An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole‐genome sequencing data

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.     

Techniques for integrating ‐omics data

The challenge for -omics research is to tackle the problem of fragmentation of knowledge by integrating several sources of heterogeneous information into a coherent entity. It is widely recognized that successful data integration is one of the keys to improve productivity for stored data. Through proper data integration tools and algorithms, researchers may correlate relationships that enable them to make better and faster decisions. The need for data integration is essential for present ‐omics community, because ‐omics data is currently spread world wide in wide variety of formats. These formats can be integrated and migrated across platforms through different techniques and one of the important techniques often used is XML. XML is used to provide a document markup language that is easier to learn, retrieve, store and transmit. It is semantically richer than HTML. Here, we describe bio warehousing, database federation, controlled vocabularies and highlighting the XML application to store, migrate and validate -omics data.     

New method for global alignment of 2 DNA sequences by the tree data structure

We introduce a new approach to investigate problem of DNA sequence alignment. The method consists of three parts: (i) simple alignment algorithm, (ii) extension algorithm for largest common substring, (iii) graphical simple alignment tree (GSA tree). The approach firstly obtains a graphical representation of scores of DNA sequences by the scoring equation R₀*R−S₀*S−T₀*(a+bk). Then a GSA tree is constructed to facilitate solving the problem for global alignment of 2 DNA sequences. Finally we give several practical examples to illustrate the utility and practicality of the approach.     

The use of singlebeam echo‐sounder depth data to produce demersal fish distribution models that are comparable to models produced using multibeam echo‐sounder depth

Seafloor characteristics can help in the prediction of fish distribution, which is required for fisheries and conservation management. Despite this, only 5%–10% of the world''s seafloor has been mapped at high resolution, as it is a time‐consuming and expensive process. Multibeam echo‐sounders (MBES) can produce high‐resolution bathymetry and a broad swath coverage of the seafloor, but require greater financial and technical resources for operation and data analysis than singlebeam echo‐sounders (SBES). In contrast, SBES provide comparatively limited spatial coverage, as only a single measurement is made from directly under the vessel. Thus, producing a continuous map requires interpolation to fill gaps between transects. This study assesses the performance of demersal fish species distribution models by comparing those derived from interpolated SBES data with full‐coverage MBES distribution models. A Random Forest classifier was used to model the distribution of Abalistes stellatus, Gymnocranius grandoculis, Lagocephalus sceleratus, Loxodon macrorhinus, Pristipomoides multidens, and Pristipomoides typus, with depth and depth derivatives (slope, aspect, standard deviation of depth, terrain ruggedness index, mean curvature, and topographic position index) as explanatory variables. The results indicated that distribution models for A. stellatus, G. grandoculis, L. sceleratus, and L. macrorhinus performed poorly for MBES and SBES data with area under the receiver operator curves (AUC) below 0.7. Consequently, the distribution of these species could not be predicted by seafloor characteristics produced from either echo‐sounder type. Distribution models for P. multidens and P. typus performed well for MBES and the SBES data with an AUC above 0.8. Depth was the most important variable explaining the distribution of P. multidens and P. typus in both MBES and SBES models. While further research is needed, this study shows that in resource‐limited scenarios, SBES can produce comparable results to MBES for use in demersal fish management and conservation.     

A machine learning method for estimating the probability of presence using presence‐background data

Estimating the prevalence or the absolute probability of the presence of a species from presence‐background data has become a controversial topic in species distribution modelling. In this paper, we propose a new method by combining both statistics and machine learning algorithms that helps overcome some of the known existing problems. We have also revisited the popular but highly controversial Lele and Keim (LK) method by evaluating its performance and assessing the RSPF condition it relies on. Simulations show that the LK method with the RSPF assumptions would render fragile estimation/prediction of the desired probabilities. Rather, we propose the local knowledge condition, which relaxes the predetermined population prevalence condition that has so often been used in much of the existing literature. Simulations demonstrate the performance of the new method utilizing the local knowledge assumption to successfully estimate the probability of presence. The local knowledge extends the local certainty or the prototypical presence location assumption, and has significant implications for demonstrating the necessary condition for identifying absolute (rather than relative) probability of presence from presence background without absence data in species distribution modelling.     

