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July 19, 2019

Single haplotype assembly of the human genome from a hydatidiform mole.

A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can improve assembly, even the longest available reads do not resolve all regions. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly. © 2014 Steinberg et al.; Published by Cold Spring Harbor Laboratory Press.

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July 19, 2019

Exploring bacterial epigenomics in the next-generation sequencing era: a new approach for an emerging frontier.

Epigenetics has an important role for the success of foodborne pathogen persistence in diverse host niches. Substantial challenges exist in determining DNA methylation to situation-specific phenotypic traits. DNA modification, mediated by restriction-modification systems, functions as an immune response against antagonistic external DNA, and bacteriophage-acquired methyltransferases (MTase) and orphan MTases – those lacking the cognate restriction endonuclease – facilitate evolution of new phenotypes via gene expression modulation via DNA and RNA modifications, including methylation and phosphorothioation. Recent establishment of large-scale genome sequencing projects will result in a significant increase in genome availability that will lead to new demands for data analysis including new predictive bioinformatics approaches that can be verified with traditional scientific rigor. Sequencing technologies that detect modification coupled with mass spectrometry to discover new adducts is a powerful tactic to study bacterial epigenetics, which is poised to make novel and far-reaching discoveries that link biological significance and the bacterial epigenome. Copyright © 2014 Elsevier Ltd. All rights reserved.

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July 19, 2019

Long-read, whole-genome shotgun sequence data for five model organisms.

Single molecule, real-time (SMRT) sequencing from Pacific Biosciences is increasingly used in many areas of biological research including de novo genome assembly, structural-variant identification, haplotype phasing, mRNA isoform discovery, and base-modification analyses. High-quality, public datasets of SMRT sequences can spur development of analytic tools that can accommodate unique characteristics of SMRT data (long read lengths, lack of GC or amplification bias, and a random error profile leading to high consensus accuracy). In this paper, we describe eight high-coverage SMRT sequence datasets from five organisms (Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, Arabidopsis thaliana, and Drosophila melanogaster) that have been publicly released to the general scientific community (NCBI Sequence Read Archive ID SRP040522). Data were generated using two sequencing chemistries (P4C2 and P5C3) on the PacBio RS II instrument. The datasets reported here can be used without restriction by the research community to generate whole-genome assemblies, test new algorithms, investigate genome structure and evolution, and identify base modifications in some of the most widely-studied model systems in biological research.

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July 19, 2019

Assessing structural variation in a personal genome-towards a human reference diploid genome.

Characterizing large genomic variants is essential to expanding the research and clinical applications of genome sequencing. While multiple data types and methods are available to detect these structural variants (SVs), they remain less characterized than smaller variants because of SV diversity, complexity, and size. These challenges are exacerbated by the experimental and computational demands of SV analysis. Here, we characterize the SV content of a personal genome with Parliament, a publicly available consensus SV-calling infrastructure that merges multiple data types and SV detection methods.We demonstrate Parliament’s efficacy via integrated analyses of data from whole-genome array comparative genomic hybridization, short-read next-generation sequencing, long-read (Pacific BioSciences RSII), long-insert (Illumina Nextera), and whole-genome architecture (BioNano Irys) data from the personal genome of a single subject (HS1011). From this genome, Parliament identified 31,007 genomic loci between 100 bp and 1 Mbp that are inconsistent with the hg19 reference assembly. Of these loci, 9,777 are supported as putative SVs by hybrid local assembly, long-read PacBio data, or multi-source heuristics. These SVs span 59 Mbp of the reference genome (1.8%) and include 3,801 events identified only with long-read data. The HS1011 data and complete Parliament infrastructure, including a BAM-to-SV workflow, are available on the cloud-based service DNAnexus.HS1011 SV analysis reveals the limits and advantages of multiple sequencing technologies, specifically the impact of long-read SV discovery. With the full Parliament infrastructure, the HS1011 data constitute a public resource for novel SV discovery, software calibration, and personal genome structural variation analysis.

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July 19, 2019

Population structure of mitochondrial genomes in Saccharomyces cerevisiae.

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

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July 19, 2019

Assembly and diploid architecture of an individual human genome via single-molecule technologies.

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

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July 19, 2019

TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors.

Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL effectors.

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July 19, 2019

Whole genome?

The reference human genome assembly is remarkable in its completeness and usefulness in research. However, the range of allelic variation in the human population is not well described by a haploid assembly with a profusion of alternative loci. Homozygous regions and the use of multiple sequencing technologies increasingly have roles in strategies for identifying regulatory and trait-associated variation.

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July 19, 2019

Selections that isolate recombinant mitochondrial genomes in animals.

