Replication Slippage Research Articles

Identification of novel intronic BRCA1 variants of uncertain significance in a Thai hereditary breast cancer family

screened for mutation in the entire coding sequence of the BRCA1 gene in 50 Thai breast cancer patients. Screening for mutations was carried out using the polymerase chain reaction-based on single-stranded conformational polymorphism (PCR-SSCP) and direct DNA sequencing. The genetic alterations were not found in patients without a family history of breast cancer. One patient (20%) with a family history of five breast cancer cases harboured a germ line mutation in the BRCA1 gene, which was a novel intronic BRCA1 mutation (IVS7+34_47delTTCTTTTCTTTTTT). In addition, two unclassified intronic BRCA1 variations (IVS7+34_47delAAGAAAAGAAAAAA in the antisense strand and IVS7+50_63delTTCTTTTTTTTTTT in the sense strand) and one unclassified intronic point mutation (IVS7+38T>C) were also identified in the patient’s healthy daughter at the age of 25 years. RNA analysis of variants of unknown significance revealed a normally occurring splicing process. These alterations were not found in other family members or unrelated healthy volunteers. Interestingly, the deletion site was in close proximity to the triplet repeat sequence of TTC·GAA. The deletion of a set of TTC repeats was identified both in the patient and the patient’s healthy daughter. It is implied that such genetic alterations could cause genomic rearrangement or an alteration to the DNA structures, possibly through a slipped strand mispairing mechanism during DNA replication.

A template-dependent dislocation mechanism potentiates K65R reverse transcriptase mutation development in subtype C variants of HIV-1.

Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C than subtype B HIV-1. We recently showed that this discrepancy lies partly in the subtype C template coding sequence that predisposes RT to pause at the site of K65R mutagenesis. However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown. Here, we report that DNA synthesis performed with subtype C templates consistently produced more K65R-containing transcripts than subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism involved is template-specific and RT-independent. In addition, a pattern of DNA synthesis characteristic of site-specific primer/template slippage and dislocation was only observed with the subtype C sequence. Analysis of RNA secondary structure suggested that the latter was unlikely to impact on K65R development between subtypes and that Streisinger strand slippage during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region might occur, resulting in misalignment of the primer and template. Consequently, slippage would lead to a deletion of the middle adenine of codon K65 and the production of a -1 frameshift mutation, which upon dislocation and realignment of the primer and template, would lead to development of the K65R mutation. These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1.

Joint Inference of Microsatellite Mutation Models, Population History and Genealogies Using Transdimensional Markov Chain Monte Carlo

We provide a framework for Bayesian coalescent inference from microsatellite data that enables inference of population history parameters averaged over microsatellite mutation models. To achieve this we first implemented a rich family of microsatellite mutation models and related components in the software package BEAST. BEAST is a powerful tool that performs Bayesian MCMC analysis on molecular data to make coalescent and evolutionary inferences. Our implementation permits the application of existing nonparametric methods to microsatellite data. The implemented microsatellite models are based on the replication slippage mechanism and focus on three properties of microsatellite mutation: length dependency of mutation rate, mutational bias toward expansion or contraction, and number of repeat units changed in a single mutation event. We develop a new model that facilitates microsatellite model averaging and Bayesian model selection by transdimensional MCMC. With Bayesian model averaging, the posterior distributions of population history parameters are integrated across a set of microsatellite models and thus account for model uncertainty. Simulated data are used to evaluate our method in terms of accuracy and precision of θ estimation and also identification of the true mutation model. Finally we apply our method to a red colobus monkey data set as an example.