Identification of two distinct subfamilies of alpha satellite DNA that are highly specific for human chromosome 15

We report the isolation of two distinct subfamilies of alpha satellite DNA (pTRA-20 and -25) from human chromosome 15. In situ hybridization experiments indicated that both subfamilies are highly specific for this chromosome. Southern analysis of a somatic hybrid cell line carrying human chromosome 15 revealed a likely higher-order genomic band of 2.5 kb for pTRA-20. Similar analysis for pTRA-25 showed multiple higher-order bands of 3.5, 4.5, and 5 kb at moderately high hybridization stringency, but a predominance of the 4.5-kb species at very high stringency. Direct comparison with human genomic DNA confirmed the authenticity of these higher-order structures and demonstrated polymorphic variations using both probes. The origin of the different alphoid subfamilies on chromosome 15 is discussed. These sequences should be useful for the construction of centromere-based genetic linkage maps for human chromosome 15 and, in conjunction with the other alphoid sequences already reported for chromosomes 13, 14, 21, and 22, should allow a concerted analysis of the evolution and the possible etiological role of these DNAs in aberrations commonly seen in these chromosomes.     

ELIXIR‐EXCELERATE: establishing Europe's data infrastructure for the life science research of the future

A new inter‐governmental research infrastructure, ELIXIR, aims to unify bioinformatics resources and life science data across Europe, thereby facilitating their mining and (re‐)use. Subject Categories: Computational Biology, Methods & Resources, S&S: Ethics

Creating knowledge by connecting and analysing large amounts of life science data is transforming our society, allowing us to start addressing major scientific and societal challenges, such as adaptation to climate change or pathogen outbreaks in an interconnected world. Modern biology is dependent on the generation, sharing and integrated analysis of digital data at scale. A deeper understanding of biological systems is now becoming possible thanks to breakthroughs in technologies that study life systematically at different scales, from molecules and single‐cell pathogens to complex animal or plant models and ecosystems as well as across temporal ranges spanning split‐second reactions to multi‐year clinical or agronomic trials, and beyond. The key to analyse and leverage this complex, fragmented and geographically dispersed life science data landscape is to ensure it is easy to find and reuse by researchers. This article comments on ELIXIR, an international organisation that brings together bioinformatics researchers and life science resources across Europe and integrates them into a single federated infrastructure.     

A multi‐data ensemble approach for predicting woodland type distribution: Oak woodland in Britain

Interactions between soil, topography, and climatic site factors can exacerbate and/or alleviate the vulnerability of oak woodland to climate change. Reducing climate‐related impacts on oak woodland habitats and ecosystems through adaptation management requires knowledge of different site interactions in relation to species tolerance. In Britain, the required thematic detail of woodland type is unavailable from digital maps. A species distribution model (SDM) ensemble, using biomod2 algorithms, was used to predict oak woodland. The model was cross‐validated (50%:50% ‐ training:testing) 30 times, with each of 15 random sets of absence data, matching the size of presence data, to maximize environmental variation while maintaining data prevalence. Four biomod2 algorithms provided stable and consistent TSS‐weighted ensemble mean results predicting oak woodland as a probability raster. Biophysical data from the Ecological Site Classification (forest site classification) for Britain were used to characterize oak woodland sites. Several forest datasets were used, each with merits and weaknesses: public forest estate subcompartment database map (PFE map) for oak‐stand locations as a training dataset; the national forest inventory (NFI) “published regional reports” of oak woodland area; and an “NFI map” of indicative forest type broad habitat. Broadleaved woodland polygons of the NFI map were filled with the biomod2 oak woodland probability raster. Ranked pixels were selected up to the published NFI regional area estimate of oak woodland and matched to the elevation distribution of oak woodland stands, from “NFI survey” sample squares. Validation using separate oak woodland data showed that the elevation filter significantly improved the accuracy of predictions from 55% (p = .53) to 83% coincidence success rate (p < .0001). The biomod2 ensemble, with masking and filtering, produced a predicted oak woodland map, from which site characteristics will be used in climate change interaction studies, supporting adaptation management recommendations for forest policy and practice.     

Group‐specific primers for amplifying DNA sequences that identify Amphipoda,Cephalopoda, Echinodermata,Gastropoda, Isopoda,Ostracoda and Thoracica

We describe seven group‐specific primer pairs that amplify small sections of ribosomal RNA genes suitable for identification of animal groups of major importance as prey items in marine ecosystems. These primer sets allow the isolation of DNA from the target animal groups from mixed pools of DNA, where DNA‐based identification using universal primers is unlikely to succeed. The primers are designed for identifying prey in animal diets, but could be used in any situation where these animal groups are to be identified by their DNA.     

