Road blocks on paleogenomes - polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA

Although the last few years have seen great progress in DNA sequence retrieval from fossil specimens, some of the characteristics of ancient DNA remain poorly understood. This is particularly true for blocking lesions, i.e. chemical alterations that cannot be bypassed by DNA polymerases and thus prevent amplification and subsequent sequencing of affected molecules. Some studies have concluded that the vast majority of ancient DNA molecules carry blocking lesions, suggesting that the removal, repair or bypass of blocking lesions might dramatically increase both the time depth and geographical range of specimens available for ancient DNA analysis. However, previous studies used very indirect detection methods that did not provide conclusive estimates on the frequency of blocking lesions in endogenous ancient DNA. We developed a new method, polymerase extension profiling (PEP), that directly reveals occurrences of polymerase stalling on DNA templates. By sequencing thousands of single primer extension products using PEP methodology, we have for the first time directly identified blocking lesions in ancient DNA on a single molecule level. Although we found clear evidence for blocking lesions in three out of four ancient samples, no more than 40% of the molecules were affected in any of the samples, indicating that such modifications are far less frequent in ancient DNA than previously thought

leeHom: adaptor trimming and merging for Illumina sequencing reads

The sequencing of libraries containing molecules shorter than the read length, such as in ancient or forensic applications, may result in the production of reads that include the adaptor, and in paired reads that overlap one another. Challenges for the processing of such reads are the accurate identification of the adaptor sequence and accurate reconstruction of the original sequence most likely to have given rise to the observed read(s). We introduce an algorithm that removes the adaptors and reconstructs the original DNA sequences using a Bayesian maximum a posteriori probability approach. Our algorithm is faster, and provides a more accurate reconstruction of the original sequence for both simulated and ancient DNA data sets, than other approaches. leeHom is released under the GPLv3 and is freely available from: https://bioinf.eva.mpg.de/leehom

freeIbis: An efficient basecaller with calibrated quality scores for Illumina sequencers

Motivation: The conversion of the raw intensities obtained from next-generation sequencing platforms into nucleotide sequences with well-calibrated quality scores is a critical step in the generation of good sequence data. While recent model-based approaches can yield highly accurate calls, they require a substantial amount of processing time and/or computational resources. We previously introduced Ibis, a fast and accurate basecaller for the Illumina platform. We have continued active development of Ibis to take into account developments in the Illumina technology, as well as to make Ibis fully open source. Results: We introduce here freeIbis, which offers significant improvements in sequence accuracy owing to the use of a novel multiclass support vector machine (SVM) algorithm. Sequence quality scores are now calibrated based on empirically observed scores, thus providing a high correlation to their respective error rates. These improvements result in downstream advantages including improved genotyping accuracy. Availability and implementation: FreeIbis is freely available for use under the GPL (http://bioinf.eva.mpg.de/freeibis/). It requires a Python interpreter and a C++ compiler. Tailored versions of LIBOCAS and LIBLINEAR are distributed along with the package. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

deML: Robust demultiplexing of Illumina sequences using a likelihood-based approach

Motivation: Pooling multiple samples increases the efficiency and lowers the cost of DNA sequencing. One approach to multiplexing is to use short DNA indices to uniquely identify each sample. After sequencing, reads must be assigned in silico to the sample of origin, a process referred to as demultiplexing. Demultiplexing software typically identifies the sample of origin using a fixed number of mismatches between the read index and a reference index set. This approach may fail or misassign reads when the sequencing quality of the indices is poor. Results: We introduce deML, a maximum likelihood algorithm that demultiplexes Illumina sequences. deML computes the likelihood of an observed index sequence being derived from a specified sample. A quality score which reflects the probability of the assignment being correct is generated for each read. Using these quality scores, even very problematic datasets can be demultiplexed and an error threshold can be set. Availability and implementation: deML is freely available for use under the GPL (http://bioinf.eva.mpg.de/deml/). Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

Computational challenges in the analysis of ancient DNA

High-throughput sequencing technologies have opened up a new avenue for studying extinct organisms. Here we identify and quantify biases introduced by particular characteristics of ancient DNA samples. These analyses demonstrate the importance of closely related genomic sequence for correctly identifying and classifying bona fide endogenous DNA fragments. We show that more accurate genome divergence estimates from ancient DNA sequence can be attained using at least two outgroup genomes and appropriate filtering

Limb observations of the Martian atmosphere with Mars Express’ High Resolution Stereo Camera