A Complex Double Deletion in LMNA Underlies Progressive Cardiac Conduction Disease, Atrial Arrhythmias, and Sudden Death

Cardiac conduction disease is a clinically and genetically heterogeneous disorder characterized by defects in electrical impulse generation and conduction and is associated with sudden cardiac death. We studied a 4-generation family with autosomal dominant progressive cardiac conduction disease, including atrioventricular conduction block and sinus bradycardia, atrial arrhythmias, and sudden death. Genome-wide linkage analysis mapped the disease locus to chromosome 1p22-q21. Multiplex ligation-dependent probe amplification analysis of the LMNA gene, which encodes the nuclear-envelope protein lamin A/C, revealed a novel gene rearrangement involving a 24-bp inversion flanked by a 3.8-kb deletion upstream and a 7.8-kb deletion downstream. The presence of short inverted sequence homologies at the breakpoint junctions suggested a mutational event involving serial replication slippage in trans during DNA replication. We identified for the first time a complex LMNA gene rearrangement involving a double deletion in a 4-generation Dutch family with progressive conduction system disease. Our findings underscore the fact that if conventional polymerase chain reaction-based direct sequencing approaches for LMNA analysis are negative in suggestive pedigrees, mutation detection techniques capable of detecting gross genomic lesions involving deletions and insertions should be considered.

Neisseria meningitidisStrain of Unknown Serogroup, China

<i>Neisseria meningitidis</i>Strain of Unknown Serogroup, China

Complex repeat structures and novel features in the mitochondrial genomes of the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana

The mitochondrial genome of the raphid pennate diatom Phaeodactylum tricornutum has several novel features compared with the mitochondrial genomes of the centric diatom Thalassiosira pseudonana and the araphid pennate diatom Synedra acus. It is almost double the size (77,356 bp) due to a 35,454 bp sequence block consisting of an elaborate combination of direct repeats, making it the largest stramenopile (heterokont) mitochondrial genome known. In addition, the cox1 gene has a + 1 translational frameshift involving Pro codons CCC and CCT, the first translational frameshift to be detected in an algal mitochondrial genome. The nad9 and rps14 genes are fused by the insertion of an in-frame sequence and cotranscribed. The nad11 gene is split into two parts corresponding to the FeS and molybdate-binding domains, but both parts are still on the mitochondrial genome, in contrast to the brown algae where the second domain appears to have been transferred to the nucleus. In contrast to P. tricornutum, the repeat region of T. pseudonana consists of a much smaller 4790 bp string of almost identical double-hairpin elements, evidence of slipped-strand mispairing and active gene conversion. The diatom mitochondrial genomes have undergone considerable gene rearrangement since the three lineages of diatoms diverged, but all three have kept their repeat regions segregated from their relatively compact coding regions.

Partial Single Base Flipping is Sufficient to Enable Strand Slippage at DNA Duplex Termini

DNA strand slippage is a structural mechanism by which mutations are introduced during replication by DNA polymerases. Although the template-slippage mechanism was suggested by Streisinger and coworkers around 40 years ago, there is no detailed atomic-resolution understanding of the dynamic pathways by which such strand slippage can occur. Using unrestrained molecular dynamics simulations, the present study demonstrates that partial single base flipping can be sufficient to enable spontaneous strand slippage at DNA duplex termini. The strand slippage was observed in both strands at the terminal end of a DNA duplex illustrating exactly how either insertion or deletion (indel) mutations were possible depending on which strand the partially flipped base belonged to. These findings highlight the steric and non-bonded interaction stringency required of DNA polymerase active sites to prevent indel mutations. This relationship between base flipping and strand slippage and its implications for sequence dependence of indel mutations will be discussed along with planned future studies to characterize these newly discovered dynamic DNA strand slippage pathways.

Factors affecting germline mutations in a hypervariable microsatellite: A comparative analysis of six species of swallows (Aves: Hirundinidae)

