Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA

DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil–DNA–glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes

The Neandertal genome and ancient DNA authenticity

Recent advances in high-thoughput DNA sequencing have made genome-scale analyses of genomes of extinct organisms possible. With these new opportunities come new difficulties in assessing the authenticity of the DNA sequences retrieved. We discuss how these difficulties can be addressed, particularly with regard to analyses of the Neandertal genome. We argue that only direct assays of DNA sequence positions in which Neandertals differ from all contemporary humans can serve as a reliable means to estimate human contamination. Indirect measures, such as the extent of DNA fragmentation, nucleotide misincorporations, or comparison of derived allele frequencies in different fragment size classes, are unreliable. Fortunately, interim approaches based on mtDNA differences between Neandertals and current humans, detection of male contamination through Y chromosomal sequences, and repeated sequencing from the same fossil to detect autosomal contamination allow initial large-scale sequencing of Neandertal genomes. This will result in the discovery of fixed differences in the nuclear genome between Neandertals and current humans that can serve as future direct assays for contamination. For analyses of other fossil hominins, which may become possible in the future, we suggest a similar ‘boot-strap' approach in which interim approaches are applied until sufficient data for more definitive direct assays are acquired

Optimization of scarless human stem cell genome editing

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks

From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing

Current efforts to recover the Neandertal and mammoth genomes by 454 DNA sequencing demonstrate the sensitivity of this technology. However, routine 454 sequencing applications still require microgram quantities of initial material. This is due to a lack of effective methods for quantifying 454 sequencing libraries, necessitating expensive and labour-intensive procedures when sequencing ancient DNA and other poor DNA samples. Here we report a 454 sequencing library quantification method based on quantitative PCR that effectively eliminates these limitations. We estimated both the molecule numbers and the fragment size distributions in sequencing libraries derived from Neandertal DNA extracts, SAGE ditags and bonobo genomic DNA, obtaining optimal sequencing yields without performing any titration runs. Using this method, 454 sequencing can routinely be performed from as little as 50 pg of initial material without titration runs, thereby drastically reducing costs while increasing the scope of sample throughput and protocol development on the 454 platform. The method should also apply to Illumina/Solexa and ABI/SOLiD sequencing, and should therefore help to widen the accessibility of all three platforms

Mitochondrial Genomes Reveal an Explosive Radiation of Extinct and Extant Bears near the Miocene-Pliocene Boundary

Background: Despite being one of the most studied families within the Carnivora, the phylogenetic relationships among the members of the bear family (Ursidae) have long remained unclear. Widely divergent topologies have been suggested based on various data sets and methods. Results: We present a fully resolved phylogeny for ursids based on ten complete mitochondrial genome sequences from all eight living and two recently extinct bear species, the European cave bear (Ursus spelaeus) and the American giant short-faced bear (Arctodus simus). The mitogenomic data yield a well-resolved topology for ursids, with the sloth bear at the basal position within the genus Ursus. The sun bear is the sister taxon to both the American and Asian black bears, and this clade is the sister clade of cave bear, brown bear and polar bear confirming a recent study on bear mitochondrial genomes. Conclusion: Sequences from extinct bears represent the third and fourth Pleistocene species for which complete mitochondrial genomes have been sequenced. Moreover, the cave bear specimen demonstrates that mitogenomic studies can be applied to Pleistocene fossils that have not been preserved in permafrost, and therefore have a broad application within ancient DNA research. Molecular dating of the mtDNA divergence times suggests a rapid radiation of bears in both the Old and New Worlds around 5 million years ago, at the Miocene-Pliocene boundary. This coincides with major global changes, such as the Messinian crisis and the first opening of the Bering Strait, and suggests a global influence of such events on species radiations

The Sloan Digital Sky Survey Quasar Catalog I. Early Data Release

We present the first edition of the Sloan Digital Sky Survey (SDSS) Quasar Catalog. The catalog consists of the 3814 objects (3000 discovered by the SDSS) in the initial SDSS public data release that have at least one emission line with a full width at half maximum larger than 1000 km/s, luminosities brighter than M_i^* = -23, and highly reliable redshifts. The area covered by the catalog is 494 square degrees; the majority of the objects were found in SDSS commissioning data using a multicolor selection technique. The quasar redshifts range from 0.15 to 5.03. For each object the catalog presents positions accurate to better than 0.2" rms per coordinate, five band (ugriz) CCD-based photometry with typical accuracy of 0.05 mag, radio and X-ray emission properties, and information on the morphology and selection method. Calibrated spectra of all objects in the catalog, covering the wavelength region 3800 to 9200 Angstroms at a spectral resolution of 1800-2100, are also available. Since the quasars were selected during the commissioning period, a time when the quasar selection algorithm was undergoing frequent revisions, the sample is not homogeneous and is not intended for statistical analysis.Comment: 27 pages, 4 figures, 4 tables, accepted by A

Colors of 2625 Quasars at 0<z<5 Measured in the Sloan Digital Sky Survey Photometric System

