Repetitive DNA Regions Research Articles

Generation of genome-wide SNP markers from minimally invasive sampling in endangered animals and applications in species ecology and conservation.

High-density genotyping methods have revolutionized the field of population and conservation genetics in the past decade. To exploit the technological and analytical advances in the field, access to high-quality genetic material is a key component. However, access to such samples in endangered and rare animals is often challenging or even impossible. Here, we used a minimally invasive sampling method (MIS) in the endangered cave salamander Proteus anguinus, the olm, to generate thousands of genetic markers using ddRADseq for population and conservation genomic analyses. Using tail clips and MIS skin swabs taken from the same individual, we investigated genotyping data properties of the two different sampling types. We found that sufficient DNA can be extracted from swab samples to generate up to 200,000 polymorphic SNPs in divergent Proteus lineages. Swab and tissue samples were highly reproducible exhibiting low SNP genotyping error rates. We found that SNPs were most frequently (~50%) located within genic regions, while the rest mapped to mostly flanking regions of repetitive DNA. The vast majority of DNA recovered from swabbing was host DNA. However, a fraction of DNA recovered from swabs contained additional ecological information on the species, including eDNA from the surrounding environment and bacterial skin fauna. Most exogenous DNA recovered from swabs were bacteria (~80%), followed by vertebrates (~20%). Our results demonstrate that MIS can be used to (i) generate tens of thousands of ddRADseq markers for conservation and population genomic analyses and (ii) inform on the species health status and ecology from exogenous DNA.

Characterization of telomere variant repeats using long reads enables allele-specific telomere length estimation.

Telomeres are regions of repetitive DNA at the ends of linear chromosomes which protect chromosome ends from degradation. Telomere lengths have been extensively studied in the context of aging and disease, though most studies use average telomere lengths which are of limited utility. We present a method for identifying all 92 telomere alleles from long read sequencing data. Individual telomeres are identified using variant repeats proximal to telomere regions, which are unique across alleles. This high-throughput and high-resolution characterization of telomeres could be foundational to future studies investigating the roles of specific telomeres in aging and disease.

First Trials of Genome Analyses in Some Onobrychis Species using Dot-Blot and Genomic in situ Hybridization Techniques

The origin and genome composition of tetraploid cultivated Onobrychis viciifolia (2n = 4x = 28) were analyzed using dot-blot and genomic in situ hybridization (GISH) techniques. Dot-blot hybridization was used to find a genomic affinity between O. viciifolia and 16 diploid Onobrychis species. The hypothesis on the origin of the O. viciifolia was tested using GISH. Dot-blot analyses suggested a genomic affinity between O. viciifolia and four diploid Onobrychis species (O. kachetica, O. supina, O. pallasii, and O. vaginalis). Hybridization signals were observed on O. viciifolia chromosomes when gDNA of O. kachetica, O. supina, O. pallasi, and O. hypargyrea were used as probes. However, the observed chromosomal distribution of hybridization signals did not resemble GISH results. The observed signals colocalized with 35S rDNA or dispearse signals on all chromosomes were observed depending on the probe. Further investigations using more comprehensive and comparative analysis with both coding and repetitive DNA regions may provide a better understanding of the genome composition and evolution of O. viciifolia.

Abstract 3009: ATRX loss blocks the differentiation of mesenchymal stem cells and promotes undifferentiated sarcoma development