Microsatellites mutate frequently by replication slippage. Empirical evidence shows that the probability of such slippage mutations may increase with the length of the repeat region as well as exposure to environmental mutagens, but the mutation rate can also differ between the male and female germline. It has been hypothesized that more intense sexual selection or sperm competition can also lead to elevated mutation rates, but the empirical evidence is inconclusive. Here, we analyzed the occurrence of germline slippage mutations in the hypervariable pentanucleotide microsatellite locus HrU10 across six species of swallow (Aves: Hirundinidae). These species exhibit marked differences in the length range of the microsatellite, as well as differences in the intensity of sperm competition. We found a strong effect of microsatellite length on the probability of mutation, but no residual effect of species or their level of sperm competition when the length effect was accounted for. Neither could we detect any difference in mutation rate between tree swallows ( Tachycineta bicolor) breeding in Hamilton Harbour, Ontario, an industrial site with previous documentation of elevated mutation rates for minisatellite DNA, and a rural reference population. However, our cross-species analysis revealed two significant patterns of sex differences in HrU10 germline mutations: (1) mutations in longer alleles occurred typically in the male germline, those in shorter alleles in the female germline, and (2) male germline mutations were more often expansions than contractions, whereas no directional bias was evident in the female germline. These results indicate some fundamental differences in male and female gametogenesis affecting the probability of slippage mutations. Our study also reflects the value of a comparative, multi-species approach for locus-specific mutation analyses, through which a wider range of influential factors can be assessed than in single-species studies.

The Conformational Dynamics of Model Slipped Strand DNA Structures are Determined by Base Pairing in the Heteroduplex Arm

Slipped strand DNA is formed from the out-of-register pairing of direct repeats into thermodynamically stable hairpin structures. The formation of slipped strand DNA is hypothesized to be an important intermediate in triplet repeat expansion, a mechanism associated with many hereditary and anticipative neurodegenerative diseases. In this study, we use three-way DNA junctions as models for slipped strand DNA structures. Each of the three-way DNA molecules have two homoduplex arms and one heteroduplex arm, and the structures and conformational dynamics of the model substrates are characterized using single-molecule fluorescence resonance energy transfer (smFRET). FRET can measure changes in the relative distances between two fluorophores that are sufficiently close together (20-80 A). In our experiments, junctions are end-labeled with donor and acceptor fluorophores, and the efficiency of energy transfer E between the fluorophores is measured as a function of time to approximate the distances between the fluorophores and to monitor the conformational dynamics of the arms. We find that the junctions have trigonal pyramidal geometry. In addition, we observe that the junctions with two or more A-A mispairs on the heteroduplex arm transition between two conformational states with conformer transition rates that are dependent on ionic strength. This investigation sheds light on the structural and dynamical properties of junctions formed by slipped strand DNA which are important for understanding how enzymes recognize and process these structures.

Tumor hypoxia and genetic alterations in sporadic cancers

The cancer genome contains many gene alterations. How cancer cells acquire these alterations is a matter for discussion. One hypothesis is that cancer cells obtain mutations in genome stability genes at an early stage of tumor development, which results in genetic instability and generates a gene pool that enhances cellular proliferation and survival. Another hypothesis puts its emphasis on the natural selection of gene mutations for fitness. Recent data for systematic cancer genome sequencing shows that mutations in stability genes are rare in human sporadic cancers. Instead, many “passenger” mutations that do not drive the carcinogenesis process have been found in the cancer genome. Both the hypotheses mentioned above fall short in explaining recent data. Recently, many studies demonstrate the role of the tumor microenvironment, especially hypoxia and reoxygenation, in genetic instability. In this review, literature will be presented which supports a third hypothesis, i.e. that hypoxia/re-oxygenation induces genetic instability.

Microsatellite markers: an overview of the recent progress in plants

In recent years, molecular markers have been utilized for a variety of applications including examination of genetic relationships between individuals, mapping of useful genes, construction of linkage maps, marker assisted selections and backcrosses, population genetics and phylogenetic studies. Among the available molecular markers, microsatellites or simple sequence repeats (SSRs) which are tandem repeats of one to six nucleotide long DNA motifs, have gained considerable importance in plant genetics and breeding owing to many desirable genetic attributes including hypervariability, multiallelic nature, codominant inheritance, reproducibility, relative abundance, extensive genome coverage including organellar genomes, chromosome specific location and amenability to automation and high throughput genotyping. High degree of allelic variation revealed by microsatellite markers results from variation in number of repeat-motifs at a locus caused by replication slippage and/or unequal crossing-over during meiosis. In spite of limited understanding of the functions of the SSR motifs within the plant genes, SSRs are being widely utilized in plant genome analysis. Microsatellites can be developed directly from genomic DNA libraries or from libraries enriched for specific microsatellites. Alternatively, microsatellites can also be found by searching public databases such as GenBank and EMBL or through cross-species transferability. At present, EST databases are an important source of candidate genes, as these can generate markers directly associated with a trait of interest and may be transferable in close relative genera. A large number of SSR based techniques have been developed and a quantum of literature has accumulated regarding the applicability of SSRs in plant genetics and genomics. In this review we discuss the recent developments (last 4–5 years) made in plant genetics using SSR markers.

