Frequent Insertions Research Articles

Yeast EndoG prevents genome instability by degrading extranuclear DNA species

In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1–3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA. Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA ( >10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45–200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability.

Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors.

CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.

What Does It Meme? English–Spanish Codeswitching and Enregisterment in Virtual Social Space

This project investigates English–Spanish codeswitching in internet memes posted to the Facebook page, We are mitú (mitú), and analyzes how lexical insertions and quotatives contribute to the enregisterment of linguistic patterns and the construction of collective identity among U.S. Latinx millennials in virtual social spaces. Data include instances of lexical insertion (n = 280) and quotative mixed codes (n = 114) drawn from a collected corpus of 765 image–text memes. The most frequent lexical insertions included food items (e.g., elote and pozole), kinship terms (e.g., abuelita and tía), and culturally specific artifacts or practices (e.g., quinceañera and lotería), which reflect biculturalism and rely on a shared set of references for the construction of a group identity. Additionally, the quotatives in the data construct Spanish-speaking characterological figures that enregister a particular brand of U.S. Latinx millennial identity that includes being bilingual, having Spanish-speaking parents, and having strong ties to Latinx culture. Overall, this work highlights not only internet memes as a vehicle for enregisterment, but also, and more importantly, how the language resources employed within them work to enregister linguistic and cultural norms of U.S. Latinx millennials, and thereby, play a role in identity construction in virtual social spaces.

Genome-wide characterization of long terminal repeat retrotransposons provides insights into trait evolution of four cucurbit species.

Cucurbits are a diverse plant family that includes economically important crops, such as cucumber, watermelon, melon, and pumpkin. Knowledge of the roles that long terminal repeat retrotransposons (LTR-RTs) have played in diversification of cucurbit species is limited; to add to understanding of the roles of LTR-RTs, we assessed their distributions in four cucurbit species. We identified 381, 578, 1086, and 623 intact LTR-RTs in cucumber (Cucumis sativus L. var. sativus cv. Chinese Long), watermelon (Citrullus lanatus subsp. vulgaris cv. 97103), melon (Cucumis melo cv. DHL92), and Cucurbita (Cucurbita moschata var. Rifu), respectively. Among these LTR-RTs, the Ale clade of the Copia superfamily was the most abundant in all the four cucurbit species. Insertion time and copy number analysis revealed that an LTR-RT burst occurred approximately 2 million years ago in cucumber, watermelon, melon, and Cucurbita, and may have contributed to their genome size variation. Phylogenetic and nucleotide polymorphism analyses suggested that most LTR-RTs were formed after species diversification. Analysis of gene insertions by LTR-RTs revealed that the most frequent insertions were of Ale and Tekay and that genes related to dietary fiber synthesis were the most commonly affected by LTR-RTs in Cucurbita. These results increase our understanding of LTR-RTs and their roles in genome evolution and trait characterization in cucurbits.

Mucoadhesive Polymers and Their Applications in Drug Delivery Systems for the Treatment of Bladder Cancer

Bladder cancer (BC) is the tenth most common type of cancer worldwide, affecting up to four times more men than women. Depending on the stage of the tumor, different therapy protocols are applied. Non-muscle-invasive cancer englobes around 70% of the cases and is usually treated using the transurethral resection of bladder tumor (TURBIT) followed by the instillation of chemotherapy or immunotherapy. However, due to bladder anatomy and physiology, current intravesical therapies present limitations concerning permeation and time of residence. Furthermore, they require several frequent catheter insertions with a reduced interval between doses, which is highly demotivating for the patient. This scenario has encouraged several pieces of research focusing on the development of drug delivery systems (DDS) to improve drug time residence, permeation capacity, and target release. In this review, the current situation of BC is described concerning the disease and available treatments, followed by a report on the main DDS developed in the past few years, focusing on those based on mucoadhesive polymers as a strategy. A brief review of methods to evaluate mucoadhesion properties is also presented; lastly, different polymers suitable for this application are discussed.

Comparative genomic analysis of clinical Candida glabrata isolates identifies multiple polymorphic loci that can improve existing multilocus sequence typing strategy.

