Assessing the enrichment performance in targeted resequencing experiments.

Target enrichment strategies are a very common approach to sequence a predefined part of an individual's genome using second-generation sequencing technologies. While highly dependent on the technology and the target sequences selected, the performance of the various assays is also variable between samples and is influenced by the way how the libraries are handled in the laboratory. Here, we show how to find detailed information about the enrichment performance using a novel software package called NGSrich, which we developed as a part of a whole-exome resequencing pipeline in a medium-sized genomics center. Our software is suitable for high-throughput use and the results can be shared using HTML and a web server. Finally, we have sequenced exome-enriched DNA libraries of 18 human individuals using three different enrichment products and used our new software for a comparative analysis of their performance.

Pipeline for large-scale microdroplet bisulfite PCR-based sequencing allows the tracking of hepitype evolution in tumors.

Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into "hepitypes" and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer.

Benchmarking of mutation diagnostics in clinical lung cancer specimens.

Treatment of EGFR-mutant non-small cell lung cancer patients with the tyrosine kinase inhibitors erlotinib or gefitinib results in high response rates and prolonged progression-free survival. Despite the development of sensitive mutation detection approaches, a thorough validation of these in a clinical setting has so far been lacking. We performed, in a clinical setting, a systematic validation of dideoxy 'Sanger' sequencing and pyrosequencing against massively parallel sequencing as one of the most sensitive mutation detection technologies available. Mutational annotation of clinical lung tumor samples revealed that of all patients with a confirmed response to EGFR inhibition, only massively parallel sequencing detected all relevant mutations. By contrast, dideoxy sequencing missed four responders and pyrosequencing missed two responders, indicating a dramatic lack of sensitivity of dideoxy sequencing, which is widely applied for this purpose. Furthermore, precise quantification of mutant alleles revealed a low correlation (r(2) = 0.27) of histopathological estimates of tumor content and frequency of mutant alleles, thereby questioning the use of histopathology for stratification of specimens for individual analytical procedures. Our results suggest that enhanced analytical sensitivity is critically required to correctly identify patients responding to EGFR inhibition. More broadly, our results emphasize the need for thorough evaluation of all mutation detection approaches against massively parallel sequencing as a prerequisite for any clinical implementation.

Positive and negative selection in murine ultraconserved noncoding elements.

There are many more selectively constrained noncoding than coding nucleotides in the mammalian genome, but most mammalian noncoding DNA is subject to weak selection, on average. One of the most striking discoveries to have emerged from comparisons among mammalian genomes is the hundreds of noncoding elements of more than 200 bp in length that show absolute conservation among mammalian orders. These elements represent the tip of the iceberg of a much larger class of conserved noncoding elements (CNEs). Much evidence suggests that CNEs are selectively constrained and not mutational cold-spots, and there is evidence that some CNEs play a role in the regulation of development. Here, we quantify negative and positive selection acting in murine CNEs by analyzing within-species nucleotide variation and between-species divergence of CNEs that we identified using a phylogenetically independent comparison. The distribution of fitness effects of new mutations in CNEs, inferred from within-species polymorphism, suggests that CNEs receive a higher number of strongly selected deleterious mutations and many fewer nearly neutral mutations than amino acid sites of protein-coding genes or regulatory elements close to genes. However, we also show that CNEs experience a far higher proportion of adaptive substitutions than any known category of genomic sites in murids. The absolute rate of adaptation of CNEs is similar to that of amino acid sites of proteins. This result suggests that there is widespread adaptation in mammalian conserved noncoding DNA elements, some of which have been implicated in the regulation of crucially important processes, including development.

Rapid formation of distinct hybrid lineages after secondary contact of two fish species (Cottus sp.).

Homoploid hybridization after secondary contact between related species can lead to mixtures of genotypes which have the potential for rapid adaptation to new environmental conditions. Here, we focus on a case where anthropogenic changes within the past 200 years have allowed the hybridization between two fish species (Cottus rhenanus and Cottus perifretum) in the Netherlands. Specifically, we address the question of the dynamics of the emergence of these hybrids and invasion of the river systems. Using a set of 81 mostly ancestry-informative SNP markers, as well as broad sample coverage in and around the area of the initial contact, we find a structured hybrid swarm with at least three distinct hybrid lineages that have emerged out of this secondary contact situation. We show that genetically coherent groups can occur at geographically distant locations, while geographically adjacent groups can be genetically different, indicating that some form of reproductive isolation between the lineages is already effective. Using a newly developed modelling approach, we test the relative influence of founding admixture, drift and migration on the allele compositions of the sampling sites. We find that the allele frequency distributions can best be explained if continued gene flow between the parental species and the hybrid lineages is invoked. Genome mapping of the invasive lineage in the Rhine shows that major chromosomal rearrangements were not involved in creating this distinct lineage. Our results show that hybridization after secondary contact can quickly lead to multiple independent new lineages that have the capacity to form hybrid species.

An algorithm for the determination and quantification of components of nucleic acid mixtures based on single sequencing reactions.

BACKGROUND: Determination and quantification of nucleic acid components in a mixture is usually accomplished by microarray approaches, where the mixtures are hybridized against specific probes. As an alternative, we propose here that a single sequencing reaction from a mixture of nucleic acids holds enough information to potentially distinguish the different components, provided it is known which components can occur in the mixture. RESULTS: We describe an algorithm that is based on a set of linear equations which can be solved when the sequencing profiles of the individual components are known and when the number of sequenced nucleotides is larger than the number of components in the mixture. We have implemented the procedure for one type of sequencing approach, pyrosequencing, which produces a stepwise output of peaks that is particularly suitable for the procedure. As an example we use signature sequences from ribosomal RNA to distinguish and quantify several different species in a mixture. Using simulations, we show that the procedure may also be applicable for dideoxy sequencing on capillary sequencers, requiring only some instrument specific adaptations of protocols and software. CONCLUSION: The parallel sequencing approach described here may become a simple and cheap alternative to microarray experiments which aim at routine re-determination and quantification of known nucleic acid components from environmental samples or tissue samples.

An invasive lineage of sculpins, Cottus sp. (Pisces, Teleostei) in the Rhine with new habitat adaptations has originated from hybridization between old phylogeographic groups.

Fish abundance surveys in the Rhine system have shown in the past two decades that there is a rapid upriver invasion of a freshwater sculpin of the genus Cottus. These fish are found in habitats that are atypical for the known species Cottus gobio, which is confined to small cold streams within the Rhine drainage. Phylogeographic analysis based on mitochondrial haplotypes and diagnostic single nucleotide polymorphisms indicates that the invasive sculpins are hybrids between two old lineages from the River Scheldt drainage and the River Rhine drainage, although it is morphologically more similar to the Scheldt sculpins. Most importantly, however, the invasive population possesses a unique ecological potential that does not occur in either of the source populations from the Rhine or the Scheldt, which allows the colonization of new habitats that have previously been free of sculpins. Microsatellite analysis shows that the new lineage is genetically intermediate between the old lineages and that it forms a distinct genetic group across its whole expansion range. We conclude that hybridization between long separated groups has lead to the fast emergence of a new, adaptationally distinct sculpin lineage.