Error-Prone DNA Repair Activity during Somatic Hypermutation in Shark B Lymphocytes

Sharks are representatives of the earliest vertebrates that possess an immune system utilizing V(D)J recombination to generate Ag receptors. Their Ab repertoire diversity is based in part on a somatic hypermutation process that introduces adjacent nucleotide substitutions of 2-5 bp. We have isolated mutant nonfunctional Ig rearrangements and intronic flank sequences to characterize the nonselected, intrinsic properties of this phenomenon; changes unique to shark were observed. Duplications and deletions were associated with N additions, suggesting participation of a DNA polymerase with some degree of template independence during the repair of DNA breaks initiated by activation-induced cytidine deaminase. Other mutations were consistent with some in vitro activities of mammalian translesion DNA polymerase η: tandem base substitutions, strand slippage, and small insertions/deletions. The nature of substitution patterns shows that DNA lesions at shark Ig genes recruit DNA repair factors with a species-specific repertoire of activities. We speculate that the tandem mutations are introduced by direct sequential misinsertions and that, in shark B cells, the mispairs tend to be extended rather than proofread. Despite extensive changes undergone by some mutants, the physical range of mutational activity remained restricted to VDJ and within the first 2-kb portion of the 6.8-kb J-C intron, perhaps a self-regulating aspect of activation-induced cytidine deaminase action that is conserved in evolution.

Multiple genetic switches spontaneously modulating bacterial mutability

BackgroundAll life forms need both high genetic stability to survive as species and a degree of mutability to evolve for adaptation, but little is known about how the organisms balance the two seemingly conflicting aspects of life: genetic stability and mutability. The DNA mismatch repair (MMR) system is essential for maintaining genetic stability and defects in MMR lead to high mutability. Evolution is driven by genetic novelty, such as point mutation and lateral gene transfer, both of which require genetic mutability. However, normally a functional MMR system would strongly inhibit such genomic changes. Our previous work indicated that MMR gene allele conversion between functional and non-functional states through copy number changes of small tandem repeats could occur spontaneously via slipped-strand mis-pairing during DNA replication and therefore may play a role of genetic switches to modulate the bacterial mutability at the population level. The open question was: when the conversion from functional to defective MMR is prohibited, will bacteria still be able to evolve by accepting laterally transferred DNA or accumulating mutations?ResultsTo prohibit allele conversion, we "locked" the MMR genes through nucleotide replacements. We then scored changes in bacterial mutability and found that Salmonella strains with MMR locked at the functional state had significantly decreased mutability. To determine the generalizability of this kind of mutability 'switching' among a wider range of bacteria, we examined the distribution of tandem repeats within MMR genes in over 100 bacterial species and found that multiple genetic switches might exist in these bacteria and may spontaneously modulate bacterial mutability during evolution.ConclusionsMMR allele conversion through repeats-mediated slipped-strand mis-pairing may function as a spontaneous mechanism to switch between high genetic stability and mutability during bacterial evolution.

