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July 7, 2019  |  

Third-generation sequencing and the future of genomics

Third-generation long-range DNA sequencing and mapping technologies are creating a renaissance in high-quality genome sequencing. Unlike second-generation sequencing, which produces short reads a few hundred base-pairs long, third-generation single-molecule technologies generate over 10,000 bp reads or map over 100,000 bp molecules. We analyze how increased read lengths can be used to address long-standing problems in de novo genome assembly, structural variation analysis and haplotype phasing.


July 7, 2019  |  

Draft genome sequences of three European laboratory derivatives from enterohemorrhagic Escherichia coli O157:H7 strain EDL933, including two plasmids.

Escherichia coliO157:H7 EDL933, isolated in 1982 in the United States, was the first enterohemorrhagicE. coli(EHEC) strain sequenced. Unfortunately, European labs can no longer receive the original strain. We checked three European EDL933 derivatives and found major genetic deviations (deletions, inversions) in two strains. All EDL933 strains contain the cryptic EHEC-plasmid, not reported before. Copyright © 2016 Fellner et al.


July 7, 2019  |  

Exploring structural variants in environmentally sensitive gene families.

Environmentally sensitive plant gene families like NBS-LRRs, receptor kinases, defensins and others, are known to be highly variable. However, most existing strategies for discovering and describing structural variation in complex gene families provide incomplete and imperfect results. The move to de novo genome assemblies for multiple accessions or individuals within a species is enabling more comprehensive and accurate insights about gene family variation. Earlier array-based genome hybridization and sequence-based read mapping methods were limited by their reliance on a reference genome and by misplacement of paralogous sequences. Variant discovery based on de novo genome assemblies overcome the problems arising from a reference genome and reduce sequence misplacement. As de novo genome sequencing moves to the use of longer reads, artifacts will be minimized, intact tandem gene clusters will be constructed accurately, and insights into rapid evolution will become feasible. Copyright © 2016 Elsevier Ltd. All rights reserved.


July 7, 2019  |  

The Atlantic salmon genome provides insights into rediploidization.

The whole-genome duplication 80 million years ago of the common ancestor of salmonids (salmonid-specific fourth vertebrate whole-genome duplication, Ss4R) provides unique opportunities to learn about the evolutionary fate of a duplicated vertebrate genome in 70 extant lineages. Here we present a high-quality genome assembly for Atlantic salmon (Salmo salar), and show that large genomic reorganizations, coinciding with bursts of transposon-mediated repeat expansions, were crucial for the post-Ss4R rediploidization process. Comparisons of duplicate gene expression patterns across a wide range of tissues with orthologous genes from a pre-Ss4R outgroup unexpectedly demonstrate far more instances of neofunctionalization than subfunctionalization. Surprisingly, we find that genes that were retained as duplicates after the teleost-specific whole-genome duplication 320 million years ago were not more likely to be retained after the Ss4R, and that the duplicate retention was not influenced to a great extent by the nature of the predicted protein interactions of the gene products. Finally, we demonstrate that the Atlantic salmon assembly can serve as a reference sequence for the study of other salmonids for a range of purposes.


July 7, 2019  |  

Conservation of the essential genome among Caulobacter and Brevundimonas species.

When the genomes of Caulobacter isolates NA1000 and K31 were compared, numerous genome rearrangements were observed. In contrast, similar comparisons of closely related species of other bacterial genera revealed nominal rearrangements. A phylogenetic analysis of the 16S rRNA indicated that K31 is more closely related to Caulobacter henricii CB4 than to other known Caulobacters. Therefore, we sequenced the CB4 genome and compared it to all of the available Caulobacter genomes to study genome rearrangements, discern the conservation of the NA1000 essential genome, and address concerns about using 16S rRNA to group Caulobacter species. We also sequenced the novel bacteria, Brevundimonas DS20, a representative of the genus most closely related to Caulobacter and used it as part of an outgroup for phylogenetic comparisons. We expected to find that there would be fewer rearrangements when comparing more closely related Caulobacters. However, we found that relatedness was not correlated with the amount of observed “genome scrambling.” We also discovered that nearly all of the essential genes previously identified for C. crescentus are present in the other Caulobacter genomes and in the Brevundimonas genomes as well. However, a few of these essential genes were only found in NA1000, and some were missing in a combination of one or more species, while other proteins were 100 % identical across species. Also, phylogenetic comparisons of highly conserved genomic regions revealed clades similar to those identified by 16S rRNA-based phylogenies, verifying that 16S rRNA sequence comparisons are a valid method for grouping Caulobacters.


