Haplotyping germline and cancer genomes with high-throughput linked-read sequencing.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

Group-based variant calling leveraging next-generation supercomputing for large-scale whole-genome sequencing studies.

MOTIVATION: Next-generation sequencing (NGS) technologies have become much more efficient, allowing whole human genomes to be sequenced faster and cheaper than ever before. However, processing the raw sequence reads associated with NGS technologies requires care and sophistication in order to draw compelling inferences about phenotypic consequences of variation in human genomes. It has been shown that different approaches to variant calling from NGS data can lead to different conclusions. Ensuring appropriate accuracy and quality in variant calling can come at a computational cost. RESULTS: We describe our experience implementing and evaluating a group-based approach to calling variants on large numbers of whole human genomes. We explore the influence of many factors that may impact the accuracy and efficiency of group-based variant calling, including group size, the biogeographical backgrounds of the individuals who have been sequenced, and the computing environment used. We make efficient use of the Gordon supercomputer cluster at the San Diego Supercomputer Center by incorporating job-packing and parallelization considerations into our workflow while calling variants on 437 whole human genomes generated as part of large association study. CONCLUSIONS: We ultimately find that our workflow resulted in high-quality variant calls in a computationally efficient manner. We argue that studies like ours should motivate further investigations combining hardware-oriented advances in computing systems with algorithmic developments to tackle emerging 'big data' problems in biomedical research brought on by the expansion of NGS technologies.

Reactive simulations of the activation barrier to dissolution of amorphous silica in water.

Molecular dynamics simulations employing reactive potentials were used to determine the activation barriers to the dissolution of the amorphous SiO2 surface in the presence of a 2 nm overlayer of water. The potential of mean force calculations of the reactions of water molecules with 15 different starting Q4 sites (Qi is the Si site with i bridging oxygen neighbors) to eventually form the dissolved Q0 site were used to obtain the barriers. Activation barriers for each step in the dissolution process, from the Q4 to Q3 to Q2 to Q1 to Q0 were obtained. Relaxation runs between each reaction step enabled redistribution of the water above the surface in response to the new Qi site configuration. The rate-limiting step observed in the simulations was in both the Q32 reaction (a Q3 site changing to a Q2 site) and the Q21 reaction, each with an average barrier of ∼14.1 kcal mol(-1). However, the barrier for the overall reaction from the Q4 site to a Q0 site, averaged over the maximum barrier for each of the 15 samples, was 15.1 kcal mol(-1). This result is within the lower end of the experimental data, which varies from 14-24 kcal mol(-1), while ab initio calculations using small cluster models obtain values that vary from 18-39 kcal mol(-1). Constraints between the oxygen bridges from the Si site and the connecting silica structure, the presence of pre-reaction strained siloxane bonds, and the location of the reacting Si site within slight concave surface contours all affected the overall activation barriers.

Lifetimes of excess protons in water using a dissociative water potential.

Molecular dynamics simulations using a dissociative water potential were applied to study transport of excess protons in water and determine the applicability of this potential to describe such behavior. While originally developed for gas-phase molecules and bulk liquid water, the potential is transferrable to nanoconfinement and interface scenarios. Applied here, it shows proton behavior consistent with ab initio calculations and empirical models specifically designed to describe proton transport. Both Eigen and Zundel complexes are observed in the simulations showing the Eigen-Zundel-Eigen-type mechanism. In addition to reproducing the short-time rattling of the excess proton between the two oxygens of Zundel complexes, a picosecond-scale lifetime was also found. These longer-lived H3O(+) ions are caused by the rapid conversion of the local solvation structure around the transferring proton from a Zundel-like form to an Eigen-like form following the transfer, effectively severing the path along which the proton can rattle. The migration of H(+) over long times (>100 ps) deviates from the conventional short-time multiexponentially decaying lifetime autocorrelation model and follows the t(-3/2) power-law behavior. The potential function employed here matches many of the features of proton transport observed in ab initio molecular dynamics simulations as well as the highly developed empirical valence bond models, yet is computationally very efficient, enabling longer time and larger systems to be studied.

Bridging oxygen as a site for proton adsorption on the vitreous silica surface.

Molecular dynamics computer simulations were used to study the protonation of bridging oxygen (Si-O-Si) sites present on the vitreous silica surface in contact with water using a dissociative water potential. In contrast to first-principles calculations based on unconstrained molecular analogs, such as H(7)Si(2)O(7)(+) molecules, the very limited flexibility of neighboring SiO(4) tetrahedra when embedded in a solid surface means that there is a relatively minor geometric response to proton adsorption, requiring sites predisposed to adsorption. Simulation results indicate that protonation of bridging oxygen occurs at predisposed sites with bridging angles in the 125 degrees-135 degrees range, well below the bulk silica mean of approximately 150 degrees, consistent with various ab initio calculations, and that a small fraction of such sites are present in all ring sizes. The energy differences between dry and protonated bridges at various angles observed in the simulations coincide completely with quantum calculations over the entire range of bridging angles encountered in the vitreous silica surface. Those sites with bridging angles near 130 degrees support adsorbed protons more stably, resulting in the proton remaining adsorbed for longer periods of time. Vitreous silica has the necessary distribution of angular strain over all ring sizes to allow protons to adsorb onto bridging oxygen at the surface, forming acidic surface groups that serve as ideal intermediate steps in proton transfer near the surface. In addition to hydronium formation and water-assisted proton transfer in the liquid, protons can rapidly move across the water-silica interface via strained bridges that are predisposed to transient proton adsorption. Thus, an excess proton at any given location on a silica surface can move by either water-assisted or strained bridge-assisted diffusion depending on the local environment. The result of this would be net migration that is faster than it would be if only one mechanism is possible. These simulation results indicate the importance of performing large size and time scale simulations of the structurally heterogeneous vitreous silica exposed to water to describe proton transport at the interface between water and the silica surface.