Introduction: Good knowledge about the aerosol distribution and compositions is essential for the understanding of thermodynamic processes in the Martian atmosphere, which in turn is important for the understanding of the Martian climate and the altitude of the upper boundary of the atmosphere. The last point is of special interest for spacecraft aerobreaking manoeuvres. The Martian atmosphere often shows horizontal layers of haze up to altitudes of about 80 km. These have been described and analysed e.g. by Jaquin et al., 1986, usingViking Orbiter images and by Montmessin et al., 2006, who used SPICAM stellar occultation data. Both showed seasonal and latitudinal changes in the vertical structure of the aerosol distribution and composition. Apart from SPICAM, the High Resolution Stereo Camera (HRSC) is also on board ESA’s robotic spacecraft Mars Express. HRSC was build and is operated by the German Aerospace Center (Neukum et al. 2004; Jaumann et al. 2007). Mars Express is orbiting Mars in an elliptical orbit, with HRSC scanning the surface of Mars, primarily for geological research. In addition to that, HRSC has been used to sample the planetary limb. We examine the HRSC planetary limb data and analyse the seasonal and latitudinal variations of the maximum altitude of the haze layer and of the occurrence of high altitude detached hazes. We make some comparisons with earlier work. In contrast to the SPICAM instrument, HRSC observes the atmosphere during daytime, which makes it possible to compare night and daytime observations. The HRSC Limb Data: HRSC is a push broom scanner with nine line sensors pointing in different directions to facilitate stereoscopic imaging. Four of the sensors have colour filters at 440 nm, 530 nm, 750 nm and 970 nm, respectively. The five other sensors all have filters centred at 650 nm. These panchromatic filters have a much wider bandpass than the four colour filters. The surface observations which are HRSC’s main purpose, are usually take while the spacecraft is nadirtracking near pericentre. Limb observations, however, are mostly made with a pointing of the spacecraft being inertially fixxed in celestial space. This leaves only a small time window to make observations of the limb during descent or ascent. Therefore, usually only a few of the nine sensors can be used for the limb observation. Due to the motion of the spacecraft, the individual image lines are taken at different geographical locations and altitudes. The position of each image pixel above the limb has to be calculated from the spacecraft positioning information (Scholten, pers. comm.). The typical difference in altitude between two neighbouring pixels is between a couple of dozen metres and 150 m. HRSC has been observing the limb occasionally throughout the mission since 2004. So far the northern hemisphere and especially the north polar region, were particularly well covered (Figure 1 and 2). In Figure 2, we give an overview of the available data, sorted by season (LS) and latitude. The channel in which the observations have been made is colourcoded. Most observations were made with the panchromatic channels. There are also many observations with the blue and green sensors and only a few were made in the red and infra red channels. We find the best data coverage in northern spring in the northern most latitudes. For obvious reasons, we do not have any data during polar nights. For most of our actual analysis we sample the five central pixels of the sensor lines. This allows for minimal horizontal averaging. Analysis: As an example, Fig. 3 shows images and profiles for the blue, nadir, and green channels from orbit 6104. Al three images show a continuously bright limb haze until an altitude of about 20 km. At higher altitudes the limb haze becomes darker and stratified consistent with the limb profiles described by Jacquin et al., 1986. As Mars Express progresses along its orbit, the limb observations are made at different locations above the surface. The locations of the three profiles in Fig. 3 are still in close proximity of each other, in fact they overlap, but none the less they show different vertical aerosol distributions. Beginning above the North Polar cap and going southward, we observe less reflectivity above 20 km and more reflectivity below 20 km, hinting at different compositions or amounts of aerosols. It is not possible to obtain and compare profiles at the same location and at the same time with different sensors, but still, averages of profiles over place and season can provide us with information about typical atmospheric conditions. In Fig. 4 we show spectra from the average profiles at three different latitudinal bands between 70�N–90�N, 30�S–30�N, and 90�S– 70�S, on the left, centre, and right, respectively. The different symbols and colours represent the different altitudes at which the spectra were sampled. The size of the symbol increases with the number of averaged profiles. There are very few observations above the South Polar region (compare Fig. 1). In the North (and South) Polar region there is almost no signal above 30 km altitude, while around the equator the limb haze remains bright until altitudes of about 60 km. At the poles, the spectrum at 10 km is reddish. At higher altitudes the spectrum gets whiter, indicating smaller particles or higher ice content. At the low latitudes the spectra are reddish up to 40 km. At 60 km we see a more or less white spectrum. Figure 5 shows the maximum altitude of the aerosols as seen by HRSC, depending on season. During aphelion (LS � 70�) the maximum altitude of the aerosols that are visible with HRSC is around 40 km. During perihelion (LS � 250�) the maximum altitude is around 70 km. Discussion: Figure 1 and 2 show that there are plenty of visual and near infra red HRSC observations of the Martian limb available. These show aerosol distributions that change with season and latitude (Fig. 3 and 4). The plots in Fig. 4 show the spectra of the average limb profiles at several altitudes for three latitudinal bands. Two important distinctions can be made between the equatorial and the polar regions. First, the altitude at which aerosol occur is higher in the equatorial region and second, the composition of the aerosols at different altitudes is different. While the spectrum is white around 20 km altitude above the north pole, it is red at the low latitudes. The seasonal variations of maximum altitude of the aerosols is in good agreement with Jaquin et al. (1986) and with Montmessin et al. (2006). The similarity between Montmessin’s results and ours is likely to be due to the large annual variation of atmospheric dust load compared to the diurnal cycle. A much closer look at the data, is forseen to analyse the daily variation of aerosols in the Martian atmosphere. The CO2 and waterice aerosols are more likely to change their vertical distribution (above the planetary boundary layer) between day and night than the mineral (dust) aerosols. Spectral information would help to discriminate between these components. HRSC can not provide it, because the observation for the different filters take place at different locations and times (see Fig. 3). An alternative is to fit aerosol models to the inverted profiles. Currently, we are preparing this work. Mars Express’ HRSC limb data present a valuable opportunity to analyse Mars daytime atmospheric dust at a high vertical resolution. This work gives a short overview of the available data and analyses some seasonal and latitudinal properties

