Crossover Locations Research Articles

A familial, telomere-to-telomere reference for human de novo mutation and recombination from a four-generation pedigree.

Using five complementary short- and long-read sequencing technologies, we phased and assembled >95% of each diploid human genome in a four-generation, 28-member family (CEPH 1463) allowing us to systematically assess de novo mutations (DNMs) and recombination. From this family, we estimate an average of 192 DNMs per generation, including 75.5 de novo single-nucleotide variants (SNVs), 7.4 non-tandem repeat indels, 79.6 de novo indels or structural variants (SVs) originating from tandem repeats, 7.7 centromeric de novo SVs and SNVs, and 12.4 de novo Y chromosome events per generation. STRs and VNTRs are the most mutable with 32 loci exhibiting recurrent mutation through the generations. We accurately assemble 288 centromeres and six Y chromosomes across the generations, documenting de novo SVs, and demonstrate that the DNM rate varies by an order of magnitude depending on repeat content, length, and sequence identity. We show a strong paternal bias (75-81%) for all forms of germline DNM, yet we estimate that 17% of de novo SNVs are postzygotic in origin with no paternal bias. We place all this variation in the context of a high-resolution recombination map (~3.5 kbp breakpoint resolution). We observe a strong maternal recombination bias (1.36 maternal:paternal ratio) with a consistent reduction in the number of crossovers with increasing paternal (r=0.85) and maternal (r=0.65) age. However, we observe no correlation between meiotic crossover locations and de novo SVs, arguing against non-allelic homologous recombination as a predominant mechanism. The use of multiple orthogonal technologies, near-telomere-to-telomere phased genome assemblies, and a multi-generation family to assess transmission has created the most comprehensive, publicly available "truth set" of all classes of genomic variants. The resource can be used to test and benchmark new algorithms and technologies to understand the most fundamental processes underlying human genetic variation.

Insights into non-crossover recombination from long-read sperm sequencing.

Meiotic recombination is a fundamental process that generates genetic diversity by creating new combinations of existing alleles. Although human crossovers have been studied at the pedigree, population and single-cell level, the more frequent non-crossover events that lead to gene conversion are harder to study, particularly at the individual level. Here we show that single high-fidelity long sequencing reads from sperm can capture both crossovers and non-crossovers, allowing effectively arbitrary sample sizes for analysis from one male. Using fifteen sperm samples from thirteen donors we demonstrate variation between and within donors for the rates of different types of recombination. Intriguingly, we observe a tendency for non-crossover gene conversions to occur upstream of nearby PRDM9 binding sites, whereas crossover locations have a slight downstream bias. We further provide evidence for two distinct non-crossover processes. One gives rise to the vast majority of non-crossovers with mean conversion tract length under 50bp, which we suggest is an outcome of standard PRDM9-induced meiotic recombination. In contrast ~2% of non-crossovers have much longer mean tract length, and potentially originate from the same process as complex events with more than two haplotype switches, which is not associated with PRDM9 binding sites and is also seen in somatic cells.

Self-assembly of DNA parallel double-crossover motifs.

DNA double-crossover motifs, including parallel and antiparallel crossovers, serve as the structural foundation for the creation of diverse nanostructures and dynamic devices in DNA nanotechnology. Parallel crossover motifs have unique advantages over the widely used antiparallel crossover design but have not developed as substantially due to the difficulties in assembly. Here we created 29 designs of parallel double-crossover motifs varying in hybridization pathways, central domain lengths, and crossover locations to investigate their assembly mechanism. Arrays were successfully formed in four distinct designs, and large tubular structures were obtained in seven designs with predefined pathways and central domains appoximately 16 nucleotides in length. The nanotubes obtained from parallel crossover design showed improved nuclease resistance than the ones from the antiparallel counterpart design. Overall, our study provides a basis for the development of generalized assembly rules of DNA parallel crossover systems and opens new opportunities for their potential use in biological systems.

Optimization Model for Rail Line Crossover Design Considering the Cost of Delay

In this study, we introduce a method to optimally select the crossover locations of an independent rail line from a set of possible crossover locations considering a fixed number of crossovers that must be used in the design. This optimal selection aims to minimize the cost of passenger delay. Previous research showed that including passenger delay in the decision of rail design choices could be beneficial from economic and societal perspectives. However, those studies were only able to evaluate a few alternatives, because the degraded schedules had to be determined manually. In this research, we introduced an integer nonlinear model to find the best crossover design. We further developed an algorithm to evaluate a set of crossovers and determine the cost of delays for all segments on a rail line given a set of potential disruptions. The monetized cost of passenger delays was used to analyze the tradeoff between the unreliability costs emerging from the delay of passengers in the case of disruptions, and the total number of required crossovers. Our model was applied on a light rail line in Bergen (Norway) resulting in 10% reduction in relation to passenger delays without increasing the number of crossovers; thus, ensuring that there were no additional costs.