Candida glabrata is the second leading cause of candidemia in many countries and is one of the most concerning yeast species of nosocomial importance due to its increasing rate of antifungal drug resistance and emerging multidrug-resistant isolates. Application of multilocus sequence typing (MLST) to clinical C. glabrata isolates revealed an association of certain sequence types (STs) with drug resistance and mortality. The current C. glabrata MLST scheme is based on single nucleotide polymorphisms (SNPs) at six loci and is therefore relatively laborious and costly. Furthermore, only a few high-quality C. glabrata reference genomes are available, limiting rapid analysis of clinical isolates by whole genome sequencing. In this study we provide long-read based assemblies for seven additional clinical strains belonging to three different STs and use this information to simplify the C. glabrata MLST scheme. Specifically, a comparison of these genomes identified highly polymorphic loci (HPL) defined by frequent insertions and deletions (indels), two of which proved to be highly resolutive for ST. When challenged with 53 additional isolates, a combination of TRP1 (a component of the current MLST scheme) with either of the two HPL fully recapitulated ST identification. Therefore, our comparative genomic analysis identified a new typing approach combining SNPs and indels and based on only two loci, thus significantly simplifying ST identification in C. glabrata. Because typing tools are instrumental in addressing numerous clinical and biological questions, our new MLST scheme can be used for high throughput typing of C. glabrata in clinical and research settings.

I/O efficient k-truss community search in massive graphs

Community detection that discovers all densely connected communities in a network has been studied a lot. In this paper, we study online community search for query-dependent communities, which is a different but practically useful task. Given a query vertex in a graph, the problem is to find meaningful communities that the vertex belongs to in an online manner. We propose a community model based on the k-truss concept, which brings nice structural and computational properties. We design a compact and elegant index structure which supports the efficient search of k-truss communities with a linear cost with respect to the community size. We also investigate the k-truss community search problem in a dynamic graph setting with frequent insertions and deletions of graph vertices and edges. In addition, to support k-truss community search over massive graphs which cannot entirely fit in main memory, we propose I/O-efficient algorithms for query processing under the semi-external model. Extensive experiments on massive real-world networks demonstrate the effectiveness of our k-truss community model, the efficiency, and the scalability of our in-memory and semi-external community search algorithms.

Retroelement Insertion in a CRISPR/Cas9 Editing Site in the Early Embryo Intensifies Genetic Mosaicism.

Continued CRISPR/Cas9-mediated editing activity that allows differential and asynchronous modification of alleles in successive cell generations expands allelic complexity. To understand the earliest events during CRISPR/Cas9 editing and the allelic selection among the progeny of subsequent cell divisions, we inspected in detail the genotypes of 4- and 8-cell embryos and embryonic stem cells (ESCs) after microinjection of a CRISPR toolkit into the zygotes. We found a higher editing frequency in 8-cell embryos than in 4-cell embryos, indicating that the CRISPR/Cas9 activity persisted through the 8-cell stage. Analysis of a CRISPR/Cas9 transgenic founder mouse revealed that four different alleles were present in its organs in different combinations and that its germline included three different mutant alleles, as shown by the genotypes of the pups. The indel depth, which measured the extent of indels at the sequence level within single embryos, decreased significantly as the embryos advanced to form ESCs, suggesting that exclusion of fatal indels occurred in the subsequent cell generations. Interestingly, we discovered that the CRISPR sites frequently contained introduced retroelement sequences and that this occurred preferentially with certain classes of retroelements. Therefore, in addition to CRISPR/Cas9's innate mechanism of separate, differential enzymatic modifications of alleles, the frequent retroelement insertions that occur in early mouse embryos during CRISPR/Cas9 editing further expand the allelic diversity and mosaicism in the resulting transgenic founders.

CN34 - Introduction of a nurse-led Totally Implantable Vascular Access Device (TIVAD) service for patients with metastatic cancer