July 7, 2019  |  

Gene duplication confers enhanced expression of 27-kDa ?-zein for endosperm modification in quality protein maize.

The maizeopaque2(o2) mutant has a high nutritional value but it develops a chalky endosperm that limits its practical use. Genetic selection foro2modifiers can convert the normally chalky endosperm of the mutant into a hard, vitreous phenotype, yielding what is known as quality protein maize (QPM). Previous studies have shown that enhanced expression of 27-kDa ?-zein in QPM is essential for endosperm modification. Taking advantage of genome-wide association study analysis of a natural population, linkage mapping analysis of a recombinant inbred line population, and map-based cloning, we identified a quantitative trait locus (q?27) affecting expression of 27-kDa ?-zein.q?27was mapped to the same region as the majoro2 modifier(o2 modifier1) on chromosome 7 near the 27-kDa ?-zein locus.q?27resulted from a 15.26-kb duplication at the 27-kDa ?-zein locus, which increases the level of gene expression. This duplication occurred before maize domestication; however, the gene structure ofq?27appears to be unstable and the DNA rearrangement frequently occurs at this locus. Because enhanced expression of 27-kDa ?-zein is critical for endosperm modification in QPM,q?27is expected to be under artificial selection. This discovery provides a useful molecular marker that can be used to accelerate QPM breeding.


July 7, 2019  |  

Single-molecule sequencing assists genome assembly improvement and structural variation inference.

Dear editor, The single-molecule real-time (SMRT) sequencing platform presented by Pacific Biosciences (PacBio) is regarded as a third-generation sequencing technology (Eid et al., 2009, Roberts et al., 2013). PacBio delivers long reads from several to tens of kilobases (kbs), which are ideal for filling unsequenced gaps due to unusual sequence contexts, such as high-GC content or repeat-rich regions (Bashir et al., 2012, Berlin et al., 2015, Chaisson et al., 2015). PacBio long reads are also favorable for detecting large DNA fragments harboring structural variations (SVs), such as inversions, translocations, duplications, and large insertions/deletions (indels) (Ritz et al., 2010, English et al., 2014). However, one drawback of PacBio is the high error rate of base calling for single pass coverage of the genome (Au et al., 2012, Koren et al., 2012). This drawback can be mitigated by increasing sequencing coverage to achieve high consensus accuracy, but the requirements may be prohibitive for the de novo assembly of large- or medium-size genomes using only PacBio when considering both budgetary and computational costs. Alternatively, PacBio may be used for assembly improvement of near-finished reference genomes, especially for filling gaps in which unsequenced bases are represented by the letter N (English et al., 2012). Here, we combined PacBio (~15x) with Illumina reads (~40x) to improve the genome assemblies of African wild (Oryza barthii) and cultivated rice (O. glaberrima), and to infer large SVs between O. barthii and O. glaberrima.


July 7, 2019  |  

Understanding the genetics of APOE and TOMM40 and role of mitochondrial structure and function in clinical pharmacology of Alzheimer’s disease.

The methodology of Genome-Wide Association Screening (GWAS) has been applied for more than a decade. Translation to clinical utility has been limited, especially in Alzheimer’s Disease (AD). It has become standard practice in the analyses of more than two dozen AD GWAS studies to exclude the apolipoprotein E (APOE) region because of its extraordinary statistical support, unique thus far in complex human diseases. New genes associated with AD are proposed frequently based on SNPs associated with odds ratio (OR) < 1.2. Most of these SNPs are not located within the associated gene exons or introns but are located variable distances away. Often pathologic hypotheses for these genes are presented, with little or no experimental support. By eliminating the analyses of the APOE-TOMM40 linkage disequilibrium region, the relationship and data of several genes that are co-located in that LD region have been largely ignored. Early negative interpretations limited the interest of understanding the genetic data derived from GWAS, particularly regarding the TOMM40 gene. This commentary describes the history and problem(s) in interpretation of the genetic interrogation of the "APOE" region and provides insight into a metabolic mitochondrial basis for the etiology of AD using both APOE and TOMM40 genetics. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.


July 7, 2019  |  

Alpha-CENTAURI: assessing novel centromeric repeat sequence variation with long read sequencing.

