Showing papers by "Matthias Meyer published in 2012"

A high-coverage genome sequence from an archaic Denisovan individual

Abstract: We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.

Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform

Abstract: Due to the increasing throughput of current DNA sequencing instruments, sample multiplexing is necessary for making economical use of available sequencing capacities. A widely used multiplexing strategy for the Illumina Genome Analyzer utilizes sample-specific indexes, which are embedded in one of the library adapters. However, this and similar multiplex approaches come with a risk of sample misidentification. By introducing indexes into both library adapters (double indexing), we have developed a method that reveals the rate of sample misidentification within current multiplex sequencing experiments. With ~0.3% these rates are orders of magnitude higher than expected and may severely confound applications in cancer genomics and other fields requiring accurate detection of rare variants. We identified the occurrence of mixed clusters on the flow as the predominant source of error. The accuracy of sample identification is further impaired if indexed oligonucleotides are cross-contaminated or if indexed libraries are amplified in bulk. Double-indexing eliminates these problems and increases both the scope and accuracy of multiplex sequencing on the Illumina platform.

Length and GC-biases during sequencing library amplification: A comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries

Abstract: High-throughput sequencing technologies frequently necessitate the use of PCR for sequencing library amplification. PCR is a sometimes enigmatic process and is known to introduce biases. Here we perform a simple amplification-sequencing assay using 10 commercially available polymerase-buffer systems to amplify libraries prepared from both modern and ancient DNA. We compare the performance of the polymerases with respect to a previously uncharacterized template length bias, as well as GC-content bias, and find that simply avoiding certain polymerase can dramatically decrease the occurrence of both. For amplification of ancient DNA, we found that some commonly used polymerases strongly bias against amplification of endogenous DNA in favor of GC-rich microbial contamination, in our case reducing the fraction of endogenous sequences to almost half.

Opsins in Onychophora (Velvet Worms) Suggest a Single Origin and Subsequent Diversification of Visual Pigments in Arthropods

Abstract: Multiple visual pigments, prerequisites for color vision, are found in arthropods, but the evolutionary origin of their diversity remains obscure. In this study, we explore the opsin genes in five distantly related species of Onychophora, using deep transcriptome sequencing and screening approaches. Surprisingly, our data reveal the presence of only one opsin gene (onychopsin) in each onychophoran species, and our behavioral experiments indicate a maximum sensitivity of onychopsin to blue-green light. In our phylogenetic analyses, the onychopsins represent the sister group to the monophyletic clade of visual r-opsins of arthropods. These results concur with phylogenomic support for the sister-group status of the Onychophora and Arthropoda and provide evidence for monochromatic vision in velvet worms and in the last common ancestor of Onychophora and Arthropoda. We conclude that the diversification of visual pigments and color vision evolved in arthropods, along with the evolution of compound eyes-one of the most sophisticated visual systems known.

Generating barcoded libraries for multiplex high-throughput sequencing.

Abstract: Molecular barcoding is an essential tool to use the high throughput of next generation sequencing platforms optimally in studies involving more than one sample. Various barcoding strategies allow for the incorporation of short recognition sequences (barcodes) into sequencing libraries, either by ligation or polymerase chain reaction (PCR). Here, we present two approaches optimized for generating barcoded sequencing libraries from low copy number extracts and amplification products typical of ancient DNA studies.

Exploiting gene families for phylogenomic analysis of myzostomid transcriptome data

Abstract: Background: In trying to understand the evolutionary relationships of organisms, the current flood of sequence data offers great opportunities, but also reveals new challenges with regard to data quality, the selection of data for subsequent analysis, and the automation of steps that were once done manually for single-gene analyses. Even though genome or transcriptome data is available for representatives of most bilaterian phyla, some enigmatic taxa still have an uncertain position in the animal tree of life. This is especially true for myzostomids, a group of symbiotic ( or parasitic) protostomes that are either placed with annelids or flatworms. Methodology: Based on similarity criteria, Illumina-based transcriptome sequences of one myzostomid were compared to protein sequences of one additional myzostomid and 29 reference metazoa and clustered into gene families. These families were then used to investigate the phylogenetic position of Myzostomida using different approaches: Alignments of 989 sequence families were concatenated, and the resulting superalignment was analyzed under a Maximum Likelihood criterion. We also used all 1,878 gene trees with at least one myzostomid sequence for a supertree approach: the individual gene trees were computed and then reconciled into a species tree using gene tree parsimony. Conclusions: Superalignments require strictly orthologous genes, and both the gene selection and the widely varying amount of data available for different taxa in our dataset may cause anomalous placements and low bootstrap support. In contrast, gene tree parsimony is designed to accommodate multilocus gene families and therefore allows a much more comprehensive data set to be analyzed. Results of this supertree approach showed a well-resolved phylogeny, in which myzostomids were part of the annelid radiation, and major bilaterian taxa were found to be monophyletic.

Single-molecule SERS detection of C60.

Abstract: Single-molecule Surface-Enhanced Raman Scattering (SERS) detection of buckminsterfullerene (C60) is achieved by using different isotopologues of the molecule with a distribution around an average isotopic substitution (12C → 13C) of ∼30%. The distribution of different isotopologues creates a broad (∼20 cm−1) average SERS signal within which single-molecule SERS spectra of individual isotopic realizations of the molecule can be distinguished. The SERS enhancement factors for SM-SERS C60 events are typically in the range of ∼108, suggesting a limitation imposed by either photobleaching or surface interactions with the (Ag) metallic colloids to reach the highest SERS hot-spots (which can typically have larger maximum enhancements). SM-SERS signals of isotopically substituted C60 also show broader peaks (FWHM ≈ 4 cm−1) than equivalent signals in natural C60. The latter feature suggests a contribution to the homogeneous broadening coming from isotopic disorder in the molecule; a feature that can only be observed with the ability to detect single-molecule spectra.

Ancient DNA extracted from Danish aurochs (Bos primigenius): Genetic diversity and preservation

Abstract: We extracted DNA from 39 Danish aurochs specimens and successfully amplified and sequenced a 252 base pair long fragment of the multivariable region I of the mitochondrial control region from 11 specimens. The sequences from these specimens dated back to 9830-2865 14Cyr BP and represent the first study of genetic variation of Danish aurochs. In addition, for all specimens we address correlations between the ability to obtain DNA sequences and various parameters such as the age of the sample, the collagen content, the museum storage period, Danish geography and whether the specimens were found in an archeological or geological context. We find that aurochs from southern Scandinavia display a star-shaped population genetic structure, that is indicative of a local and relatively recent diversification from a few ancestral haplotypes that may have originated in the ancestral Western European population before migration northwards during the retreat of the glaciers. Scenarios suggesting several invasions of genetically distinct aurochs are not supported by these analyses. Rather, our results suggest that a single continuous migration northward occurred. Our findings also suggest, although with only limited support, that aurochs in Northwestern Europe underwent a population expansion beginning shortly after the retreat of the glacial ice from Denmark and had a stable population size until the population decline that must have occurred prior to extinction. The absence of haplotypes similar to modern domestic cattle in our aurochs suggests that introgression between these species must have been limited, if it occurred at all. We found that the successful recovery of genetic material for PCR amplification correlates with sample age and local geographic conditions. However, contrary to other studies, we found no significant correlation between length of time in museum storage or the type of the locality in which a specimen was discovered (archeological or geological) and amplification success. Finally, we found large variances in our estimates of collagen content preventing an evaluation of this as an indicator of preservation quality.