BackgroundPatients with metastatic cancer frequently require Peripherally Inserted Central Catheters (PICCs) to deliver chemotherapy in the ambulatory setting. PICCs have a higher incidence of Venous Thrombo-Embolism (VTE), infections, need to be removed when patients have a treatment break and also require weekly flushes. They also interfere with daily activities such as bathing and can preclude patients from swimming or going on long holidays. At the time, TIVAD’s were not available in our trust. To reduce line complications and improve quality of life I was keen to develop a service at my hospital for patients with metastatic cancer. The process took 2 years to develop from initial idea to inserting the first TIVAD, however I am now pleased to report that I have gained competency, inserted over 30 TIVADs in the first 3 months and am now training up another nurse. MethodsI was keen to explore the potential to introduce a nurse-led TIVAD service that wouldn’t need any form of x-ray guidance using ECG and Ultrasound technology. I contacted a vascular access company who were able to supply 1:1 clinical training until competency was achieved. I also contacted a local charity who agreed to raise money for the ultrasound machine; we also used legacies donated by 2 patient’s families who had left donations to be spent on developing the service as they had felt their quality of life had been impacted upon by frequent PICC insertions. ResultsA total of 30 patients have had TIVADs inserted and we are now running independent nurse-led lists every week to ensure that all patients with metastatic cancer can have a TIVAD inserted. Plans are afoot to formally audit the service and patients receiving treatment for metastatic cancer have already been able to swim and benefit from not having to attend for weekly flushes. There has been 1 VTE and no infections in the TIVAD group, compared to the previous 30 PICC insertions where there were 5 episodes of VTE and 3line infections over the same period of time. ConclusionsThe introduction of a nurse-led TIVAD service is already delivering significant improvements for the quality of life of patients with metastatic cancer. Patients are no longer having multiple PICCs and we have already seen a reduction in VTE and infections. Legal entity responsible for the studyThe author. FundingHas not received any funding. DisclosureThe author has declared no conflicts of interest.

Transposable elements drive rapid phenotypic variation in Capsella rubella

Rapid phenotypic changes in traits of adaptive significance are crucial for organisms to thrive in changing environments. How such phenotypic variation is achieved rapidly, despite limited genetic variation in species that experience a genetic bottleneck is unknown. Capsella rubella, an annual and inbreeding forb (Brassicaceae), is a great system for studying this basic question. Its distribution is wider than those of its congeneric species, despite an extreme genetic bottleneck event that severely diminished its genetic variation. Here, we demonstrate that transposable elements (TEs) are an important source of genetic variation that could account for its high phenotypic diversity. TEs are (i) highly enriched in C. rubella compared with its outcrossing sister species Capsella grandiflora, and (ii) 4.2% of polymorphic TEs in C. rubella are associated with variation in the expression levels of their adjacent genes. Furthermore, we show that frequent TE insertions at FLOWERING LOCUS C (FLC) in natural populations of C. rubella could explain 12.5% of the natural variation in flowering time, a key life history trait correlated with fitness and adaptation. In particular, we show that a recent TE insertion at the 3' UTR of FLC affects mRNA stability, which results in reducing its steady-state expression levels, to promote the onset of flowering. Our results highlight that TE insertions can drive rapid phenotypic variation, which could potentially help with adaptation to changing environments in a species with limited standing genetic variation.

Dna2 nuclease deficiency results in large and complex DNA insertions at chromosomal breaks.

Insertions of mobile elements1-4, mitochondrial DNA5 and fragments of nuclear chromosomes6 at DNA double strand breaks (DSBs) threaten genome integrity and are common in cancer7-9. Insertions of chromosome fragments at V(D)J loci can stimulate antibody diversification10. The origin of insertions of chromosomal fragments and the mechanisms that prevent such insertions remain unknown. Here we found the first mutant, lacking evolutionarily conserved Dna2 nuclease, that shows frequent insertions of ~0.1-1.5 kb long sequences into DSBs with many events carrying multiple DNA fragments joined together. Sequencing of ~500 DNA inserts revealed that they originate from Ty retrotransposons (~8%), rDNA (~15%) and from throughout the genome with preference for fragile regions such as origins of replication, R-loops, centromeres, telomeres or replication fork barriers. Inserted fragments are not lost from their original loci and therefore represent duplications. These duplications depend on nonhomologous end-joining (NHEJ) and Pol4. We propose a model in which alternative processing of DNA structures arising in Dna2-deficient cells can result in the release of DNA fragments and their capture at DSBs.Similar DNA insertions at DSBs are expected in any cells with linear extrachromosomal DNA fragments.

Order of removal of conventional and nonconventional introns from nuclear transcripts of Euglena gracilis.