Abstract Soft tissue sarcomas (STSs) are a collection of rare tumor types originated from the mesenchymal tissues. Among the over 70 histological subtypes of STS, undifferentiated sarcoma (US), which lacks any discernible morphological features of differentiated tissues, accounts for over 20% of all STSs and is correlated with high tumor grade and poor prognosis. Recent genomic profiling in US has identified p53 and ATRX among the most frequently mutated or deleted genes. ATRX is a SWI/SNF2 type of chromatin remodeling factor which functions in a histone variant H3.3-specific chaperone complex to orchestrates replication-independent nucleosome assembly at repetitive heterochromatic DNA regions including telomeres. Loss-of-function mutations occur frequently in many tumor types and is associated with the development of alternative lengthening of telomere (ALT). However, how loss of ATRX function may contribute to tumorigenesis is largely unknown. Here, we demonstrated that ATRX is required for the terminal differentiation of mouse mesenchymal stem cells (MSCs) into multiple lineages, such as adipocytes and osteocytes. Targeted deletion of Atrx in mouse MSCs, but not terminally differentiated mesenchymal lineage cells, accelerated sarcoma initiation induced by loss of p53 and significantly shortened the overall survival. Atrx-deficient tumors express high level of MSC markers such as CD44 and CD29 and exhibit pathological features highly reminiscent of human high-grade US. Transcriptomic analysis indicates that Atrx deficiency is associated with enhanced proliferation. Together, our data indicates that ATRX deficiency contributes to undifferentiated sarcoma development by inhibiting mesenchymal stem cell differentiation. Citation Format: Yiming Cai, Jun Yao, Dean N. Pan, Yanqiang Jin, James M. You, Wantong Yao, Haoqiang Ying. ATRX loss blocks the differentiation of mesenchymal stem cells and promotes undifferentiated sarcoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3009.

Tigerfish designs oligonucleotide-based in situ hybridization probes targeting intervals of highly repetitive DNA at the scale of genomes

Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate > 100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific probes against such regions presents significant technical challenges. Consequently, many open questions remain about the organization and function of highly repetitive sequences. Here, we introduce Tigerfish, a software tool for the genome-scale design of oligo probes against repetitive DNA intervals. We showcase Tigerfish by designing a panel of 24 interval-specific repeat probes specific to each of the 24 human chromosomes and imaging this panel on metaphase spreads and in interphase nuclei. Tigerfish extends the powerful toolkit of oligo-based FISH to highly repetitive DNA.

Sex and early-life conditions shape telomere dynamics in an ectotherm.

Telomeres, the repetitive DNA regions that protect the ends of chromosomes, and their shortening have been linked to key life history trade-offs among growth, reproduction and lifespan. In contrast to most endotherms, many ectotherms can compensate for telomere shortening throughout life by upregulation of telomerase in somatic tissues. However, during development, marked by rapid growth and an increased sensitivity to extrinsic factors, the upregulation of telomerase may be overwhelmed, resulting in long-term impacts on telomere dynamics. In ectotherms, one extrinsic factor that may play a particularly important role in development is temperature. Here, we investigated the influence of developmental temperature and sex on early-life telomere dynamics in an oviparous ectotherm, Lacerta agilis. While there was no effect of developmental temperature on telomere length at hatching, there were subsequent effects on telomere maintenance capacity, with individuals incubated at warm temperatures exhibiting less telomere maintenance compared with cool-incubated individuals. Telomere dynamics were also sexually dimorphic, with females having longer telomeres and greater telomere maintenance compared with males. We suggest that selection drives this sexual dimorphism in telomere maintenance, in which females maximise their lifetime reproductive success by investing in traits promoting longevity such as maintenance, while males invest in short-term reproductive gains through a polygynous mating behaviour. These early-life effects, therefore, have the potential to mediate life-long changes to life histories.

Comparative genomics analysis of three conserved plasmid families in the Western Hemisphere soft tick-borne relapsing fever borreliae provides insight into variation in genome structure and antigenic variation systems.

Borrelia spirochetes, causative agents of Lyme disease and relapsing fever (RF), have uniquely complex genomes, consisting of a linear chromosome and both circular and linear plasmids. The plasmids harbor genes important for the vector-host life cycle of these tick-borne bacteria. The role of plasmids from Lyme disease causing spirochetes is more refined compared to RF Borrelia because of limited plasmid-resolved genome assemblies for the latter. We recently addressed this limitation and found that three linear plasmid families (F6, F27, and F28) were syntenic across all the RF Borrelia species that we examined. Given this conservation, we further investigated the three plasmid families. The F6 family, also known as the megaplasmid, contained regions of repetitive DNA. The F27 was the smallest, encoding genes with unknown function. The F28 family encoded the putative expression locus for antigenic variation in all species except Borrelia hermsii and Borrelia anserina. Taken together, this work provides a foundation for future investigations to identify essential plasmid-localized genes that drive the vector-host life cycle of RF Borrelia. IMPORTANCE Borrelia spp. spirochetes are arthropod-borne bacteria found globally that infect humans and other vertebrates. RF borreliae are understudied and misdiagnosed pathogens because of the vague clinical presentation of disease and the elusive feeding behavior of argasid ticks. Consequently, genomics resources for RF spirochetes have been limited. Analyses of Borrelia plasmids have been challenging because they are often highly fragmented and unassembled in most available genome assemblies. By utilizing Oxford Nanopore Technologies, we recently generated plasmid-resolved genome assemblies for seven Borrelia spp. found in the Western Hemisphere. This current study is an in-depth investigation into the linear plasmids that were conserved and syntenic across species. We identified differences in genome structure and, importantly, in antigenic variation systems between species. This work is an important step in identifying crucial plasmid-localized genetic elements essential for the life cycle of RF spirochetes.