Variable Numbers of Tandem Repeats in Plasmodium falciparum Genes

Genome variation studies in Plasmodium falciparum have focused on SNPs and, more recently, large-scale copy number polymorphisms and ectopic rearrangements. Here, we examine another source of variation: variable number tandem repeats (VNTRs). Interspersed low complexity features, including the well-studied P. falciparum microsatellite sequences, are commonly classified as VNTRs; however, this study is focused on longer coding VNTR polymorphisms, a small class of copy number variations. Selection against frameshift mutation is a main constraint on tandem repeats (TRs) in coding regions, while limited propagation of TRs longer than 975nt total length is a minor restriction in coding regions. Comparative analysis of three P. falciparum genomes reveals that more than 9% of all P. falciparum ORFs harbor VNTRs, much more than has been reported for any other species. Moreover, genotyping of VNTR loci in a drug-selected line, progeny of a genetic cross, and 334 field isolates demonstrates broad variability in these sequences. Functional enrichment analysis of ORFs harboring VNTRs identifies stress and DNA damage responses along with chromatin modification activities, suggesting an influence on genome mutability and functional variation. Analysis of the repeat units and their flanking regions in both P. falciparum and Plasmodium reichenowi sequences implicates a replication slippage mechanism in the generation of TRs from an initially unrepeated sequence. VNTRs can contribute to rapid adaptation by localized sequence duplication. They also can confound SNP-typing microarrays or mapping short-sequence reads and therefore must be accounted for in such analyses.

Mechanisms behind variation in the Clostridium difficile 16S–23S rRNA intergenic spacer region

Clostridium difficile infection is an increasing problem in hospitals worldwide, mainly due to the recent emergence of a hypervirulent C. difficile strain. C. difficile PCR ribotyping, based on size variation of the 16S–23S rRNA intergenic spacer region (16S–23S ISR), is widely used in Europe for molecular epidemiological investigation. The mechanism underlying the 16S–23S ISR size variations in the genome of C. difficile is currently not completely understood. To elucidate this mechanism, isolates of six different PCR ribotypes were analysed by cloning and sequencing the 16S–23S ISR. A direct repeat, IB, of 9 bp was detected up to five times in the 16S–23S ISR in all 47 clones investigated. Thirty-five clones displayed differences either by ribotype or by nucleotide sequence. The sequences of the 16S–23S ISR of C. difficile showed a uniformly organized structure, composed of a tRNAAla gene and spacers of 33 and 53 bp separated by the 9 bp direct repeat IB. The results of the study support the hypothesis that this composition is responsible for the length variations seen in the 16S–23S ISR, and indicate that these length variations result from slipped-strand mispairing and intra- and possibly interchromosomal homologous recombination.

Formation of Complex and Unstable Chromosomal Translocations in Yeast

Genome instability, associated with chromosome breakage syndromes and most human cancers, is still poorly understood. In the yeast Saccharomyces cerevisiae, numerous genes with roles in the preservation of genome integrity have been identified. DNA-damage-checkpoint-deficient yeast cells that lack Sgs1, a RecQ-like DNA helicase related to the human Bloom's-syndrome-associated helicase BLM, show an increased rate of genome instability, and we have previously shown that they accumulate recurring chromosomal translocations between three similar genes, CAN1, LYP1 and ALP1. Here, the chromosomal location, copy number and sequence similarity of the translocation targets ALP1 and LYP1 were altered to gain insight into the formation of complex translocations. Among 844 clones with chromosomal rearrangements, 93 with various types of simple and complex translocations involving CAN1, LYP1 and ALP1 were identified. Breakpoint sequencing and mapping showed that the formation of complex translocation types is strictly dependent on the location of the initiating DNA break and revealed that complex translocations arise via a combination of interchromosomal translocation and template-switching, as well as from unstable dicentric intermediates. Template-switching occurred between sequences on the same chromosome, but was inhibited if the genes were transferred to different chromosomes. Unstable dicentric translocations continuously gave rise to clones with multiple translocations in various combinations, reminiscent of intratumor heterogeneity in human cancers. Base substitutions and evidence of DNA slippage near rearrangement breakpoints revealed that translocation formation can be accompanied by point mutations, and their presence in different translocation types within the same clone provides evidence that some of the different translocation types are derived from each other rather than being formed de novo. These findings provide insight into eukaryotic genome instability, especially the formation of translocations and the sources of intraclonal heterogeneity, both of which are often associated with human cancers.

