Many tools have been developed for haplotype assembly-the reconstruction of individual haplotypes using reads mapped to a reference genome sequence. Due to increasing interest in obtaining ...haplotype-resolved human genomes, a range of new sequencing protocols and technologies have been developed to enable the reconstruction of whole-genome haplotypes. However, existing computational methods designed to handle specific technologies do not scale well on data from different protocols. We describe a new algorithm, HapCUT2, that extends our previous method (HapCUT) to handle multiple sequencing technologies. Using simulations and whole-genome sequencing (WGS) data from multiple different data types-dilution pool sequencing, linked-read sequencing, single molecule real-time (SMRT) sequencing, and proximity ligation (Hi-C) sequencing-we show that HapCUT2 rapidly assembles haplotypes with best-in-class accuracy for all data types. In particular, HapCUT2 scales well for high sequencing coverage and rapidly assembled haplotypes for two long-read WGS data sets on which other methods struggled. Further, HapCUT2 directly models Hi-C specific error modalities, resulting in significant improvements in error rates compared to HapCUT, the only other method that could assemble haplotypes from Hi-C data. Using HapCUT2, haplotype assembly from a 90× coverage whole-genome Hi-C data set yielded high-resolution haplotypes (78.6% of variants phased in a single block) with high pairwise phasing accuracy (∼98% across chromosomes). Our results demonstrate that HapCUT2 is a robust tool for haplotype assembly applicable to data from diverse sequencing technologies.
Focal oncogene amplification and rearrangements drive tumor growth and evolution in multiple cancer types. We present AmpliconArchitect (AA), a tool to reconstruct the fine structure of focally ...amplified regions using whole genome sequencing (WGS) and validate it extensively on multiple simulated and real datasets, across a wide range of coverage and copy numbers. Analysis of AA-reconstructed amplicons in a pan-cancer dataset reveals many novel properties of copy number amplifications in cancer. These findings support a model in which focal amplifications arise due to the formation and replication of extrachromosomal DNA. Applying AA to 68 viral-mediated cancer samples, we identify a large fraction of amplicons with specific structural signatures suggestive of hybrid, human-viral extrachromosomal DNA. AA reconstruction, integrated with metaphase fluorescence in situ hybridization (FISH) and PacBio sequencing on the cell-line UPCI:SCC090 confirm the extrachromosomal origin and fine structure of a Forkhead box E1 (FOXE1)-containing hybrid amplicon.
Recent reports have demonstrated that oncogene amplification on extrachromosomal DNA (ecDNA) is a frequent event in cancer, providing new momentum to explore a phenomenon first discovered several ...decades ago. The direct consequence of ecDNA gains in these cases is an increase in DNA copy number of the oncogenes residing on the extrachromosomal element. A secondary effect, perhaps even more important, is that the unequal segregation of ecDNA from a parental tumour cell to offspring cells rapidly increases tumour heterogeneity, thus providing the tumour with an additional array of responses to microenvironment-induced and therapy-induced stress factors and perhaps providing an evolutionary advantage. This Perspectives article discusses the current knowledge and potential implications of oncogene amplification on ecDNA in cancer.
Motivation: The goal of the haplotype assembly problem is to reconstruct the two haplotypes (chromosomes) for an individual using a mix of sequenced fragments from the two chromosomes. This problem ...has been shown to be computationally intractable for various optimization criteria. Polynomial time algorithms have been proposed for restricted versions of the problem. In this article, we consider the haplotype assembly problem in the most general setting, i.e. fragments of any length and with an arbitrary number of gaps. Results: We describe a novel combinatorial approach for the haplotype assembly problem based on computing max-cuts in certain graphs derived from the sequenced fragments. Levy et al. have sequenced the complete genome of a human individual and used a greedy heuristic to assemble the haplotypes for this individual. We have applied our method HapCUTto infer haplotypes from this data and demonstrate that the haplotypes inferred using HapCUT are significantly more accurate (20–25% lower maximum error correction scores for all chromosomes) than the greedy heuristic and a previously published method, Fast Hare. We also describe a maximum likelihood based estimator of the absolute accuracy of the sequence-based haplotypes using population haplotypes from the International HapMap project. Availability: A program implementing HapCUT is available on request. Contact: vibansal@cs.ucsd.edu
Proteogenomics has emerged as a field at the junction of genomics and proteomics. It is a loose collection of technologies that allow the search of tandem mass spectra against genomic databases to ...identify and characterize protein-coding genes. Proteogenomic peptides provide invaluable information for gene annotation, which is difficult or impossible to ascertain using standard annotation methods. Examples include confirmation of translation, reading-frame determination, identification of gene and exon boundaries, evidence for post-translational processing, identification of splice-forms including alternative splicing, and also, prediction of completely novel genes. For proteogenomics to deliver on its promise, however, it must overcome a number of technological hurdles, including speed and accuracy of peptide identification, construction and search of specialized databases, correction of sampling bias, and others. This article reviews the state of the art of the field, focusing on the current successes, and the role of computation in overcoming these challenges. We describe how technological and algorithmic advances have already enabled large-scale proteogenomic studies in many model organisms, including arabidopsis, yeast, fly, and human. We also provide a preview of the field going forward, describing early efforts in tackling the problems of complex gene structures, searching against genomes of related species, and immunoglobulin gene reconstruction.