Analyzing geolocator data for birds that roost in cavities year‐round

The use of light‐level geolocators for monitoring migration has been limited to non‐cavity roosting species because light transitions for cavity‐roosting species are obscured. Using Northern Flickers (Colaptes auratus), nocturnal cavity‐roosting woodpeckers, as a model, I describe a method for analyzing geolocator data that initially adjusts light transitions to account for differences between the time of minimum light threshold and when a bird enters or exits a cavity. Using known locations from the breeding grounds, I assessed the precision of this adjustment method for estimating location by examining the associated error, the repeatability of the length of time individuals roosted in cavities, and by conducting a sensitivity analysis to assess uncertainty. Mean location error decreased from 1417 ± 277 km (SD) to 129 ± 194 km when sunrise and sunset times were adjusted and locations from >25 d were averaged. Sensitivity analysis showed that if an adjusted sunrise or sunset time was “incorrect” by 10 min, the error was 121–137 km from the actual location. This adjustment method significantly improved location estimates at known sites, suggesting that adjusting light transitions based off a calibration is a good initial step for determining location. However, to account for behavioral changes in entrance and emergence times, applying state‐space Kalman filter models can further improve the accuracy of location estimates. The combination of adjusting transitions and applying a state‐space Kalman filter thus allows location estimates to be obtained from cavity‐roosting species using geolocator data.     

A novel DNA damage and repair‐related gene signature to improve predictive capacity of overall survival for patients with gliomas

Gliomas, as the most lethal and malignant brain tumours in adults, remain a major challenge worldwide. DNA damage and repair‐related genes (DDRRGs) appear to play a significant role in gliomas, but the studies of DDRRGs are still insufficient. Herein, we systematically explored and analysed 1547 DDRRGs in 938 glioma samples from TCGA and CGGA datasets. Using least absolute shrinkage and selection operator (LASSO) Cox regression analysis, we identified a 16‐DDRRG signature, characterized by high‐risk and low‐risk patterns. This risk model harbours robust predictive capability for overall survival of glioma patients. We found the high‐risk score is strongly associated with well‐known malignant features of gliomas, such as the mesenchymal subtype, IDH‐wildtype, 1p/19q non‐codeletion and MGMT promoter unmethylated status. In addition, we found that the high‐risk score is also linked with multiple oncogenic pathways and therapeutic resistance. Significantly, we found the high‐risk group has higher enrichment of immunosuppressive cells (M2‐type macrophages, Tregs and MDSCs) and immune inhibition biomarkers (PD‐1, PD‐L1 and CTLA‐4). Lastly, we proved that SMC4, which has the highest positive regression coefficient in our risk model, is strongly linked with malignant progression and TMZ resistance of gliomas in a E2F1‐dependent manner.     

Holocene chloroplast genetic variation of shrubs (Alnus alnobetula,Betula nana,Salix sp.) at the siberian tundra‐taiga ecotone inferred from modern chloroplast genome assembly and sedimentary ancient DNA analyses

Climate warming alters plant composition and population dynamics of arctic ecosystems. In particular, an increase in relative abundance and cover of deciduous shrub species (shrubification) has been recorded. We inferred genetic variation of common shrub species (Alnus alnobetula, Betula nana, Salix sp.) through time. Chloroplast genomes were assembled from modern plants (n = 15) from the Siberian forest‐tundra ecotone. Sedimentary ancient DNA (sedaDNA; n = 4) was retrieved from a lake on the southern Taymyr Peninsula and analyzed by metagenomics shotgun sequencing and a hybridization capture approach. For A. alnobetula, analyses of modern DNA showed low intraspecies genetic variability and a clear geographical structure in haplotype distribution. In contrast, B. nana showed high intraspecies genetic diversity and weak geographical structure. Analyses of sedaDNA revealed a decreasing relative abundance of Alnus since 5,400 cal yr BP, whereas Betula and Salix increased. A comparison between genetic variations identified in modern DNA and sedaDNA showed that Alnus variants were maintained over the last 6,700 years in the Taymyr region. In accordance with modern individuals, the variants retrieved from Betula and Salix sedaDNA showed higher genetic diversity. The success of the hybridization capture in retrieving diverged sequences demonstrates the high potential for future studies of plant biodiversity as well as specific genetic variation on ancient DNA from lake sediments. Overall, our results suggest that shrubification has species‐specific trajectories. The low genetic diversity in A. alnobetula suggests a local population recruitment and growth response of the already present communities, whereas the higher genetic variability and lack of geographical structure in B. nana may indicate a recruitment from different populations due to more efficient seed dispersal, increasing the genetic connectivity over long distances.     

Empirical calibrated radiocarbon sampler: a tool for incorporating radiocarbon‐date and calibration error into Bayesian phylogenetic analyses of ancient DNA

Studies of DNA from ancient samples provide a valuable opportunity to gain insight into past evolutionary and demographic processes. Bayesian phylogenetic methods can estimate evolutionary rates and timescales from ancient DNA sequences, with the ages of the samples acting as calibrations for the molecular clock. Sample ages are often estimated using radiocarbon dating, but the associated measurement error is rarely taken into account. In addition, the total uncertainty quantified by converting radiocarbon dates to calendar dates is typically ignored. Here, we present a tool for incorporating both of these sources of uncertainty into Bayesian phylogenetic analyses of ancient DNA. This empirical calibrated radiocarbon sampler (ECRS) integrates the age uncertainty for each ancient sequence over the calibrated probability density function estimated for its radiocarbon date and associated error. We use the ECRS to analyse three ancient DNA data sets. Accounting for radiocarbon‐dating and calibration error appeared to have little impact on estimates of evolutionary rates and related parameters for these data sets. However, analyses of other data sets, particularly those with few or only very old radiocarbon dates, might be more sensitive to using artificially precise sample ages and should benefit from use of the ECRS.     

