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July 19, 2019

Introduction: The host-associated microbiome: Pattern, process and function.

An explosion of studies in recent years has established the ubiquity of host-associated microbes and their centrality to host biology (McFall-Ngai et al., 2013; Russell, Dubilier, & Rudgers, 2014). Microbes aid in digestion, modulate development, contribute to host immunity, mediate abiotic stress and more. While relationships with host-associated microbes are ubiquitous and important, they are cer- tainly not monolithic. Characterizing the microbial diversity associ- ated with an ever-broadening array of hosts (diverse animals, plants, algae and protists) has shown that essential functions can be per- formed by microbes that are integrated with the host to varying degrees, ranging from embedded endosymbionts to a variable cast of transient microbes acquired from the environment. The maturing host–microbiome field is now developing a mechanistic understand- ing of host/microbe relationships across this spectrum and the cross- talk mediating these interactions. Similarly, studies across systems are illuminating the ecological and evolutionary factors that shape host–microbe interactions today and providing hints into the origins of specific relationships.

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July 19, 2019

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing.

Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc.

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July 19, 2019

De novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a multisystem disorder, caused by expansion of a CTG trinucleotide repeat in the 3′-untranslated region of the DMPK gene. The repeat expansion is somatically unstable and tends to increase in length with time, contributing to disease progression. In some individuals, the repeat array is interrupted by variant repeats such as CCG and CGG, stabilising the expansion and often leading to milder symptoms. We have characterised three families, each including one person with variant repeats that had arisen de novo on paternal transmission of the repeat expansion. Two individuals were identified for screening due to an unusual result in the laboratory diagnostic test, and the third due to exceptionally mild symptoms. The presence of variant repeats in all three expanded alleles was confirmed by restriction digestion of small pool PCR products, and allele structures were determined by PacBio sequencing. Each was different, but all contained CCG repeats close to the 3′-end of the repeat expansion. All other family members had inherited pure CTG repeats. The variant repeat-containing alleles were more stable in the blood than pure alleles of similar length, which may in part account for the mild symptoms observed in all three individuals. This emphasises the importance of somatic instability as a disease mechanism in DM1. Further, since patients with variant repeats may have unusually mild symptoms, identification of these individuals has important implications for genetic counselling and for patient stratification in DM1 clinical trials.

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July 19, 2019

A Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat.

Borrelia burgdorferi evades the host immune system by switching the surface antigen. VlsE, in a process known as antigenic variation. The DNA mechanisms and genetic elements present on the vls locus that participate in the switching process remain to be elucidated. Manipulating the vls locus has been difficult due to its instability on Escherichia coli plasmids. In this study, we generated for the first time a mini-vls system composed of a single silent vlsE variable region (silent cassette 2) through the vlsE gene by performing some cloning steps directly in a highly transformable B. burgdorferi strain. Variants of the mini system were constructed with or without the long inverted repeat (IR) located upstream of vlsE and on both circular and linear plasmids to investigate the importance of the IR and plasmid topology on recombinational switching at vlsE. Amplicon sequencing using PacBio long read technology and analysis of the data with our recently reported pipeline and VAST software showed that the system undergoes switching in mice in both linear and circular versions and that the presence of the hairpin does not seem to be crucial in the linear version, however it is required when the topology is circular.© 2018 John Wiley & Sons Ltd.

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July 19, 2019

Adaptation and conservation insights from the koala genome.

The koala, the only extant species of the marsupial family Phascolarctidae, is classified as ‘vulnerable’ due to habitat loss and widespread disease. We sequenced the koala genome, producing a complete and contiguous marsupial reference genome, including centromeres. We reveal that the koala’s ability to detoxify eucalypt foliage may be due to expansions within a cytochrome P450 gene family, and its ability to smell, taste and moderate ingestion of plant secondary metabolites may be due to expansions in the vomeronasal and taste receptors. We characterized novel lactation proteins that protect young in the pouch and annotated immune genes important for response to chlamydial disease. Historical demography showed a substantial population crash coincident with the decline of Australian megafauna, while contemporary populations had biogeographic boundaries and increased inbreeding in populations affected by historic translocations. We identified genetically diverse populations that require habitat corridors and instituting of translocation programs to aid the koala’s survival in the wild.

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July 19, 2019

Fern genomes elucidate land plant evolution and cyanobacterial symbioses.