Nuclear genes of euglenids and marine diplonemids harbor atypical, nonconventional introns which are not observed in the genomes of other eukaryotes. Nonconventional introns do not have the conserved borders characteristic for spliceosomal introns or the sequence complementary to U1 snRNA at the 5' end. They form a stable secondary structure bringing together both exon/intron junctions, nevertheless, this conformation does not resemble the form of self-splicing or tRNA introns. In the genes studied so far, frequent nonconventional introns insertions at new positions have been observed, whereas conventional introns have been either found at the conserved positions, or simply lost. In this work, we examined the order of intron removal from Euglena gracilis transcripts of the tubA and gapC genes, which contain two types of introns: nonconventional and spliceosomal. The relative order of intron excision was compared for pairs of introns belonging to different types. Furthermore, intermediate products of splicing were analyzed using the PacBio Next Generation Sequencing system. The analysis led to the main conclusion that nonconventional introns are removed in a rapid way but later than spliceosomal introns. Moreover, the observed accumulation of transcripts with conventional introns removed and nonconventional present may suggest the existence of a time gap between the two types of splicing.

Immune signatures correlate with L1 retrotransposition in gastrointestinal cancers.

Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons are normally suppressed in somatic tissues mainly due to DNA methylation and antiviral defense. However, the mechanism to suppress L1s may be disrupted in cancers, thus allowing L1s to act as insertional mutagens and cause genomic rearrangement and instability. Whereas the frequency of somatic L1 insertions varies greatly among individual tumors, much remains to be learned about underlying genetic, cellular, or environmental factors. Here, we report multiple correlates of L1 activity in stomach, colorectal, and esophageal tumors through an integrative analysis of cancer whole-genome and matched RNA-sequencing profiles. Clinical indicators of tumor progression, such as tumor grade and patient age, showed positive association. A potential L1 expression suppressor, TP53, was mutated in tumors with frequent L1 insertions. We characterized the effects of somatic L1 insertions on mRNA splicing and expression, and demonstrated an increased risk of gene disruption in retrotransposition-prone cancers. In particular, we found that a cancer-specific L1 insertion in an exon of MOV10, a key L1 suppressor, caused exon skipping and decreased expression of the affected allele due to nonsense-mediated decay in a tumor with a high L1 insertion load. Importantly, tumors with high immune activity, for example, those associated with Epstein–Barr virus infection or microsatellite instability, tended to carry a low number of L1 insertions in genomes with high expression levels of L1 suppressors such as APOBEC3s and SAMHD1. Our results indicate that cancer immunity may contribute to genome stability by suppressing L1 retrotransposition in gastrointestinal cancers.

Abstract 5755: Cancer immunity contributes to genome stability by suppressing L1 retrotransposition in gastrointestinal cancers

Abstract Long interspersed nuclear element-1 (L1) retrotransposons are normally suppressed in somatic tissues, mainly by DNA methylation and antiviral defense. However, L1s can be de-suppressed in cancers to act as insertional mutagens and cause genomic instability by creating DNA double-strand breaks and chromosomal rearrangements. Whereas the frequency of somatic L1 insertions varies greatly among individual tumors, much remains to be learned about underlying genetic, cellular, or environmental factors. Here, our pan-gastrointestinal cancer genome analyses for stomach, colorectal, and esophageal tumors identified multiple correlates of L1 activity. Clinical indicators of tumor progression, such as tumor grade and patient age, showed positive association. Potential L1 expression suppressors such as TP53 and DNMT1, a DNA methyltransferase, were inactivated in tumors with frequent L1 insertions. Importantly, tumors with high immune activity, for example, due to viral infection or high tumor-antigen load, tended to carry a low number of L1 insertions in their genomes with high expression levels of L1 suppressors such as APOBEC3s and SAMHD1. Our analysis of the transcriptional effects of intragenic retrotransposon insertions demonstrated an increased risk of gene disruption in retrotransposition-prone cancers. In particular, we found a splicing-disrupting L1 insertion in an exon of MOV10, a key L1 suppressor, which caused exon skipping with evidence of nonsense-mediated decay in a tumor with a high L1 insertion load. Our results indicate that cancer immunity may contribute to genome stability by suppressing L1 retrotransposition, particularly in gastrointestinal cancers. Citation Format: Hyunchul Jung, Jung Kyoon Choi, Eunjung Alice Lee. Cancer immunity contributes to genome stability by suppressing L1 retrotransposition in gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5755.

A Flow Procedure for Linearization of Genome Sequence Graphs.