Unique B-1 cells specific for both N-pyrrolated proteins and DNA evolve with apolipoprotein E deficiency

Lysine N-pyrrolation, a posttranslational modification, which converts lysine residues to N^ε-pyrrole-L-lysine, imparts electronegative properties to proteins, causing them to mimic DNA. Apolipoprotein E (apoE) has been identified as a soluble receptor for pyrrolated proteins (pyrP), and accelerated lysine N-pyrrolation has been observed in apoE-deficient (apoE^−/−) hyperlipidemic mice. However, the impact of pyrP accumulation consequent to apoE deficiency on the innate immune response remains unclear. Here, we investigated B-1a cells known to produce germline-encoded immunoglobulin M (IgM) from mice deficient in apoE and identified a particular cell population that specifically produces IgM antibodies against pyrP and DNA. We demonstrated an expansion of B-1a cells involved in IgM production in the peritoneal cavity of apoE^−/− mice compared with wild-type mice, consistent with a progressive increase of IgM response in the mouse sera. We found that pyrP exhibited preferential binding to B-1a cells and facilitated the production of IgM. B cell receptor analysis of pyrP-specific B-1a cells showed restricted usage of gene segments selected from the germline gene set; most sequences contained high levels of non-templated-nucleotide additions (N-additions) that could contribute to junctional diversity of B cell receptors. Finally, we report that a subset of monoclonal IgM antibodies against pyrP/DNA established from the apoE^−/− mice also contained abundant N-additions. These results suggest that the accumulation of pyrP due to apoE deficiency may influence clonal diversity in the pyrP-specific B cell repertoire. The discovery of these unique B-1a cells for pyrP/DNA provides a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.     

Nuclear DNA level and life cycle of kelps: Evidence for sex‐specific polyteny in Macrocystis (Laminariales,Phaeophyceae)

Giant kelp, Macrocystis pyrifera (Linnaeus) C. Agardh, is the subject of intense breeding studies for marine biomass production and conservation of natural resources. In this context, six gametophyte pairs and a sporophyte offspring of Macrocystis from South America were analyzed by flow cytometry. Minimum relative DNA content per cell (1C) was found in five males. Unexpectedly, nuclei of all female gametophytes contained approximately double the DNA content (2C) of males; the male gametophyte from one locality also contained 2C, likely a spontaneous natural diploid variant. The results illustrate a sex‐specific difference in nuclear DNA content among Macrocystis gametophytes, with the chromosomes of the females in a polytenic condition. This correlates with significantly larger cell sizes in female gametophytes compared to males and resource allocation in oogamous reproduction. The results provide key information for the interpretation of DNA measurements in kelp life cycle stages and prompt further research on the regulation of the cell cycle, metabolic activity, sex determination, and sporophyte development.     

Novel polymerase chain reaction primers for the specific detection of bacterial copper P‐type ATPases gene sequences in environmental isolates and metagenomic DNA

Aims: In the last decades, the worldwide increase in copper wastes release by industrial activities like mining has driven environmental metal contents to toxic levels. For this reason, the study of the biological copper‐resistance mechanisms in natural environments is important. Therefore, an appropriate molecular tool for the detection and tracking of copper‐resistance genes was developed. Methods and Results: In this work, we designed a PCR primer pair to specifically detect copper P‐type ATPases gene sequences. These PCR primers were tested in bacterial isolates and metagenomic DNA from intertidal marine environments impacted by copper pollution. As well, T‐RFLP fingerprinting of these gene sequences was used to compare the genetic composition of such genes in microbial communities, in normal and copper‐polluted coastal environments. New copper P‐type ATPases gene sequences were found, and a high degree of change in the genetic composition because of copper exposure was also determined. Conclusions: This PCR based method is useful to track bacterial copper‐resistance gene sequences in the environment. Significance and Impact of the Study: This study is the first to report the design and use of a PCR primer pair as a molecular marker to track bacterial copper‐resistance determinants, providing an excellent tool for long‐term analysis of environmental communities exposed to metal pollution.     

Deletion of the <Emphasis Type="Italic">Clostridium thermocellum recA</Emphasis> gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥?60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ?recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.