Ferns are the closest sister group to all seed plants, yet little is known about their genomes other than that they are generally colossal. Here, we report on the genomes of Azolla filiculoides and Salvinia cucullata (Salviniales) and present evidence for episodic whole-genome duplication in ferns-one at the base of ‘core leptosporangiates’ and one specific to Azolla. One fern-specific gene that we identified, recently shown to confer high insect resistance, seems to have been derived from bacteria through horizontal gene transfer. Azolla coexists in a unique symbiosis with N2-fixing cyanobacteria, and we demonstrate a clear pattern of cospeciation between the two partners. Furthermore, the Azolla genome lacks genes that are common to arbuscular mycorrhizal and root nodule symbioses, and we identify several putative transporter genes specific to Azolla-cyanobacterial symbiosis. These genomic resources will help in exploring the biotechnological potential of Azolla and address fundamental questions in the evolution of plant life.

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July 19, 2019

Loss of maternal EED results in postnatal overgrowth.

Investigating how epigenetic information is transmitted through the mammalian germline is the key to understanding how this information impacts on health and disease susceptibility in offspring. EED is essential for regulating the repressive histone modification, histone 3 lysine 27 tri-methylation (H3K27me3) at many developmental genes.In this study, we used oocyte-specific Zp3-Cre recombinase (Zp3Cre) to delete Eed specifically in mouse growing oocytes, permitting the study of EED function in oocytes and the impact of depleting EED in oocytes on outcomes in offspring. As EED deletion occurred only in growing oocytes and females were mated to normal wild type males, this model allowed the study of oocyte programming without confounding factors such as altered in utero environment. Loss of EED from growing oocytes resulted in a significant overgrowth phenotype that persisted into adult life. Significantly, this involved increased adiposity (total fat) and bone mineral density in offspring. Similar overgrowth occurs in humans with Cohen-Gibson (OMIM 617561) and Weaver (OMIM 277590) syndromes, that result from de novo germline mutations in EED or its co-factor EZH2, respectively. Consistent with a role for EZH2 in human oocytes, we demonstrate that de novo germline mutations in EZH2 occurred in the maternal germline in some cases of Weaver syndrome. However, deletion of Ezh2 in mouse oocytes resulted in a distinct phenotype compared to that resulting from oocyte-specific deletion of Eed.This study provides novel evidence that altering EED-dependent oocyte programming leads to compromised offspring growth and development in the next generation.

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July 19, 2019

A high-quality, long-read de novo genome assembly to aid conservation of Hawaii’s last remaining crow species

Genome-level data can provide researchers with unprecedented precision to examine the causes and genetic consequences of population declines, which can inform conservation management. Here, we present a high-quality, long-read, de novo genome assembly for one of the world’s most endangered bird species, the ?Alala (Corvus hawaiiensis; Hawaiian crow). As the only remaining native crow species in Hawai?i, the ?Alala survived solely in a captive-breeding program from 2002 until 2016, at which point a long-term reintroduction program was initiated. The high-quality genome assembly was generated to lay the foundation for both comparative genomics studies and the development of population-level genomic tools that will aid conservation and recovery efforts. We illustrate how the quality of this assembly places it amongst the very best avian genomes assembled to date, comparable to intensively studied model systems. We describe the genome architecture in terms of repetitive elements and runs of homozygosity, and we show that compared with more outbred species, the ?Alala genome is substantially more homozygous. We also provide annotations for a subset of immunity genes that are likely to be important in conservation management, and we discuss how this genome is currently being used as a roadmap for downstream conservation applications.

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July 19, 2019

Extensive intraspecific gene order and gene structural variations between Mo17 and other maize genomes.

Maize is an important crop with a high level of genome diversity and heterosis. The genome sequence of a typical female line, B73, was previously released. Here, we report a de novo genome assembly of a corresponding male representative line, Mo17. More than 96.4% of the 2,183?Mb assembled genome can be accounted for by 362 scaffolds in ten pseudochromosomes with 38,620 annotated protein-coding genes. Comparative analysis revealed large gene-order and gene structural variations: approximately 10% of the annotated genes were mutually nonsyntenic, and more than 20% of the predicted genes had either large-effect mutations or large structural variations, which might cause considerable protein divergence between the two inbred lines. Our study provides a high-quality reference-genome sequence of an important maize germplasm, and the intraspecific gene order and gene structural variations identified should have implications for heterosis and genome evolution.

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July 19, 2019

Identification and analysis of adenine N6-methylation sites in the rice genome.