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Variable number tandem repeats (VNTRs) account for significant genetic variation in many organisms. In humans, VNTRs have been implicated in both Mendelian and complex disorders, but are largely ...ignored by genomic pipelines due to the complexity of genotyping and the computational expense. We describe adVNTR-NN, a method that uses shallow neural networks to genotype a VNTR in 18 seconds on 55X whole genome data, while maintaining high accuracy. We use adVNTR-NN to genotype 10,264 VNTRs in 652 GTEx individuals. Associating VNTR length with gene expression in 46 tissues, we identify 163 "eVNTRs". Of the 22 eVNTRs in blood where independent data is available, 21 (95%) are replicated in terms of significance and direction of association. 49% of the eVNTR loci show a strong and likely causal impact on the expression of genes and 80% have maximum effect size at least 0.3. The impacted genes are involved in diseases including Alzheimer's, obesity and familial cancers, highlighting the importance of VNTRs for understanding the genetic basis of complex diseases.
Genetic adaptation to external stimuli occurs through the combined action of mutation and selection. A central problem in genetics is to identify loci responsive to specific selective constraints. ...Many tests have been proposed to identify the genomic signatures of natural selection by quantifying the skew in the site frequency spectrum (SFS) under selection relative to neutrality. We build upon recent work that connects many of these tests under a common framework, by describing how selective sweeps affect the scaled SFS. We show that the specific skew depends on many attributes of the sweep, including the selection coefficient and the time under selection. Using supervised learning on extensive simulated data, we characterize the features of the scaled SFS that best separate different types of selective sweeps from neutrality. We develop a test, SFselect, that consistently outperforms many existing tests over a wide range of selective sweeps. We apply SFselect to polymorphism data from a laboratory evolution experiment of Drosophila melanogaster adapted to hypoxia and identify loci that strengthen the role of the Notch pathway in hypoxia tolerance, but were missed by previous approaches. We further apply our test to human data and identify regions that are in agreement with earlier studies, as well as many novel regions.
Oncogene amplification, a major driver of cancer pathogenicity, is often mediated through focal amplification of genomic segments. Recent results implicate extrachromosomal DNA (ecDNA) as the primary ...driver of focal copy number amplification (fCNA) - enabling gene amplification, rapid tumor evolution, and the rewiring of regulatory circuitry. Resolving an fCNA's structure is a first step in deciphering the mechanisms of its genesis and the fCNA's subsequent biological consequences. We introduce a computational method, AmpliconReconstructor (AR), for integrating optical mapping (OM) of long DNA fragments (>150 kb) with next-generation sequencing (NGS) to resolve fCNAs at single-nucleotide resolution. AR uses an NGS-derived breakpoint graph alongside OM scaffolds to produce high-fidelity reconstructions. After validating its performance through multiple simulation strategies, AR reconstructed fCNAs in seven cancer cell lines to reveal the complex architecture of ecDNA, a breakage-fusion-bridge and other complex rearrangements. By reconstructing the rearrangement signatures associated with an fCNA's generative mechanism, AR enables a more thorough understanding of the origins of fCNAs.
The ability to inexpensively describe taxonomic diversity is critical in this era of rapid climate and biodiversity changes. The recent genome-skimming approach extends current barcoding practices ...beyond short markers by applying low-pass sequencing and recovering whole organelle genomes computationally. This approach discards the nuclear DNA, which constitutes the vast majority of the data. In contrast, we suggest using all unassembled reads. We introduce an assembly-free and alignment-free tool, Skmer, to compute genomic distances between the query and reference genome skims. Skmer shows excellent accuracy in estimating distances and identifying the closest match in reference datasets.
The cost of sequencing the genome is dropping at a much faster rate compared to assembling and finishing the genome. The use of lightly sampled genomes (genome-skims) could be transformative for ...genomic ecology, and results using k-mers have shown the advantage of this approach in identification and phylogenetic placement of eukaryotic species. Here, we revisit the basic question of estimating genomic parameters such as genome length, coverage, and repeat structure, focusing specifically on estimating the k-mer repeat spectrum. We show using a mix of theoretical and empirical analysis that there are fundamental limitations to estimating the k-mer spectra due to ill-conditioned systems, and that has implications for other genomic parameters. We get around this problem using a novel constrained optimization approach (Spline Linear Programming), where the constraints are learned empirically. On reads simulated at 1X coverage from 66 genomes, our method, REPeat SPECTra Estimation (RESPECT), had 2.2% error in length estimation compared to 27% error previously achieved. In shotgun sequenced read samples with contaminants, RESPECT length estimates had median error 4%, in contrast to other methods that had median error 80%. Together, the results suggest that low-pass genomic sequencing can yield reliable estimates of the length and repeat content of the genome. The RESPECT software will be publicly available at https://urldefense.proofpoint.com/v2/url?u=https-3A__github.com_shahab-2Dsarmashghi_RESPECT.git&d=DwIGAw&c=-35OiAkTchMrZOngvJPOeA&r=ZozViWvD1E8PorCkfwYKYQMVKFoEcqLFm4Tg49XnPcA&m=f-xS8GMHKckknkc7Xpp8FJYw_ltUwz5frOw1a5pJ81EpdTOK8xhbYmrN4ZxniM96&s=717o8hLR1JmHFpRPSWG6xdUQTikyUjicjkipjFsKG4w&e=.
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