Mechanostimulation of Medicago truncatula leads to enhanced levels of jasmonic acid

Wounding of plants leads to endogenous rise of jasmonic acid (JA) accompanied with the expression of a distinct set of genes. Among them are those coding for the allene oxide cyclase (AOC) that catalyses a regulatory step in JA biosynthesis, and for 1-deoxy-D-xylulose 5-phosphate synthase 2 (DXS2), an enzyme involved in isoprenoid biosynthesis. To address the question how roots and shoots of Medicago truncatula respond to mechanostimulation and wounding, M. truncatula plants were analysed in respect to JA levels as well as MtAOC1 and MtDXS2-1 transcript accumulation. Harvest-caused mechanostimulation resulted in a strong, but transient increase in JA level in roots and shoots followed by a transient increase in MtAOC1 transcript accumulation. Additional wounding of either shoots or roots led to further increased JA and MtAOC1 transcript levels in shoots, but not in roots. In situ hybridization revealed a cell-specific transcript accumulation of MtAOC1 after mechanostimulation in companion cells of the vascular tissue of the stem. AOC protein, however, was found to occur constitutively in vascular bundles. Further, transcript accumulation of MtDXS2-1 was similar to that of MtAOC1 in shoots, but its transcript levels were not enhanced in roots. Repeated touching of shoots increased MtAOC1 transcript levels and led to significantly shorter shoots and increased biomass. In conclusion, M. truncatula plants respond very sensitively to mechanostimulation with enhanced JA levels and altered transcript accumulation, which might contribute to the altered phenotype after repeated touching of plants

Hadron beam test of a scintillating fibre tracker system for elastic scattering and luminosity measurement in ATLAS

A scintillating fibre tracker is proposed to measure elastic proton scattering at very small angles in the ATLAS experiment at CERN. The tracker will be located in so-called Roman Pot units at a distance of 240 m on each side of the ATLAS interaction point. An initial validation of the design choices was achieved in a beam test at DESY in a relatively low energy electron beam and using slow off-the-shelf electronics. Here we report on the results from a second beam test experiment carried out at CERN, where new detector prototypes were tested in a high energy hadron beam, using the first version of the custom designed front-end electronics. The results show an adequate tracking performance under conditions which are similar to the situation at the LHC. In addition, the alignment method using so-called overlap detectors was studied and shown to have the expected precision.Comment: 12 pages, 8 figures. Submitted to Journal of Instrumentation (JINST

Linear Response Calculations of Spin Fluctuations

A variational formulation of the time--dependent linear response based on the Sternheimer method is developed in order to make practical ab initio calculations of dynamical spin susceptibilities of solids. Using gradient density functional and a muffin-tin-orbital representation, the efficiency of the approach is demonstrated by applications to selected magnetic and strongly paramagnetic metals. The results are found to be consistent with experiment and are compared with previous theoretical calculations.Comment: 11 pages, RevTex; 3 Figures, postscript, high-resolution printing (~1200dpi) is desire