Comparative chromosome mapping of repetitive DNA in four minnow fishes (Cyprinidae, Cypriniformes)

The present study focused on the repetitive DNA of the chromosome in four minnow fishes from the genera Danio Hamilton, 1822, Devario Heckel, 1843 and Rasbora Bleeker, 1859. Chromosomes were analysed using fluorescence in situ hybridization (FISH) with microsatellite probes including (CA)15, (CAC)10, (CGG)10, (GC)15 and (TA)15 staining. All species retained the diploid chromosome number 2n = 50 in male and female. The microsatellite sequences were mapped in the chromosomes of Danio albolineatus (Blyth, 1860), Devario regina (Fowler, 1934), Rasbora aurotaenia Tirant, 1885 and R. paviana Tirant, 1885. In most cases, the microsatellite was dispersed in the chromosome with conspicuous markings in the telomeric region and the whole genome, which suggests that sequences contribute to chromosome structure and may have played a role in the relationship of this fish group. The comparative genome mapping data presented here provide novel information on the structure and organisation of the repetitive DNA region of the minnow’s genome and contribute to a better understanding of the genomes of these minnows.

Insights on Microsatellite Characteristics, Evolution, and Function From the Social Amoeba Dictyostelium discoideum.

Microsatellites are repetitive sequences commonly found in the genomes of higher organisms. These repetitive sequences are prone to expansion or contraction, and when microsatellite expansion occurs in the regulatory or coding regions of genes this can result in a number of diseases including many neurodegenerative diseases. Unlike in humans and other organisms, the social amoeba Dictyostelium discoideum contains an unusually high number of microsatellites. Intriguingly, many of these microsatellites fall within the coding region of genes, resulting in nearly 10,000 homopolymeric repeat proteins within the Dictyostelium proteome. Surprisingly, among the most common of these repeats are polyglutamine repeats, a type of repeat that causes a class of nine neurodegenerative diseases in humans. In this minireview, we summarize what is currently known about homopolymeric repeats and microsatellites in Dictyostelium discoideum and discuss the potential utility of Dictyostelium for identifying novel mechanisms that utilize and regulate regions of repetitive DNA.

Effect of Physical Activity, Smoking, and Sleep on Telomere Length: A Systematic Review of Observational and Intervention Studies.

Aging is a risk factor for several pathologies, restricting one’s health span, and promoting chronic diseases (e.g., cardiovascular and neurodegenerative diseases), as well as cancer. Telomeres are regions of repetitive DNA located at chromosomal ends. Telomere length has been inversely associated with chronological age and has been considered, for a long time, a good biomarker of aging. Several lifestyle factors have been linked with telomere shortening or maintenance. However, the consistency of results is hampered by some methodological issues, including study design, sample size, measurement approaches, and population characteristics, among others. Therefore, we aimed to systematically review the current literature on the effects of three relevant lifestyle factors on telomere length in human adults: physical activity, smoking, and sleep. We conducted a qualitative systematic review of observational and intervention studies using the Preferred Reporting Item for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The systematic literature search covered articles published in MEDLINE and EMBASE databases (from 2010 to 2020). A total of 1400 studies were identified; 83 were included after quality control. Although fewer sedentary activities, optimal sleep habits, and non- or ex-smoker status have been associated with less telomere shortening, several methodological issues were detected, including the need for more targeted interventions and standardized protocols to better understand how physical activity and sleep can impact telomere length and aging. We discuss the main findings and current limitations to gain more insights into the influence of these lifestyle factors on the healthy aging process.

