 1 of C9orf72 causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD. , the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r(G4C2)exp RNA gain- and loss-of-function mechanisms. The r(G4C2)exp RNA folds into both a hairpin structure with repeating 1 × 1 nucleotide GG internal loops and a G-quadruplex structure. Here, we report the identification of a small molecule (CB253) that selectively binds the hairpin form of r(G4C2)exp. Interestingly, the small molecule binds to a previously unobserved conformation in which the RNA forms 2 × 2 nucleotide GG internal loops, as revealed by a series of binding and structural studies. NMR and molecular dynamics simulations suggest that the r(G4C2)exp hairpin interconverts between 1 × 1 and 2 × 2 internal loops through the process of strand slippage. We provide experimental evidence that CB253 binding indeed shifts the equilibrium toward the 2 × 2 GG internal loop conformation, inhibiting mechanisms that drive c9ALS/FTD pathobiology, such as repeat-associated non-ATG translation formation of stress granules and defective nucleocytoplasmic transport in various cellular models of c9ALS/FTD.

Evaluation of ArmedXpert software tools, MixtureAce and Mixture Interpretation, to analyze MPS-STR data

Massively parallel sequencing (MPS) technologies have revolutionized studies of genomic variations and transformed DNA analysis in multiple fields. Assays based on MPS must be capable of discriminating variations introduced by the method, i.e. artifacts from true polymorphisms. In PCR-MPS methods targeting microsatellite markers, artifacts can arise from PCR mis-incorporation, PCR strand slippage (stutter), and sequencing error. Reliable detection of artifacts in mixed DNA samples is a significant challenge that must be addressed in forensic DNA analysis. The ArmedXpert (NicheVision) software tools, MixtureAce™ and Mixture Interpretation, can analyze MPS data by categorizing sequence reads in alleles, stutter, and non-stutter artifacts and analyzing autosomal STR loci of mixed samples. In this study, we evaluated the ArmedXpert tools for the analysis of STR profiles of single-sourced and mixed samples generated by the ForenSeq™ DNA Signature Prep kit (Verogen). Data from eight experimental runs (240 samples) were analyzed: one benchmark run, two runs testing sensitivity with down to 50 pg DNA input, one run testing artificially degraded samples and DNA derived from bones, blood cards and teeth, as well as four runs with mixed DNA samples of varying ratios, sex, and different number of contributors (two to six). The MixtureAce stutter thresholds were initially set following the recommendations from Verogen, plus a non-stutter artifact threshold was set at 5% of allele read counts. A benchmark run, of 30 samples, plus two controls, containing 2310 total alleles, revealed over 5000 artifacts, above an analytical threshold of 10. A total of 4869 artifacts were correctly classified, while 435 were mis-classified as alleles due to exceedance of initial threshold settings. False positives must be resolved by an analyst, which can be time consuming. Stutter thresholds were adjusted based on the benchmark data and the samples were re-tested, resulting in only 57 false positive allele calls. The revised settings were then used in the analysis of the remaining seven experimental runs. Results show that MixtureAce can accurately classify artifacts and alleles when laboratory-specific threshold settings are used. The Mixture Interpretation tool was applied on two- and three-person mixtures. This tool utilized the analyzed data from MixtureAce to calculate, based on the number of alleles at a locus and their read counts, possible deconvolution outcomes with their respective ratios.

Modelling segmental duplications in the human genome

BackgroundSegmental duplications (SDs) are long DNA sequences that are repeated in a genome and have high sequence identity. In contrast to repetitive elements they are often unique and only sometimes have multiple copies in a genome. There are several well-studied mechanisms responsible for segmental duplications: non-allelic homologous recombination, non-homologous end joining and replication slippage. Such duplications play an important role in evolution, however, we do not have a full understanding of the dynamic properties of the duplication process.ResultsWe study segmental duplications through a graph representation where nodes represent genomic regions and edges represent duplications between them. The resulting network (the SD network) is quite complex and has distinct features which allow us to make inference on the evolution of segmantal duplications. We come up with the network growth model that explains features of the SD network thus giving us insights on dynamics of segmental duplications in the human genome. Based on our analysis of genomes of other species the network growth model seems to be applicable for multiple mammalian genomes.ConclusionsOur analysis suggests that duplication rates of genomic loci grow linearly with the number of copies of a duplicated region. Several scenarios explaining such a preferential duplication rates were suggested.

Novel tRNA gene rearrangements in the mitochondrial genomes of praying mantises (Mantodea: Mantidae): Translocation, duplication and pseudogenization

Gene rearrangements have been found in several mitochondrial genomes of Mantodea, located in the gene blocks CR-I-Q-M-ND2, COX1-K-D-ATP8 and ND3-A-R-N-S-E-F-ND5. We have sequenced one mitogenome of Amelidae (Yersinia mexicana) and six mitogenomes of Mantidae to discuss the mitochondrial gene rearrangement and the phylogenetic relationship within Mantidae. These mitogenomes showed rearrangements of tRNA genes except for Asiadodis yunnanensis and Hierodula zhangi. These novel gene rearrangements of Mantidae were primarily concentrated in the region of CR-I-Q-M-ND2, including gene translocation, duplication and pseudogenization. For the occurrences of these rearrangements, the tandem duplication-random loss (TDRL) model and slipped-strand mispairing model were suitable to explain. Large non-coding regions (LNCRs) located in the region of CR-I-Q-M-ND2 were detected in most Mantidae species, whereas some LNCRs had high similarity to the control region (CR). Both BI and ML phylogenetic analyses supported the monophyly of Mantidae and the paraphyly of Mantinae. The phylogenetic results with the gene order and the location of NCRs acted as forceful evidence that specific gene rearrangements and special LNCRs may be synapomorphies for several groups of mantises.

Genome-wide characterization of microsatellite DNA in fishes: survey and analysis of their abundance and frequency in genome-specific regions

BackgroundMicrosatellite repeats are ubiquitous in organism genomes and play an important role in the chromatin organization, regulation of gene activity, recombination and DNA replication. Although microsatellite distribution patterns have been studied in most phylogenetic lineages, they are unclear in fish species.ResultsHere, we present the first systematic examination of microsatellite distribution in coding and non-coding regions of 14 fish genomes. Our study showed that the number and type of microsatellites displayed nonrandom distribution for both intragenic and intergenic regions, suggesting that they have potential roles in transcriptional or translational regulation and DNA replication slippage theories alone were insufficient to explain the distribution patterns. Our results showed that microsatellites are dominant in non-coding regions. The total number of microsatellites ranged from 78,378 to 1,012,084, and the relative density varied from 4925.76 bp/Mb to 25,401.97 bp/Mb. Overall, (A + T)-rich repeats were dominant. The dependence of repeat abundance on the length of the repeated unit (1–6 nt) showed a great similarity decrease, whereas more tri-nucleotide repeats were found in exonic regions than tetra-nucleotide repeats of most species. Moreover, the incidence of different repeated types appeared species- and genomic-specific. These results highlight potential mechanisms for maintaining microsatellite distribution, such as selective forces and mismatch repair systems.ConclusionsOur data could be beneficial for the studies of genome evolution and microsatellite DNA evolutionary dynamics, and facilitate the exploration of microsatellites structural, function, composition mode and molecular markers development in these species.