Repetitive sequences in the lamprey mitochondrial DNA control region and speciation of Lethenteron

The sequence of the mitochondrial DNA control region was examined in four species of lamprey in the genus Lethenteron. The 3′ half of the control region contains highly variable repeat sequences, showing variation in both copy number and nucleotide sequence, even within local populations. Detailed analyses of the sequences of the repeats allowed us to deduce that slipped-strand mispairing during DNA replication, accompanied by a high rate of substitutions and indels, was primarily responsible for the variation in the repeats. We also found that some cases might be better explained by gene conversion, due to intermolecular recombination. Based on the observed variable nature of the mitochondrial control region, we searched for molecular markers in mitochondrial DNA, because there are few fixed genetic markers for distinguishing between Lethenteron japonicum and Lethenteron kessleri. However, we found no reliable markers in the control region. No fixed substitution was observed in intron sequences of the nuclear gene SoxD. Thus, these two species likely diverged quite recently and may possess only a limited number of fixed genetic loci.

TRStalker: an efficient heuristic for finding fuzzy tandem repeats

Motivation: Genomes in higher eukaryotic organisms contain a substantial amount of repeated sequences. Tandem Repeats (TRs) constitute a large class of repetitive sequences that are originated via phenomena such as replication slippage and are characterized by close spatial contiguity. They play an important role in several molecular regulatory mechanisms, and also in several diseases (e.g. in the group of trinucleotide repeat disorders). While for TRs with a low or medium level of divergence the current methods are rather effective, the problem of detecting TRs with higher divergence (fuzzy TRs) is still open. The detection of fuzzy TRs is propaedeutic to enriching our view of their role in regulatory mechanisms and diseases. Fuzzy TRs are also important as tools to shed light on the evolutionary history of the genome, where higher divergence correlates with more remote duplication events.Results: We have developed an algorithm (christened TRStalker) with the aim of detecting efficiently TRs that are hard to detect because of their inherent fuzziness, due to high levels of base substitutions, insertions and deletions. To attain this goal, we developed heuristics to solve a Steiner version of the problem for which the fuzziness is measured with respect to a motif string not necessarily present in the input string. This problem is akin to the ‘generalized median string’ that is known to be an NP-hard problem. Experiments with both synthetic and biological sequences demonstrate that our method performs better than current state of the art for fuzzy TRs and that the fuzzy TRs of the type we detect are indeed present in important biological sequences.Availability: TRStalker will be integrated in the web-based TRs Discovery Service (TReaDS) at bioalgo.iit.cnr.it.Contact: marco.pellegrini@iit.cnr.itSupplementary information: Supplementary data are available at Bioinformatics online.

Stopping the stutter: Improvements in sequence quality from regions with mononucleotide repeats can increase the usefulness of non–coding regions for DNA barcoding