Long arrays of near-identical tandem repeats are a common feature of centromeric and subtelomeric regions in complex genomes. These sequences present a source of repeat structure diversity that is commonly ignored by standard genomic tools. Unlike reads shorter than the underlying repeat structure that rely on indirect inference methods, e.g. assembly, long reads allow direct inference of satellite higher order repeat structure. To automate characterization of local centromeric tandem repeat sequence variation we have designed Alpha-CENTAURI (ALPHA satellite CENTromeric AUtomated Repeat Identification), that takes advantage of Pacific Bioscience long-reads from whole-genome sequencing datasets. By operating on reads prior to assembly, our approach provides a more comprehensive set of repeat-structure variants and is not impacted by rearrangements or sequence underrepresentation due to misassembly.We demonstrate the utility of Alpha-CENTAURI in characterizing repeat structure for alpha satellite containing reads in the hydatidiform mole (CHM1, haploid-like) genome. The pipeline is designed to report local repeat organization summaries for each read, thereby monitoring rearrangements in repeat units, shifts in repeat orientation and sites of array transition into non-satellite DNA, typically defined by transposable element insertion. We validate the method by showing consistency with existing centromere high order repeat references. Alpha-CENTAURI can, in principle, run on any sequence data, offering a method to generate a sequence repeat resolution that could be readily performed using consensus sequences available for other satellite families in genomes without high-quality reference assemblies.Documentation and source code for Alpha-CENTAURI are freely available at http://github.com/volkansevim/alpha-CENTAURI CONTACT: ali.bashir@mssm.eduSupplementary information: Supplementary data are available at Bioinformatics online.© The Author 2016. Published by Oxford University Press.


July 7, 2019  |  

Next-generation sequencing-based detection of germline L1-mediated transductions.

While active LINE-1 (L1) elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking.Here we present the computational approach TIGER (Transduction Inference in GERmline genomes), enabling the discovery of non-reference L1-mediated transductions by combining L1 discovery with detection of unique insertion sequences and detailed characterization of insertion sites. We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome. We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species.By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis.


July 7, 2019  |  

Population scale mapping of transposable element diversity reveals links to gene regulation and epigenomic variation.

Variation in the presence or absence of transposable elements (TEs) is a major source of genetic variation between individuals. Here, we identified 23,095 TE presence/absence variants between 216 Arabidopsis accessions. Most TE variants were rare, and we find these rare variants associated with local extremes of gene expression and DNA methylation levels within the population. Of the common alleles identified, two thirds were not in linkage disequilibrium with nearby SNPs, implicating these variants as a source of novel genetic diversity. Many common TE variants were associated with significantly altered expression of nearby genes, and a major fraction of inter-accession DNA methylation differences were associated with nearby TE insertions. Overall, this demonstrates that TE variants are a rich source of genetic diversity that likely plays an important role in facilitating epigenomic and transcriptional differences between individuals, and indicates a strong genetic basis for epigenetic variation.


July 7, 2019  |  

Microevolution of monophasic Salmonella Typhimurium during epidemic, United Kingdom, 2005-2010.

Microevolution associated with emergence and expansion of new epidemic clones of bacterial pathogens holds the key to epidemiologic success. To determine microevolution associated with monophasic Salmonella Typhimurium during an epidemic, we performed comparative whole-genome sequencing and phylogenomic analysis of isolates from the United Kingdom and Italy during 2005-2012. These isolates formed a single clade distinct from recent monophasic epidemic clones previously described from North America and Spain. The UK monophasic epidemic clones showed a novel genomic island encoding resistance to heavy metals and a composite transposon encoding antimicrobial drug resistance genes not present in other Salmonella Typhimurium isolates, which may have contributed to epidemiologic success. A remarkable amount of genotypic variation accumulated during clonal expansion that occurred during the epidemic, including multiple independent acquisitions of a novel prophage carrying the sopE gene and multiple deletion events affecting the phase II flagellin locus. This high level of microevolution may affect antigenicity, pathogenicity, and transmission.


July 7, 2019  |  

A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer.

Although human LINE-1 (L1) elements are actively mobilized in many cancers, a role for somatic L1 retrotransposition in tumor initiation has not been conclusively demonstrated. Here, we identify a novel somatic L1 insertion in the APC tumor suppressor gene that provided us with a unique opportunity to determine whether such insertions can actually initiate colorectal cancer (CRC), and if so, how this might occur. Our data support a model whereby a hot L1 source element on Chromosome 17 of the patient’s genome evaded somatic repression in normal colon tissues and thereby initiated CRC by mutating the APC gene. This insertion worked together with a point mutation in the second APC allele to initiate tumorigenesis through the classic two-hit CRC pathway. We also show that L1 source profiles vary considerably depending on the ancestry of an individual, and that population-specific hot L1 elements represent a novel form of cancer risk. © 2016 Scott et al.; Published by Cold Spring Harbor Laboratory Press.


July 7, 2019  |  

Extensive sequencing of seven human genomes to characterize benchmark reference materials.

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.


July 7, 2019  |  

Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102.

Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture.The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90.Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.


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