Machine-learning predicts genomic determinants of meiosis-driven structural variation in a eukaryotic pathogen

Species harbor extensive structural variation underpinning recent adaptive evolution. However, the causality between genomic features and the induction of new rearrangements is poorly established. Here, we analyze a global set of telomere-to-telomere genome assemblies of a fungal pathogen of wheat to establish a nucleotide-level map of structural variation. We show that the recent emergence of pesticide resistance has been disproportionally driven by rearrangements. We use machine learning to train a model on structural variation events based on 30 chromosomal sequence features. We show that base composition and gene density are the major determinants of structural variation. Retrotransposons explain most inversion, indel and duplication events. We apply our model to Arabidopsis thaliana and show that our approach extends to more complex genomes. Finally, we analyze complete genomes of haploid offspring in a four-generation pedigree. Meiotic crossover locations are enriched for new rearrangements consistent with crossovers being mutational hotspots. The model trained on species-wide structural variation accurately predicts the position of >74% of newly generated variants along the pedigree. The predictive power highlights causality between specific sequence features and the induction of chromosomal rearrangements. Our work demonstrates that training sequence-derived models can accurately identify regions of intrinsic DNA instability in eukaryotic genomes.

Increased Ice Thinning over Svalbard Measured by ICESat/ICESat-2 Laser Altimetry

A decade-long pronounced increase in temperatures in the Arctic, especially in the Barents Sea region, resulted in a global warming hotspot over Svalbard. Associated changes in the cryosphere are the consequence and lead to a demand for monitoring of the glacier changes. This study uses spaceborne laser altimetry data from the ICESat and ICESat-2 missions to obtain ice elevation and mass change rates between 2003–2008 and 2019. Elevation changes are derived at orbit crossover locations throughout the study area, and regional volume and mass changes are estimated using a hypsometric approach. A Svalbard-wide annual elevation change rate of −0.30 ± 0.15 m yr−1 was found, which corresponds to a mass loss rate of −12.40 ± 4.28 Gt yr−1. Compared to the ICESat period (2003–2009), thinning has increased over most regions, including the highest negative rates along the west coast and areas bordering the Barents Sea. The overall negative regime is expected to be linked to Arctic warming in the last decades and associated changes in glacier climatic mass balance. Further, observed increased thinning rates and pronounced changes at the eastern side of Svalbard since the ICESat period are found to correlate with atmospheric and oceanic warming in the respective regions.

Cryo-Electron Microscopy and Mass Analysis of Oligolysine-Coated DNA Nanostructures.

Cationic coatings can enhance the stability of synthetic DNA objects in low ionic strength environments such as physiological fluids. Here, we used single-particle cryo-electron microscopy (cryo-EM), pseudoatomic model fitting, and single-molecule mass photometry to study oligolysine and polyethylene glycol (PEG)-oligolysine-coated multilayer DNA origami objects. The coatings preserve coarse structural features well on a resolution of multiple nanometers but can also induce deformations such as twisting and bending. Higher-density coatings also led to internal structural deformations in the DNA origami test objects, in which a designed honeycomb-type helical lattice was deformed into a more square-lattice-like pattern. Under physiological ionic strength, where the uncoated objects disassembled, the coated objects remained intact but they shrunk in the helical direction and expanded in the direction perpendicular to the helical axis. Helical details like major/minor grooves and crossover locations were not discernible in cryo-EM maps that we determined of DNA origami coated with oligolysine and PEG-oligolysine, whereas these features were visible in cryo-EM maps determined from the uncoated reference objects. Blunt-ended double-helical interfaces remained accessible underneath the coating and may be used for the formation of multimeric DNA origami assemblies that rely on stacking interactions between blunt-ended helices. The ionic strength requirements for forming multimers from coated DNA origami differed from those needed for uncoated objects. Using single-molecule mass photometry, we found that the mass of coated DNA origami objects prior to and after incubation in low ionic strength physiological conditions remained unchanged. This finding indicated that the coating effectively prevented strand dissociation but also that the coating itself remained stable in place. Our results validate oligolysine coatings as a powerful stabilization method for DNA origami but also reveal several potential points of failure that experimenters should watch to avoid working with false premises.