ATRX proximal protein associations boast roles beyond histone deposition.

The ATRX ATP-dependent chromatin remodelling/helicase protein associates with the DAXX histone chaperone to deposit histone H3.3 over repetitive DNA regions. Because ATRX-protein interactions impart functions, such as histone deposition, we used proximity-dependent biotinylation (BioID) to identify proximal associations for ATRX. The proteomic screen captured known interactors, such as DAXX, NBS1, and PML, but also identified a range of new associating proteins. To gauge the scope of their roles, we examined three novel ATRX-associating proteins that likely differed in function, and for which little data were available. We found CCDC71 to associate with ATRX, but also HP1 and NAP1, suggesting a role in chromatin maintenance. Contrastingly, FAM207A associated with proteins involved in ribosome biosynthesis and localized to the nucleolus. ATRX proximal associations with the SLF2 DNA damage response factor help inhibit telomere exchanges. We further screened for the proteomic changes at telomeres when ATRX, SLF2, or both proteins were deleted. The loss caused important changes in the abundance of chromatin remodelling, DNA replication, and DNA repair factors at telomeres. Interestingly, several of these have previously been implicated in alternative lengthening of telomeres. Altogether, this study expands the repertoire of ATRX-associating proteins and functions.

Structural and functional characterization of the bacterial biofilm activator RemA

Bacillus subtilis can form structurally complex biofilms on solid or liquid surfaces, which requires expression of genes for matrix production. The transcription of these genes is activated by regulatory protein RemA, which binds to poorly conserved, repetitive DNA regions but lacks obvious DNA-binding motifs or domains. Here, we present the structure of the RemA homologue from Geobacillus thermodenitrificans, showing a unique octameric ring with the potential to form a 16-meric superstructure. These results, together with further biochemical and in vivo characterization of B. subtilis RemA, suggests that the protein can wrap DNA around its ring-like structure through a LytTR-related domain.

Thousands of high-quality sequencing samples fail to show meaningful correlation between 5S and 45S ribosomal DNA arrays in humans

The ribosomal RNA genes (rDNA) are tandemly arrayed in most eukaryotes and exhibit vast copy number variation. There is growing interest in integrating this variation into genotype–phenotype associations. Here, we explored a possible association of rDNA copy number variation with autism spectrum disorder and found no difference between probands and unaffected siblings. Because short-read sequencing estimates of rDNA copy number are error prone, we sought to validate our 45S estimates. Previous studies reported tightly correlated, concerted copy number variation between the 45S and 5S arrays, which should enable the validation of 45S copy number estimates with pulsed-field gel-verified 5S copy numbers. Here, we show that the previously reported strong concerted copy number variation may be an artifact of variable data quality in the earlier published 1000 Genomes Project sequences. We failed to detect a meaningful correlation between 45S and 5S copy numbers in thousands of samples from the high-coverage Simons Simplex Collection dataset as well as in the recent high-coverage 1000 Genomes Project sequences. Our findings illustrate the challenge of genotyping repetitive DNA regions accurately and call into question the accuracy of recently published studies of rDNA copy number variation in cancer that relied on diverse publicly available resources for sequence data.

A Traceable Cancer Model: DNA Damage, Fragile Site-SMGs, Mitotic Slippage, 4n-Genome-Reduction to Fitness-Gained, Initiating, 2n First Cells

We have known since 1976 that cancer evolves clonally from one initiated normal human cell, the first cell. Today we see that this fact has been overshadowed from federal funding choice of the mutation theory (MT), which not yet has shown tumorigenesis-initiation in normal human cells. Our suggested, death signaled, stress model from time delayed S-period (replication slowness), causing repair instability from under-replicated lesions in repetitive DNAs, herein has the objective of revealing, significant literature support from a mini-review. We reasoned that early versus late S-period stress would have different outcomes: early the slowness affecting mitotic slippage with diploid re-replication to 4n cells whereas late-S, with milder stress effect, producing diploid cells. In cancer burden, near-half is diploid, but tetraploid solid tumors have the attention. The initial 4n cells were special with orderly genomic reductive division to diploid first cells with measurable fitness-gain from hours-reduced total cell cycle time. Experimental data from Coxsakie-B3 virus infected normal fibroblasts, reiterated 4n cell production from death-signaled recovery-cells with progressive cell-phenotypic changes to polygonal and roundness cell-shapes, indistinguishable from diagnostic/prognostic cancer morphology. The 4n cells showed a self-inflicted 90° turn of the 4n nucleus before division, affecting a perpendicular orientation of the fitness-gained first cells relative to neighboring cells. In an illustrated cell cycle drawing with early and late S-period stress, it became clear that coding genes on borders of repair unstable satellite, repetitive DNA regions, could become mutated. We found these mutations to be tumor SMGs (significantly mutated genes). Evidential material was presented for loss of function genetics driving tumorigenesis to a parasitic lifestyle.

Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability

BackgroundThe U937 cell line is widely employed as a research tool. It has a complex karyotype. A PICALM-MLLT10 fusion gene formed by the recurrent t(10;11) translocation is present, and the myeloid common deleted region at 20q12 has been lost from its near-triploid karyotype. We carried out a detailed investigation of U937 genome reorganisation including the chromosome 20 rearrangements and other complex rearrangements.ResultsSNP array, G-banding and Multicolour FISH identified chromosome segments resulting from unbalanced and balanced rearrangements. The organisation of the abnormal chromosomes containing these segments was then reconstructed with the strategic use of targeted metaphase FISH. This provided more accurate karyotype information for the evolving karyotype. Rearrangements involving the homologues of a chromosome pair could be differentiated in most instances. Centromere capture was demonstrated in an abnormal chromosome containing parts of chromosomes 16 and 20 which were stabilised by joining to a short section of chromosome containing an 11 centromere. This adds to the growing number of examples of centromere capture, which to date have a high incidence in complex karyotypes where the centromeres of the rearranged chromosomes are identified. There were two normal copies of one chromosome 20 homologue, and complex rearrangement of the other homologue including loss of the 20q12 common deleted region. This confirmed the previously reported loss of heterozygosity of this region in U937, and defined the rearrangements giving rise to this loss.ConclusionsCentromere capture, stabilising chromosomes pieced together from multiple segments, may be a common feature of complex karyotypes. However, it has only recently been recognised, as this requires deliberate identification of the centromeres of abnormal chromosomes. The approach presented here is invaluable for studying complex reorganised genomes such as those produced by chromothripsis, and provides a more complete picture than can be obtained by microarray, karyotyping or FISH studies alone. One major advantage of SNP arrays for this process is that the two homologues can usually be distinguished when there is more than one rearrangement of a chromosome pair. Tracking the fate of each homologue and of highly repetitive DNA regions such as centromeres helps build a picture of genome evolution. Centromere- and telomere-containing elements are important to deducing chromosome structure. This study confirms and highlights ongoing evolution in cultured cell lines.

Telomere Length Measurement by Molecular Combing.

Telomeres are repetitive regions of DNA bound by specialized proteins at the termini of linear chromosomes that prevent the natural chromosome ends from being recognized as DNA double strand breaks. Telomeric DNA is gradually eroded with each round of cell division, resulting in the accumulation of critically short or dysfunctional telomeres that eventually trigger cellular senescence. Consequently, telomere length is indicative of the proliferative capacity of a cell. Multiple methods exist to measure telomere length and telomere content, but a simple and reliable technique to accurately measure individual telomere lengths is currently lacking. We have developed the Telomere length Combing Assay (TCA) to measure telomere length on stretched DNA fibers. We used TCA to measure telomere erosion in primary human fibroblasts, and to detect telomere lengthening in response to activation of telomere maintenance pathways. TCA was also used to accurately measure telomere length in healthy individuals, and to identify critically short telomeres in patients with telomere biology disorders. TCA is performed on isolated DNA, negating the need for cycling cells. TCA is amenable to semi-automated image analysis, and can be fully automated using the Genomic Vision molecular combing platform. This not only precludes sampling bias, but also provides the potential for high-throughput applications and clinical development. TCA is a simple and versatile technique to measure the distribution of individual telomere lengths in a cell population, offering improved accuracy, and more detailed biological insight for telomere length measurement applications.