Efforts to incorporate human genetic variation into the reference human genome have converged on the idea of a graph representation of genetic variation within a species, a genome sequence graph. A sequence graph represents a set of individual haploid reference genomes as paths in a single graph. When that set of reference genomes is sufficiently diverse, the sequence graph implicitly contains all frequent human genetic variations, including translocations, inversions, deletions, and insertions. In representing a set of genomes as a sequence graph, one encounters certain challenges. One of the most important is the problem of graph linearization, essential both for efficiency of storage and access, and for natural graph visualization and compatibility with other tools. The goal of graph linearization is to order nodes of the graph in such a way that operations such as access, traversal, and visualization are as efficient and effective as possible. A new algorithm for the linearization of sequence graphs, called the flow procedure (FP), is proposed in this article. Comparative experimental evaluation of the FP against other algorithms shows that it outperforms its rivals in the metrics most relevant to sequence graphs.

Twist in the Tail: Escape from HIV Neutralising Antibodies at a Single Site Confers Broad Susceptibility to Others

An efficacious vaccine for HIV remains elusive. Numerous groups have isolated antibodies from HIV infected individuals that can bind and neutralise antigenically diverse HIV strains, so-called broadly neutralising antibodies (bNAbs) (Wibmer et al., 2015). HIV bNAbs target several conserved regions on the viral envelope (Env; a heterotrimer composed of gp120 and gp41 subunits) including the CD4 binding site, peptidoglycan associated with either the V1/V2 or the V3 loops, and the gp120-gp41 interface or the membrane proximal external region (MPER) of gp41. Possessing broad and potent antiviral activity, bNAbs may have utility in HIV immunotherapy, and are also an attractive template to guide the rational design of vaccines to generate analogous humoral immunity. However, bNAbs arise in only a minority of infected individuals and have been associated with extended periods of viremic infection and more recently, perturbations in the follicular and regulatory CD4 T cell compartment (Moody et al., 2016). Analysis of isolated bNAb lineages reveals genetic and structural features that likely contribute to their scarcity, including very high rates of somatic mutation, restricted germline selection, frequent genetic insertions and deletions, extended CDR-H3 regions and a propensity for poly- or autoreactivity. The complicated immunological contexts that underpin bNAb development are unlikely to be recapitulated by vaccination. Indeed, generating serum antibody responses able to combat neutralisation-resistant viral isolates (so-called tier 2 viruses) has not been consistently demonstrated by immunisation. Clearer insights into what governs neutralisation sensitivity to bNAbs should help speed further development of bNAb-based immunisation strategies and HIV immunotherapy.

Abstract 5230: Large scale whole genome sequencing with imputation into GWAS improves our understanding of the genetic architecture of colorectal cancer

Abstract Whole-genome sequencing (WGS) has started a new era in human genetics in which data can be used to more fully understand the role of genetic variation in common complex diseases, including the role of less frequent and rare variants and structural variation. To explore the impact of these variants on colorectal cancer risk we conducted the first large scale WGS study for colorectal cancer (CRC) including 1,961 CRC cases and 981 controls. These WGS data as well as those from the Haplotype Reference Consortium were imputed in 13,104 CRC cases and 15,521 controls with genome-wide association study (GWAS) data that are part of the Colorectal Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). Focusing on rare and less frequent variants, insertions and deletions we observed potentially novel variants: a less frequent variant (MAF = 0.026) on chromosome 5 located in NREP/STARD4-AS1 (p = value 4E-08); and a novel rare multi-allelic variant (MAF = 0.003) on chromosome 9 near KLF9 and TRPM3 (p-value 2E-09; the other allele of this multi-allelic variant had a MAF of 0.0003 and p-value of 0.55). Furthermore, we observed an independent locus close to the known region 8q24 that was located upstream of GSDMC (MAF = 0.16, p-value 5E-08). Within the known region 8q23/EIF3H we identified several low frequency variants with similar MAF (0.0181 to 0.0204) including a 6bp deletion with p-values between 4E-08 and 1E-09 that were independent of the common variant signal in this region. In addition, we identified statistically significant (p<5E-08) deletions, insertions, and an essential splice site within known GWAS loci that present interesting candidates for functional studies. We will follow up these findings in independent samples from the Colorectal Cancer Transdisciplinary Study (CORECT) and CCFR, as well as additional samples currently genotyped in GECCO. In conclusion, next generation sequencing combined with imputation in large GWAS data sets has the potential to identify novel low frequency and rare genetic variants, aid fine-mapping of known CRC susceptibility loci and point to interesting functional candidates. Citation Format: Jeroen Huyghe, Sai Chen, Hyun M. Kang, Tabitha A. Harrison, Sonja I. Berndt, Stephane Bézieau, Hermann Brenner, Graham Casey, Andrew T. Chan, Jenny Chang-Claude, Gallinger J. Steven, Stephen B. Gruber, Andrea Gsur, Michael Hoffmeister, Thomas J. Hudson, Loic Le Marchand, Polly A. Newcomb, John D. Potter, Conghui Qu, Martha L. Slattery, Joshua D. Smith, Emily White, Li Hsu, Goncalo R. Abecasis, Deborah A. Nickerson, Ulrike Peters. Large scale whole genome sequencing with imputation into GWAS improves our understanding of the genetic architecture of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5230.