Inflow characteristics for generating axis-switching phenomenon of heated rectangular jets

Flow characteristics due to the axis-switching phenomenon are numerically investigated for heated rectangular jets with typical inlet conditions and three aspect ratios (AR), namely 1.0, 1.5, and 2.0. To examine various inlets and their relation to the axis-switching flow, an inflow generator is newly devised, wherein jet flows from an orifice plate and a long pipe nozzle can be realized. Depending on the inlet conditions, the 45° and 90° axis-switching phenomena for the rectangular jet are observed to appear differently. As the aspect ratio increases and jet temperature decreases, the axis-switching positions are delayed downstream. It is observed that the strong and weak states of the axis-switching flows can be interpreted by the crossover locations of the jet half-widths and the saddle-shaped velocity profile is not the only main factor for axis-switching flow. As the jet temperature increases, a well-mixed state is quickly obtained, and the mixing performance of the square jet is better than that of the rectangular jet. For both isothermal and heated jets, the flow mixing is improved by the axis-switching flow. Various analyses are attempted to find the specific inlet structure for the axis-switching flow. From the results, the special features for inflow criterion producing axis-switching flow can be explained by factors such as the deformation parameter, interrelations between shear strain and normal rates, turbulence-driven and geometry-driven streamwise vortices, and normal Reynolds stresses.

Automatic identification of crossovers in cryo-EM images of murine amyloid protein A fibrils with machine learning.

Detecting crossovers in cryo-electron microscopy images of protein fibrils is an important step towards determining the morphological composition of a sample. Currently, the crossover locations are picked by hand, which introduces errors and is a time-consuming procedure. With the rise of deep learning in computer vision tasks, the automation of such problems has become more and more applicable. However, because of insufficient quality of raw data and missing labels, neural networks alone cannot be applied successfully to target the given problem. Thus, we propose an approach combining conventional computer vision techniques and deep learning to automatically detect fibril crossovers in two-dimensional cryo-electron microscopy image data and apply it to murine amyloid protein A fibrils, where we first use direct image processing methods to simplify the image data such that a convolutional neural network can be applied to the remaining segmentation problem. LAY DESCRIPTION: The ability of protein to form fibrillary structures underlies important cellular functions but can also give rise to disease, such as in a group of disorders, termed amyloid diseases. These diseases are characterised by the formation of abnormal protein filaments, so-called amyloid fibrils, that deposit inside the tissue. Many amyloid fibrils are helically twisted, which leads to periodic variations in the apparent width of the fibril, when observing amyloid fibrils using microscopy techniques like cryogenic electron microscopy (cryo-EM). Due to the two-dimensional projection, parts of the fibril orthogonal to the projection plane appear narrower than parts parallel to the plane. The parts of small width are called crossovers. The distance between two adjacent crossovers is an important characteristic for the analysis of amyloid fibrils, because it is informative about the fibril morphology and because it can be determined from raw data by eye. A given protein can typically form different fibril morphologies. The morphology can vary depending on the chemical and physical conditions of fibril formation, but even when fibrils are formed under identical solution conditions, different morphologies may be present in a sample. As the crossovers allow to define fibril morphologies in a heterogeneous sample, detecting crossovers is an important first step in the sample analysis. In the present paper, we introduce a method for the automated detection of fibril crossovers in cryo-EM image data. The data consists of greyscale images, each showing an unknown number of potentially overlapping fibrils. In a first step, techniques from image analysis and pattern detection are employed to detect single fibrils in the raw data. Then, a convolutional neural network is used to find the locations of crossovers on each single fibril. As these predictions may contain errors, further postprocessing steps assess the quality and may slightly alter or reject the predictedcrossovers.

New Results from Strapdown Airborne Gravimetry Using Temperature Stabilisation

In recent years, the use of a strapdown Inertial Measurement Unit (IMU) for airborne gravimetry has proven itself to be an accurate and resilient measurement system, improving the operational flexibility. The main concern is erroneous long-wavelength information in the resulting estimates, which is suspected to originate from uncompensated long-term drift of the accelerometers, probably originating from temperature variation. For this reason, iMAR navigation has designed a temperature stabilisation box, which allows for temperature stabilisation of their IMU systems. On a regional airborne gravity survey over the Kattegat Sea between Denmark and Sweden, such a temperature stabilised strapdown IMU was operated alongside a traditional spring-type platform-stabilised gravity system from ZLS. An analysis of the difference in gravity estimates at cross-over locations yielded a mean value of −0.3 mGal for the iMAR system with an indicated accuracy of 1.0 mGal. The temperature stabilisation unit therefore effectively limits the accelerometer drift and improves the long-wavelength information. However, a straightforward merging approach, adjusting the line-based mean values of the iMAR estimates to match that of the ZLS estimates, improved the accuracy to 0.8 mGal. This indicates that the long-wavelength information of the stabilised-platform system is still superior to that of the strapdown system.