Paternally Inherited P-Element Copy Number Affects the Magnitude of Hybrid Dysgenesis in Drosophila simulans and D.melanogaster.

Transposable elements (TEs) are repetitive regions of DNA that are able to self-replicate and reinsert themselves throughout host genomes. Since the discovery of TEs, a prevalent question has been whether increasing TE copy number has an effect on the fitness of their hosts. P-elements (PEs) in Drosophila are a well-studied TE that has strong phenotypic effects. When a female without PEs (M) is crossed to a male with them (P), the resulting females are often sterile, a phenomenon called hybrid dysgenesis (HD). Here, we used short- and long-read sequencing to infer the number of PEs in the genomes of dozens of isofemale lines from two Drosophila species and measured whether the magnitude of HD was correlated with the number of PEs in the paternal genome. Consistent with previous reports, we find evidence for a positive correlation between the paternal PE copy number and the magnitude of HD in progeny from ♀M × ♂ P crosses for both species. Other crosses are not affected by the number of PE copies. We also find that the correlation between the strength of HD and PE copy number differs between species, which suggests that there are genetic differences that might make some genomes more resilient to the potentially deleterious effects of TEs. Our results suggest that PE copy number interacts with other factors in the genome and the environment to cause HD and that the importance of these interactions is species specific.

Detection of Toxoplasma gondii oocysts in fresh vegetables and berry fruits

BackgroundToxoplasma gondii is the third most important contributor to health burden caused by food-borne illness. Ingestion of tissue cysts from undercooked meat is an important source of horizontal transmission to humans. However, there is an increasing awareness of the consumption of fresh fruit and vegetables, as a possible source for oocyst transmission, since this stage of the parasite can persist and remain infective in soil and water for long time. Herein, we outline findings related with detection of T. gondii oocysts in vegetables and berry fruits, which are usually raw consumed. The procedure includes the estimation of the number of oocysts.MethodsFood samples were collected from local producers and supermarket suppliers. Toxoplasma gondii oocysts were concentrated after washing the samples by applying high resolution water filtration and immunomagnetic separation (method 1623.1: EPA 816-R-12-001-Jan 2012), in order to (i) remove potential Cryptosporidium spp. oocysts and Giardia spp. cysts present in the samples; and (ii) select T. gondii oocysts. Toxoplasma gondii oocyst detection and an estimation of their numbers was performed by conventional PCR and real time qPCR, using specific primers for a 183-bp sequence of the T. gondii repetitive DNA region. All PCR-positive DNA samples were purified and sequenced. Restriction enzyme digestion with EcoRV endonuclease confirmed the presence of the T. gondii DNA fragment. In addition, the presence of the parasite was observed by fluorescent microscopy, taking advantage of the oocysts autofluorescence under UV light.ResultsForty percent of the analysed samples (95% CI: 25.5–56.5%) presented the expected PCR and digested DNA fragments. These fragments were confirmed by sequencing. Microscopic autofluorescence supported the presence of T. gondii-like oocysts. The estimated mean (± SE) oocyst concentration was 23.5 ± 12.1 oocysts/g, with a range of 0.6–179.9 oocysts/g.ConclusionsOur findings provide relevant evidence of contamination of fresh vegetables and berry fruits with T. gondii oocysts.

Expanding applications for nanopore sequencing.

Two new studies report advances for nanopore-based long-read sequencing for characterizing complex human transcriptomes and for analysing human repetitive DNA regions.

Re-sequencing and optical mapping reveals misassemblies and real inversions on Corynebacterium pseudotuberculosis genomes

The number of draft genomes deposited in Genbank from the National Center for Biotechnology Information (NCBI) is higher than the complete ones. Draft genomes are assemblies that contain fragments of misassembled regions (gaps). Such draft genomes present a hindrance to the complete understanding of the biology and evolution of the organism since they lack genomic information. To overcome this problem, strategies to improve the assembly process are developed continuously. Also, the greatest challenge to the assembly progress is the presence of repetitive DNA regions. This article highlights the use of optical mapping, to detect and correct assembly errors in Corynebacterium pseudotuberculosis. We also demonstrate that choosing a reference genome should be done with caution to avoid assembly errors and loss of genetic information.