Structural homology guided alignment of cysteine rich proteins.

BackgroundCysteine rich protein families are notoriously difficult to align due to low sequence identity and frequent insertions and deletions.ResultsHere we present an alignment method that ensures homologous cysteines align by assigning a unique 10 amino acid barcode to those identified as structurally homologous by the DALI webserver. The free inter-cysteine regions of the barcoded sequences can then be aligned using any standard algorithm. Finally the barcodes are replaced with the original columns to yield an alignment which requires the minimum of manual refinement.ConclusionsUsing structural homology information to constrain sequence alignments allows the alignment of highly divergent, repetitive sequences that are poorly dealt with by existing algorithms. Tools are provided to perform this method online using the CysBar web-tool (http://CysBar.science.latrobe.edu.au) and offline (python script available from http://github.com/ts404/CysBar).Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1609-z) contains supplementary material, which is available to authorized users.

DYZ1 arrays show sequence variation between the monozygotic males

BackgroundMonozygotic twins (MZT) are an important resource for genetical studies in the context of normal and diseased genomes. In the present study we used DYZ1, a satellite fraction present in the form of tandem arrays on the long arm of the human Y chromosome, as a tool to uncover sequence variations between the monozygotic males.ResultsWe detected copy number variation, frequent insertions and deletions within the sequences of DYZ1 arrays amongst all the three sets of twins used in the present study. MZT1b showed loss of 35 bp compared to that in 1a, whereas 2a showed loss of 31 bp compared to that in 2b. Similarly, 3b showed 10 bp insertion compared to that in 3a. MZT1a germline DNA showed loss of 5 bp and 1b blood DNA showed loss of 26 bp compared to that of 1a blood and 1b germline DNA, respectively. Of the 69 restriction sites detected in DYZ1 arrays, MboII, BsrI, TspEI and TaqI enzymes showed frequent loss and or gain amongst all the 3 pairs studied. MZT1 pair showed loss/gain of VspI, BsrDI, AgsI, PleI, TspDTI, TspEI, TfiI and TaqI restriction sites in both blood and germline DNA. All the three sets of MZT showed differences in the number of DYZ1 copies. FISH signals reflected somatic mosaicism of the DYZ1 copies across the cells.ConclusionsDYZ1 showed both sequence and copy number variation between the MZT males. Sequence variation was also noticed between germline and blood DNA samples of the same individual as we observed at least in one set of sample. The result suggests that DYZ1 faithfully records all the genetical changes occurring after the twining which may be ascribed to the environmental factors.

A steady-state non-equilibrium molecular dynamics approach for the study of evaporation processes

Two non-equilibrium methods (called bubble method and splitting method, respectively) have been developed and tested to study the steady state evaporation of a droplet surrounded by its vapor, where the evaporation continuously occurs at the vapor-liquid interface while the droplet size remains constant. In the bubble method, gas molecules are continuously reinserted into a free volume (represented by a bubble) located at the centre of mass of the droplet to keep the droplet size constant. In the splitting method, a molecule close to the centre of mass of the droplet is split into two: In this way, the droplet size is also maintained during the evaporation. By additional local thermostats confined to the area of insertion, the effect of frequent insertions on properties such as density and temperature can be limited to the immediate insertion area. Perturbations are not observed in other parts of the droplet. In the end, both the bubble method and the splitting method achieve steady-state droplet evaporation. Although these methods have been developed using an isolated droplet, we anticipate that they will find a wide range of applications in the study of the evaporation of isolated films and droplets or thin films on heated substrates or under confinement. They can in principle also be used to study the steady-state of other physical processes, such as the diffusion or permeation of gas molecules or ions in a pressure gradient or a concentration gradient.