Retinal vascular junction detection and classification via deep neural networks

Background and ObjectivesThe retinal fundus contains intricate vascular trees, some of which are mutually intersected and overlapped. The intersection and overlapping of retinal vessels represent vascular junctions (i.e. bifurcation and crossover) in 2D retinal images. These junctions are important for analyzing vascular diseases and tracking the morphology of vessels. In this paper, we propose a two-stage pipeline to detect and classify the junction points. MethodsIn the detection stage, a RCNN-based Junction Proposal Network is utilized to search the potential bifurcation and crossover locations directly on color retinal images, which is followed by a Junction Refinement Network to eliminate the false detections. In the classification stage, the detected junction points are identified as crossover or bifurcation using the proposed Junction Classification Network that shares the same model structure with the refinement network. ResultsOur approach achieves 70% and 60% F1-score on DRIVE and IOSTAR dataset respectively which outperform the state-of-the-art methods by 4.5% and 1.7%, with a high and balanced precision and recall values. ConclusionsThis paper proposes a new junction detection and classification method which performs directly on color retinal images without any vessel segmentation nor skeleton preprocessing. The superior performance demonstrates that the effectiveness of our approach.

Assessing the potential for measuring Europa's tidal Love number h2 using radar sounder and topographic imager data

The tidal Love number h2 is a key geophysical measurement for the characterization of Europa's interior, especially of its outer ice shell if a subsurface ocean is present. We performed numerical simulations to assess the potential for estimating h2 using altimetric measurements with a combination of radar sounding and stereo imaging data. The measurement principle exploits both delay and Doppler information in the radar surface return in combination with topography from a digital terrain model (DTM). The resulting radar range measurements at cross-over locations can be used in combination with radio science Doppler data for an improved trajectory solution and for estimating the h2 Love number. Our simulation results suggest that the absolute accuracy of h2 from the joint analysis of REASON (Radar for Europa Assessment and Sounding: Ocean to Near-surface) surface return and EIS (Europa Imaging System) DTM data will be in the range of 0.04–0.17 assuming full radio link coverage. The error is controlled by the SNR budget and DTM quality, both dependent on the surface properties of Europa. We estimate that this would unambiguously confirm (or reject) the global ocean hypothesis and, in combination with a nominal radio-science based measurement of the tidal Love number k2, constrain the thickness of Europa's outer ice shell to up to ±15 km.

Temporal scaling phenomena in groundwater-floodplain systems using robust detrended fluctuation analysis

In order to determine objectively the fractal behaviour of a time series, and to facilitate potential future attempts to assess model performance by incorporating fractal behaviour, a multi-order robust detrended fluctuation analysis (r-DFAn) procedure is developed herein. The r-DFAn procedure allows for robust and automated quantification of mono-fractal behaviour. The fractal behaviour is quantified with three parts: a global scaling exponent, crossovers, and local scaling exponents. The robustness of the r-DFAn procedure is established by the systematic use of robust regression, piecewise linear regression, Analysis of Covariance (ANCOVA) and Multiple Comparison Procedure to determine statistically significant scaling exponents and optimum crossover locations. The MATLAB code implementing the r-DFAn procedure has also been open sourced to enable reproducible results.r-DFAn will be illustrated on a synthetic signal after which is used to analyse high-resolution hydrologic data; although the r-DFAn procedure is not limited to hydrological or geophysical time series. The hydrological data are 4year-long datasets (January 2012 to January 2016) of 1-min groundwater level, river stage, groundwater and river temperature, and 15-min precipitation and air temperature, at Wallingford, UK. The datasets are analysed in both time and fractal domains. The study area is a shallow riparian aquifer in hydraulic connection to River Thames, which traverses the site. The unusually high resolution datasets, along with the responsive nature of the aquifer, enable detailed examination of the various data and their interconnections in both time- and fractal-domains.