We present an empirical investigation of the colors of quasars in the Sloan Digital Sky Survey (SDSS) photometric system. The sample studied includes 2625 quasars with SDSS photometry. The quasars are distributed in a 2.5 degree wide stripe centered on the Celestial Equator covering $\sim529$ square degrees. Positions and SDSS magnitudes are given for the 898 quasars known prior to SDSS spectroscopic commissioning. New SDSS quasars represent an increase of over 200% in the number of known quasars in this area of the sky. The ensemble average of the observed colors of quasars in the SDSS passbands are well represented by a power-law continuum with $\alpha_{\nu} = -0.5$ ($f_{\nu} \propto \nu^{\alpha}$). However, the contributions of the $3000 {\rm \AA}$ bump and other strong emission lines have a significant effect upon the colors. The color-redshift relation exhibits considerable structure, which may be of use in determining photometric redshifts for quasars. The range of colors can be accounted for by a range in the optical spectral index with a distribution $\alpha_{\nu}=-0.5\pm0.65$ (95% confidence), but there is a red tail in the distribution. This tail may be a sign of internal reddening. Finally, we show that there is a continuum of properties between quasars and Seyfert galaxies and we test the validity of the traditional division between the two classes of AGN.Comment: 66 pages, 15 figures (3 color), accepted by A

Galaxy Clustering in Early SDSS Redshift Data

We present the first measurements of clustering in the Sloan Digital Sky Survey (SDSS) galaxy redshift survey. Our sample consists of 29,300 galaxies with redshifts 5,700 km/s < cz < 39,000 km/s, distributed in several long but narrow (2.5-5 degree) segments, covering 690 square degrees. For the full, flux-limited sample, the redshift-space correlation length is approximately 8 Mpc/h. The two-dimensional correlation function \xi(r_p,\pi) shows clear signatures of both the small-scale, ``fingers-of-God'' distortion caused by velocity dispersions in collapsed objects and the large-scale compression caused by coherent flows, though the latter cannot be measured with high precision in the present sample. The inferred real-space correlation function is well described by a power law, \xi(r)=(r/6.1+/-0.2 Mpc/h)^{-1.75+/-0.03}, for 0.1 Mpc/h < r < 16 Mpc/h. The galaxy pairwise velocity dispersion is \sigma_{12} ~ 600+/-100 km/s for projected separations 0.15 Mpc/h < r_p < 5 Mpc/h. When we divide the sample by color, the red galaxies exhibit a stronger and steeper real-space correlation function and a higher pairwise velocity dispersion than do the blue galaxies. The relative behavior of subsamples defined by high/low profile concentration or high/low surface brightness is qualitatively similar to that of the red/blue subsamples. Our most striking result is a clear measurement of scale-independent luminosity bias at r < 10 Mpc/h: subsamples with absolute magnitude ranges centered on M_*-1.5, M_*, and M_*+1.5 have real-space correlation functions that are parallel power laws of slope ~ -1.8 with correlation lengths of approximately 7.4 Mpc/h, 6.3 Mpc/h, and 4.7 Mpc/h, respectively.Comment: 51 pages, 18 figures. Replaced to match accepted ApJ versio

The Multi-Object, Fiber-Fed Spectrographs for SDSS and the Baryon Oscillation Spectroscopic Survey

We present the design and performance of the multi-object fiber spectrographs for the Sloan Digital Sky Survey (SDSS) and their upgrade for the Baryon Oscillation Spectroscopic Survey (BOSS). Originally commissioned in Fall 1999 on the 2.5-m aperture Sloan Telescope at Apache Point Observatory, the spectrographs produced more than 1.5 million spectra for the SDSS and SDSS-II surveys, enabling a wide variety of Galactic and extra-galactic science including the first observation of baryon acoustic oscillations in 2005. The spectrographs were upgraded in 2009 and are currently in use for BOSS, the flagship survey of the third-generation SDSS-III project. BOSS will measure redshifts of 1.35 million massive galaxies to redshift 0.7 and Lyman-alpha absorption of 160,000 high redshift quasars over 10,000 square degrees of sky, making percent level measurements of the absolute cosmic distance scale of the Universe and placing tight constraints on the equation of state of dark energy. The twin multi-object fiber spectrographs utilize a simple optical layout with reflective collimators, gratings, all-refractive cameras, and state-of-the-art CCD detectors to produce hundreds of spectra simultaneously in two channels over a bandpass covering the near ultraviolet to the near infrared, with a resolving power R = \lambda/FWHM ~ 2000. Building on proven heritage, the spectrographs were upgraded for BOSS with volume-phase holographic gratings and modern CCD detectors, improving the peak throughput by nearly a factor of two, extending the bandpass to cover 360 < \lambda < 1000 nm, and increasing the number of fibers from 640 to 1000 per exposure. In this paper we describe the original SDSS spectrograph design and the upgrades implemented for BOSS, and document the predicted and measured performances.Comment: 43 pages, 42 figures, revised according to referee report and accepted by AJ. Provides background for the instrument responsible for SDSS and BOSS spectra. 4th in a series of survey technical papers released in Summer 2012, including arXiv:1207.7137 (DR9), arXiv:1207.7326 (Spectral Classification), and arXiv:1208.0022 (BOSS Overview