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germ line and revealed somatic recombination. When the recombination partner was a diverged D. melanogaster genome or a genome from a different species such as D. yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome.

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July 19, 2019

Emergence of ebola virus escape variants in infected nonhuman primates treated with the MB-003 antibody cocktail.

MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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July 19, 2019

Genetic variation and the de novo assembly of human genomes.

The discovery of genetic variation and the assembly of genome sequences are both inextricably linked to advances in DNA-sequencing technology. Short-read massively parallel sequencing has revolutionized our ability to discover genetic variation but is insufficient to generate high-quality genome assemblies or resolve most structural variation. Full resolution of variation is only guaranteed by complete de novo assembly of a genome. Here, we review approaches to genome assembly, the nature of gaps or missing sequences, and biases in the assembly process. We describe the challenges of generating a complete de novo genome assembly using current technologies and the impact that being able to perfectly sequence the genome would have on understanding human disease and evolution. Finally, we summarize recent technological advances that improve both contiguity and accuracy and emphasize the importance of complete de novo assembly as opposed to read mapping as the primary means to understanding the full range of human genetic variation.

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July 19, 2019

A supergene determines highly divergent male reproductive morphs in the ruff.

Three strikingly different alternative male mating morphs (aggressive ‘independents’, semicooperative ‘satellites’ and female-mimic ‘faeders’) coexist as a balanced polymorphism in the ruff, Philomachus pugnax, a lek-breeding wading bird. Major differences in body size, ornamentation, and aggressive and mating behaviors are inherited as an autosomal polymorphism. We show that development into satellites and faeders is determined by a supergene consisting of divergent alternative, dominant and non-recombining haplotypes of an inversion on chromosome 11, which contains 125 predicted genes. Independents are homozygous for the ancestral sequence. One breakpoint of the inversion disrupts the essential CENP-N gene (encoding centromere protein N), and pedigree analysis confirms the lethality of homozygosity for the inversion. We describe new differences in behavior, testis size and steroid metabolism among morphs and identify polymorphic genes within the inversion that are likely to contribute to the differences among morphs in reproductive traits.

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July 19, 2019

Major improvements to the Heliconius melpomene genome assembly used to confirm 10 chromosome fusion events in 6 million years of butterfly evolution.

The Heliconius butterflies are a widely studied adaptive radiation of 46 species spread across Central and South America, several of which are known to hybridize in the wild. Here, we present a substantially improved assembly of the Heliconius melpomene genome, developed using novel methods that should be applicable to improving other genome assemblies produced using short read sequencing. First, we whole-genome-sequenced a pedigree to produce a linkage map incorporating 99% of the genome. Second, we incorporated haplotype scaffolds extensively to produce a more complete haploid version of the draft genome. Third, we incorporated ~20x coverage of Pacific Biosciences sequencing, and scaffolded the haploid genome using an assembly of this long-read sequence. These improvements result in a genome of 795 scaffolds, 275 Mb in length, with an N50 length of 2.1 Mb, an N50 number of 34, and with 99% of the genome placed, and 84% anchored on chromosomes. We use the new genome assembly to confirm that the Heliconius genome underwent 10 chromosome fusions since the split with its sister genus Eueides, over a period of about 6 million yr. Copyright © 2016 Davey et al.

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July 19, 2019

Long-read sequence assembly of the gorilla genome.

Accurate sequence and assembly of genomes is a critical first step for studies of genetic variation. We generated a high-quality assembly of the gorilla genome using single-molecule, real-time sequence technology and a string graph de novo assembly algorithm. The new assembly improves contiguity by two to three orders of magnitude with respect to previously released assemblies, recovering 87% of missing reference exons and incomplete gene models. Although regions of large, high-identity segmental duplications remain largely unresolved, this comprehensive assembly provides new biological insight into genetic diversity, structural variation, gene loss, and representation of repeat structures within the gorilla genome. The approach provides a path forward for the routine assembly of mammalian genomes at a level approaching that of the current quality of the human genome. Copyright © 2016, American Association for the Advancement of Science.

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July 19, 2019

An incomplete understanding of human genetic variation.

Deciphering the genetic basis of human disease requires a comprehensive knowledge of genetic variants irrespective of their class or frequency. Although an impressive number of human genetic variants have been catalogued, a large fraction of the genetic difference that distinguishes two human genomes is still not understood at the base-pair level. This is because the emphasis has been on single-nucleotide variation as opposed to less tractable and more complex genetic variants, including indels and structural variants. The latter, we propose, will have a large impact on human phenotypes but require a more systematic assessment of genomes at deeper coverage and alternate sequencing and mapping technologies. Copyright © 2016 by the Genetics Society of America.

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