Optimizing Intermediate Crossovers and Ramp Closures for Hurricane Evacuation

Current hurricane evacuation plans of coastal states in the United States do not actively coordinate the provision of contraflow access points and ramp closure locations on evacuation routes. In this paper, contraflow and ramp closure techniques are integrated and optimized to achieve higher evacuation efficiency. A simulation-based optimization framework for identifying the intermediate crossover between regular and contraflow lanes and ramp closure locations is proposed. The framework is applied to a case study of Hampton Roads, Virginia; using models developed based on evacuee behavioral data and transportation network and traffic control data. Two heuristic solution approaches, Genetic algorithm and Tabu search, are applied to determine optimal intermediate crossover and ramp closure locations for the case study network. The importance of each crossover and ramp closure location included in the optimal solution was also determined. Keywords: hurricane evacuation, intermediate crossovers, contraflow, heuristic algorithm, dynamic traffic assignment Copyright c 2016 SERSC Language: en

Fine-Scale Crossover Rate Variation on the Caenorhabditis elegans X Chromosome.

Meiotic recombination creates genotypic diversity within species. Recombination rates vary substantially across taxa, and the distribution of crossovers can differ significantly among populations and between sexes. Crossover locations within species have been found to vary by chromosome and by position within chromosomes, where most crossover events occur in small regions known as recombination hotspots. However, several species appear to lack hotspots despite significant crossover heterogeneity. The nematode Caenorhabditis elegans was previously found to have the least fine-scale variation in crossover distribution among organisms studied to date. It is unclear whether this pattern extends to the X chromosome given its unique compaction through the pachytene stage of meiotic prophase in hermaphrodites. We generated 798 recombinant nested near-isogenic lines (NILs) with crossovers in a 1.41 Mb region on the left arm of the X chromosome to determine if its recombination landscape is similar to that of the autosomes. We find that the fine-scale variation in crossover rate is lower than that of other model species, and is inconsistent with hotspots. The relationship of genomic features to crossover rate is dependent on scale, with GC content, histone modifications, and nucleosome occupancy being negatively associated with crossovers. We also find that the abundances of 4- to 6-bp DNA motifs significantly explain crossover density. These results are consistent with recombination occurring at unevenly distributed sites of open chromatin.

High-Resolution Mapping of Crossover and Non-crossover Recombination Events by Whole-Genome Re-sequencing of an Avian Pedigree.

Recombination is an engine of genetic diversity and therefore constitutes a key process in evolutionary biology and genetics. While the outcome of crossover recombination can readily be detected as shuffled alleles by following the inheritance of markers in pedigreed families, the more precise location of both crossover and non-crossover recombination events has been difficult to pinpoint. As a consequence, we lack a detailed portrait of the recombination landscape for most organisms and knowledge on how this landscape impacts on sequence evolution at a local scale. To localize recombination events with high resolution in an avian system, we performed whole-genome re-sequencing at high coverage of a complete three-generation collared flycatcher pedigree. We identified 325 crossovers at a median resolution of 1.4 kb, with 86% of the events localized to <10 kb intervals. Observed crossover rates were in excellent agreement with data from linkage mapping, were 52% higher in male (3.56 cM/Mb) than in female meiosis (2.28 cM/Mb), and increased towards chromosome ends in male but not female meiosis. Crossover events were non-randomly distributed in the genome with several distinct hot-spots and a concentration to genic regions, with the highest density in promoters and CpG islands. We further identified 267 non-crossovers, whose location was significantly associated with crossover locations. We detected a significant transmission bias (0.18) in favour of ‘strong’ (G, C) over ‘weak’ (A, T) alleles at non-crossover events, providing direct evidence for the process of GC-biased gene conversion in an avian system. The approach taken in this study should be applicable to any species and would thereby help to provide a more comprehensive portray of the recombination landscape across organism groups.

On Measurements of the Tide at Churchill, Hudson Bay

Since the late 1990s the semi-diurnal tide at Churchill, on the western shore of Hudson Bay, has been decreasing in amplitude, with M2 amplitudes falling from approximately 154 cm in 1998 to 146 cm in 2012 and 142 cm in 2014. There has been a corresponding small increase in phase lag. Mean low water, decreasing throughout most of the twentieth century, has levelled off. Although the tidal changes could reflect merely a malfunctioning tide gauge, the fact that there are no other measurements in the region and the possibility that the tide is revealing important environmental changes calls for serious investigation. Satellite altimeter measurements of the tide in Hudson Bay are complicated by the seasonal ice cover; at most locations less than 40% of satellite passes return valid ocean heights and even those can be impacted by errors from sea ice. Because the combined TOPEX/Poseidon, Jason-1, and Jason-2 time series is more than 23 years long, it is now possible to obtain sufficient data at crossover locations near Churchill to search for tidal changes. The satellites sense no changes in M2 that are comparable to the changes seen at the Churchill gauge. The changes appear to be localized to the harbour, or to the Churchill River, or to the gauge itself.