DNA barcoding is now well established in animals based on sequences from a 648 base pair (bp) portion of the mito chondrial coding gene cytochrome oxidase 1 (COl) (Hebert & al., 2003). In contrast, it has proved more difficult to identify a suitable DNA barcoding locus (or loci) for land plants (reviewed by Hollingsworth, 2008). The generally low substitution rate of plant mitochondrial DNA (e.g., Fazekas & al., 2008) has led to investigations into the relative performance and information content of different loci from the plastid genome as alternative DNA barcodes for plants (e.g., Chase & al., 2007; Kress & Er ickson, 2007; Fazekas & al., 2008; Lahaye & al., 2008; CBOL Plant Working Group, 2009; Ford & al., 2009; Hollingsworth & al, 2009). At the third International Barcode of Life Conference in Mexico City (November 2009), the Consortium for the Barcode of Life (CBOL) announced that the standard core DNA bar code for land plants would consist of a two locus combination comprising portions of the protein-coding plastid genes rbcL and matK, to be supplemented with additional loci as required. In making this recommendation, two potential problems were recognized. Firstly, successful amplification and sequencing of the matK barcoding region can be difficult in some taxa with existing primers, and further primer and protocol development is required for this locus. Secondly, the rbcL+matK barcode will not lead to 100% species discrimination in many plant groups, and additional loci beyond this core-barcode will be needed to increase levels of species discrimination in these cases. In recognition of these problems, an 18-month review period on the performance of the rbcL + matK barcode has been established (completion due in mid-2011). During this review period, the executive committee of CBOL recommended that the plant barcoding community continue to collect data from other strongly performing candidate barcoding loci such as the non-coding plastid intergenic spacer trnH-psbA and the internal transcribed spacers (ITS) of nuclear ribosomal DNA. One concern that has been raised regarding the use of non coding plastid regions such as trnH-psbA as DNA barcodes is the presence of microsatellite repeats which can make it dif ficult to obtain bi-directional sequences in some samples (Faze kas & al, 2008; CBOL Plant Working Group, 2009; Devey & al, 2009). Verification of the sequenced strand through bidi rectional sequencing is desirable for the maintenance of data quality standards, as recommended by the CBOL Database Working Group for sequences destined to receive annotation with the reserved keyword 'Barcode' by the INSDC (DDBJ, EMBL & GenBank). However, PCR amplicons derived from regions containing microsatellites can produce poor qual ity sequence chromatograms. Slippage of the polymerase at microsatellite regions during PCR results in a 'stutter' effect observed in the chromatogram, with decreasing sequence qual ity associated with increasing repeat length. The reduction in sequence quality from regions with mononucleotide runs with less than ten repeats is generally moderate and ambigu ous base calls by the software are usually few, requiring little editing by hand. However, as the repeat number increases, the number of ambiguous bases increases disproportionately. This results in a longer amount of time required for editing (with a corresponding reduction in confidence of the true sequence) to the point where sequence data cannot be used at all past the repeat. The net effect is often a contig with overlapping data only at the repeat, and a shortened read length due to missing data at the ends (Fig. 1). In more extreme cases, where mul tiple mononucleotide repeats occur within a given amplicon, disruption of forward and reverse sequencing reads at differ ent mononucleotide runs can lead to only partial sequences of the region with no overlap, thus preventing the construction of a sequence contig. The problem caused by mononucleotide repeats in non-coding regions was noted by the CBOL Plant Working Group (2009) when evaluating the attributes of differ ent barcoding loci for plants, and this contributed towards the selection of an entirely coding core plant barcode. Fixing the 'mononucleotide repeat problem' was considered less tractable than the challenge of improving primer universality for matK. Recently, however, an evaluation of PCR methods and DNA polymerases, focusing on the reduction of the stutter ef fects resulting from mononucleotide repeats, has demonstrated that the use of particular polymerases can reduce the effect of slipped strand mispairing in PCR (Fazekas & al., 2010). This recent study tested the performance of different PCR profiles, reaction chemistries and polymerases on sequences from the trnH-psbA spacer from 25 plant samples. These samples were specifically selected because they contain mononucleotide re peats which had previously resulted in ambiguous base calls and low-quality sequence traces with conventional PCR and

Simple Sequence Repeats and Genome Plasticity inStreptococcus agalactiae

Simple sequence repeats (SSRs) and their role in phase variation have been extensively studied in Gram-negative organisms, where they have been associated with antigenic variation and other adaptation strategies. In this study, we apply comparative genomics in order to find evidence of slipped-strand mispairing in the human Gram-positive pathogen Streptococcus agalactiae. In two consecutive screenings, 2,233 (650 + 1,583) SSRs were identified in our reference genome 2603V/R, and these loci were examined in seven other S. agalactiae genomes. A total of 56 SSR loci were found to exhibit variation, where gain or loss of repeat units was observed in at least one other genome, resulting in aberrant genotypes. Homopolymeric adenine tracts predominated among the repeats that varied. Positional analysis revealed that long polyadenine tracts were overrepresented in the 5' ends of open reading frames (ORFs) and underrepresented in the 3' ends. Repeat clustering in ORFs was also examined, and the highest degree of clustering was observed for a capsule biosynthesis gene and a pilus sortase. A statistical analysis of observed over expected ratios suggested a selective pressure against long homopolymeric tracts. Altered phenotypes were verified for three genes encoding surface-attached proteins, in which frameshifts or fusions led to truncation of proteins and/or affected surface localization through loss or gain of the cell wall sorting signal. The data suggest that SSRs contributes to genome plasticity in S. agalactiae but that the bet-hedging strategy is different from Gram-negative organisms.