DNA N6-methyladenine (6mA) is a non-canonical DNA modification that is present at low levels in different eukaryotes1-8, but its prevalence and genomic function in higher plants are unclear. Using mass spectrometry, immunoprecipitation and validation with analysis of single-molecule real-time sequencing, we observed that about 0.2% of all adenines are 6mA methylated in the rice genome. 6mA occurs most frequently at GAGG motifs and is mapped to about 20% of genes and 14% of transposable elements. In promoters, 6mA marks silent genes, but in bodies correlates with gene activity. 6mA overlaps with 5-methylcytosine (5mC) at CG sites in gene bodies and is complementary to 5mC at CHH sites in transposable elements. We show that OsALKBH1 may be potentially involved in 6mA demethylation in rice. The results suggest that 6mA is complementary to 5mC as an epigenomic mark in rice and reinforce a distinct role for 6mA as a gene expression-associated epigenomic mark in eukaryotes.

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July 19, 2019

Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease.

Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 ‘GGGGCC’ (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences’ (PacBio) and Oxford Nanopore Technologies’ (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinION was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8× coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained >?800× coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual’s repeat region was >?99% G4C2 content, though we cannot rule out small interruptions.Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

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July 19, 2019

Single copy transgene integration in a transcriptionally active site for recombinant protein synthesis.

For the biomanufacturing of protein biologics, establishing stable cell lines with high transgene transcription is critical for high productivity. Modern genome engineering tools can direct transgene insertion to a specified genomic locus and can potentially become a valuable tool for cell line generation. In this study, the authors survey transgene integration sites and their transcriptional activity to identify characteristics of desirable regions. A lentivirus containing destabilized Green Fluorescent Protein (dGFP) is used to infect Chinese hamster ovary cells at a low multiplicity of infection, and cells with high or low GFP fluorescence are isolated. RNA sequencing and Assay for Transposase Accessible Chromatin using sequencing data shows integration sites with high GFP expression are in larger regions of high transcriptional activity and accessibility, but not necessarily within highly transcribed genes. This method is used to obtain high Immunoglobulin G (IgG) expressing cell lines with a single copy of the transgene integrated into transcriptionally active and accessible genomic regions. Dual recombinase-mediated cassette exchange is then employed to swap the IgG transgene for erythropoietin or tumor necrosis factor receptor-Fc. This work thus highlights a strategy to identify desirable sites for transgene integration and to streamline the development of new product producing cell lines.© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 19, 2019

Characterization of a human-specific tandem repeat associated with bipolar disorder and schizophrenia.

Bipolar disorder (BD) and schizophrenia (SCZ) are highly heritable diseases that affect more than 3% of individuals worldwide. Genome-wide association studies have strongly and repeatedly linked risk for both of these neuropsychiatric diseases to a 100 kb interval in the third intron of the human calcium channel gene CACNA1C. However, the causative mutation is not yet known. We have identified a human-specific tandem repeat in this region that is composed of 30 bp units, often repeated hundreds of times. This large tandem repeat is unstable using standard polymerase chain reaction and bacterial cloning techniques, which may have resulted in its incorrect size in the human reference genome. The large 30-mer repeat region is polymorphic in both size and sequence in human populations. Particular sequence variants of the 30-mer are associated with risk status at several flanking single-nucleotide polymorphisms in the third intron of CACNA1C that have previously been linked to BD and SCZ. The tandem repeat arrays function as enhancers that increase reporter gene expression in a human neural progenitor cell line. Different human arrays vary in the magnitude of enhancer activity, and the 30-mer arrays associated with increased psychiatric disease risk status have decreased enhancer activity. Changes in the structure and sequence of these arrays likely contribute to changes in CACNA1C function during human evolution and may modulate neuropsychiatric disease risk in modern human populations. Copyright © 2018. Published by Elsevier Inc.

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July 19, 2019

Reference grade characterization of polymorphisms in full-length HLA class I and II genes with short-read sequencing on the Ion PGM system and long-reads generated by Single Molecule, Real-time Sequencing on the PacBio platform

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

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July 19, 2019

Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch.

Crop diversification required to meet demands for food security and industrial use is often challenged by breeding time and amenability of varieties to genome modification. Cassava is one such crop. Grown for its large starch-rich storage roots, it serves as a staple food and a commodity in the multibillion-dollar starch industry. Starch is composed of the glucose polymers amylopectin and amylose, with the latter strongly influencing the physicochemical properties of starch during cooking and processing. We demonstrate that CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated targeted mutagenesis of two genes involved in amylose biosynthesis, PROTEIN TARGETING TO STARCH (PTST1) or GRANULE BOUND STARCH SYNTHASE (GBSS), can reduce or eliminate amylose content in root starch. Integration of the Arabidopsis FLOWERING LOCUS T gene in the genome-editing cassette allowed us to accelerate flowering-an event seldom seen under glasshouse conditions. Germinated seeds yielded S1, a transgene-free progeny that inherited edited genes. This attractive new plant breeding technique for modified cassava could be extended to other crops to provide a suite of novel varieties with useful traits for food and industrial applications.

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