Prediction and efficient control of vibration mitigation using floating slabs: practical application at Athens metro lines 2 and 3

This paper describes the finite element modelling and the in situ evaluation of anti-vibration mitigation measures recently introduced in the latest two extensions on lines 2 and 3 of the Athens Metro: (1) extension of Line 3 from Aigaleo to Haidari and (2) extension of Line 2 from Agios Antonios to Anthoupoli. Appropriate mitigation measures, guaranteeing the allowable ground-borne noise and vibration levels, especially in crossover (CO) locations, were examined, within a noise and vibration study. To do this, the alignment was divided into homogeneous sections, i.e. sections along which the tunnel and soil types, depth and distance from nearby buildings, with particular emphasis on section with COs on direct fixation. Floating slab (FS) track solutions were then evaluated at COs locations ensuring a complete ground-borne vibration attenuation with an insertion loss of −20 dBV with respect to the predominant excitation (40–125 Hz). The relevant in situ measurement campaign regarding both the dynamic characteristics of FS and the vibration velocity at the relevant receptors proves that the ground-borne noise in the nearest – to FS – buildings at all CO locations was significantly lower than the maximum allowable limits proving the high efficiency of the mitigation measure.

TroX: a new method to learn about the genesis of aneuploidy from trisomic products of conception.

An estimated 10-30% of clinically recognized conceptions are aneuploid, leading to spontaneous miscarriages, in vitro fertilization failures and, when viable, severe developmental disabilities. With the ongoing reduction in the cost of genotyping and DNA sequencing, the use of high-density single nucleotide polymorphism (SNP) markers for clinical diagnosis of aneuploidy and biomedical research into its causes is becoming common practice. A reliable, flexible and computationally feasible method for inferring the sources of aneuploidy is thus crucial. We propose a new method, TroX, for analyzing human trisomy data using high density SNP markers from a trisomic individual or product of conception and one parent. Using a hidden Markov model, we infer the stage of the meiotic error (I or II) and the individual in which non-disjunction event occurred, as well as the crossover locations on the trisomic chromosome. A novel and important feature of the method is its reliance on data from the proband and only one parent, reducing the experimental cost by a third and enabling a larger set of data to be used. We evaluate our method by applying it to simulated trio data as well as to genotype data for 282 trios that include a child trisomic for chromosome 21. The analyses show the method to be highly reliable even when data from only one parent are available. With the increasing availability of DNA samples from mother and fetus, application of approaches such as ours should yield unprecedented insights into the genetic risk factors for aneuploidy. An R package implementing TroX is available for download at http://przeworski.uchicago.edu/.

Mobility Benefits of Intermediate Crossovers on Contraflow Facilities during Hurricane Evacuation

A contraflow facility, in which the direction of traffic on one or more travel lanes is reversed to increase outbound capacity, is a major component of hurricane evacuation plans in many coastal states. Contraflow facilities typically are implemented when significant evacuation demand is expected (e.g., during a severe hurricane). Typical contraflow plans have one upstream crossover that routes vehicles into the contraflow lanes and one downstream crossover that routes vehicles back into the regular lanes. How can contraflow facilities best be used to optimize evacuation performance? The impact on mobility of additional intermediate crossovers between the beginning and termination points of contraflow is investigated. The proposed heuristic algorithm is the first attempt in the literature to provide a systematic approach–-with iterative elimination–-to determine the critical intermediate crossover locations that provide maximum improvement in evacuation performance. Results of a case study of I-64 in Hampton Roads, Virginia, indicate that adding intermediate crossovers improves network performance for medium-and high-demand evacuation situations. For low-demand situations, the addition of intermediate crossovers does not improve network performance. Therefore, any consideration of intermediate crossovers for low-demand evacuations must be solely to provide access to roadside facilities such as gas, food, and emergency services. For medium- and high-demand situations, the average travel time improved 28% when four intermediate crossovers of the 44 potential crossover locations were deployed in the I-64 corridor.