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July 19, 2019

Dissecting the causal mechanism of X-linked Dystonia-Parkinsonism by integrating genome and transcriptome assembly.

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders. Copyright © 2018 Elsevier Inc. All rights reserved.

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July 19, 2019

Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations.

Recently, the Mucin-1 (MUC1) gene has been identified as a causal gene of autosomal dominant tubulointerstitial kidney disease (ADTKD). Most causative mutations are buried within a GC-rich 60 basepair variable number of tandem repeat (VNTR), which escapes identification by massive parallel sequencing methods due to the complexity of the VNTR. We established long read single molecule real time sequencing (SMRT) targeted to the MUC1-VNTR as an alternative strategy to the snapshot assay. Our approach allows complete VNTR assembly, thereby enabling the detection of all variants residing within the VNTR and simultaneous determination of VNTR length. We present high resolution data on the VNTR architecture for a cohort of snapshot positive (n?=?9) and negative (n?=?7) ADTKD families. By SMRT sequencing we could confirm the diagnosis in all previously tested cases, reconstruct both VNTR alleles and determine the exact position of the causative variant in eight of nine families. This study demonstrates that precise positioning of the causative mutation(s) and identification of other coding and noncoding sequence variants in ADTKD-MUC1 is feasible. SMRT sequencing could provide a powerful tool to uncover potential factors encoded within the VNTR that associate with intra- and interfamilial phenotype variability of MUC1 related kidney disease.

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July 19, 2019

Phasevarions of bacterial pathogens: Methylomics sheds new light on old enemies.

A wide variety of bacterial pathogens express phase-variable DNA methyltransferases that control expression of multiple genes via epigenetic mechanisms. These randomly switching regulons – phasevarions – regulate genes involved in pathogenesis, host adaptation, and antibiotic resistance. Individual phase-variable genes can be identified in silico as they contain easily recognized features such as simple sequence repeats (SSRs) or inverted repeats (IRs) that mediate the random switching of expression. Conversely, phasevarion-controlled genes do not contain any easily identifiable features. The study of DNA methyltransferase specificity using Single-Molecule, Real-Time (SMRT) sequencing and methylome analysis has rapidly advanced the analysis of phasevarions by allowing methylomics to be combined with whole-transcriptome/proteome analysis to comprehensively characterize these systems in a number of important bacterial pathogens. Copyright © 2018 Elsevier Ltd. All rights reserved.

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July 19, 2019

Coupling of single molecule, long read sequencing with IMGT/HighV-QUEST analysis expedites identification of SIV gp140-specific antibodies from scFv phage display libraries.

The simian immunodeficiency virus (SIV)/macaque model of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome pathogenesis is critical for furthering our understanding of the role of antibody responses in the prevention of HIV infection, and will only increase in importance as macaque immunoglobulin (IG) gene databases are expanded. We have previously reported the construction of a phage display library from a SIV-infected rhesus macaque (Macaca mulatta) using oligonucleotide primers based on human IG gene sequences. Our previous screening relied on Sanger sequencing, which was inefficient and generated only a few dozen sequences. Here, we re-analyzed this library using single molecule, real-time (SMRT) sequencing on the Pacific Biosciences (PacBio) platform to generate thousands of highly accurate circular consensus sequencing (CCS) reads corresponding to full length single chain fragment variable. CCS data were then analyzed through the international ImMunoGeneTics information system®(IMGT®)/HighV-QUEST (www.imgt.org) to identify variable genes and perform statistical analyses. Overall the library was very diverse, with 2,569 different IMGT clonotypes called for the 5,238 IGHV sequences assigned to an IMGT clonotype. Within the library, SIV-specific antibodies represented a relatively limited number of clones, with only 135 different IMGT clonotypes called from 4,594 IGHV-assigned sequences. Our data did confirm that the IGHV4 and IGHV3 gene usage was the most abundant within the rhesus antibodies screened, and that these genes were even more enriched among SIV gp140-specific antibodies. Although a broad range of VH CDR3 amino acid (AA) lengths was observed in the unpanned library, the vast majority of SIV gp140-specific antibodies demonstrated a more uniform VH CDR3 length (20 AA). This uniformity was far less apparent when VH CDR3 were classified according to their clonotype (range: 9-25 AA), which we believe is more relevant for specific antibody identification. Only 174 IGKV and 588 IGLV clonotypes were identified within the VL sequences associated with SIV gp140-specific VH. Together, these data strongly suggest that the combination of SMRT sequencing with the IMGT/HighV-QUEST querying tool will facilitate and expedite our understanding of polyclonal antibody responses during SIV infection and may serve to rapidly expand the known scope of macaque V genes utilized during these responses.

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July 19, 2019

Piercing the dark matter: bioinformatics of long-range sequencing and mapping.

Several new genomics technologies have become available that offer long-read sequencing or long-range mapping with higher throughput and higher resolution analysis than ever before. These long-range technologies are rapidly advancing the field with improved reference genomes, more comprehensive variant identification and more complete views of transcriptomes and epigenomes. However, they also require new bioinformatics approaches to take full advantage of their unique characteristics while overcoming their complex errors and modalities. Here, we discuss several of the most important applications of the new technologies, focusing on both the currently available bioinformatics tools and opportunities for future research.

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July 19, 2019

Survey on the use of whole-genome sequencing for infectious diseases surveillance: Rapid expansion of European national capacities, 2015-2016.

Whole-genome sequencing (WGS) has become an essential tool for public health surveillance and molecular epidemiology of infectious diseases and antimicrobial drug resistance. It provides precise geographical delineation of spread and enables incidence monitoring of pathogens at genotype level. Coupled with epidemiological and environmental investigations, it delivers ultimate resolution for tracing sources of epidemic infections. To ascertain the level of implementation of WGS-based typing for national public health surveillance and investigation of prioritized diseases in the European Union (EU)/European Economic Area (EEA), two surveys were conducted in 2015 and 2016. The surveys were designed to determine the national public health reference laboratories’ access to WGS and operational WGS-based typing capacity for national surveillance of selected foodborne pathogens, antimicrobial-resistant pathogens, and vaccine-preventable diseases identified as priorities for European genomic surveillance. Twenty-eight and twenty-nine out of the 30 EU/EEA countries participated in the survey in 2015 and 2016, respectively. National public health reference laboratories in 22 and 25 countries had access to WGS-based typing for public health applications in 2015 and 2016, respectively. Reported reasons for limited or no access were lack of funding, staff, and expertise. Illumina technology was the most frequently used followed by Ion Torrent technology. The access to bioinformatics expertise and competence for routine WGS data analysis was limited. By mid-2016, half of the EU/EEA countries were using WGS analysis either as first- or second-line typing method for surveillance of the pathogens and antibiotic resistance issues identified as EU priorities. The sampling frame as well as bioinformatics analysis varied by pathogen/resistance issue and country. Core genome multilocus allelic profiling, also called cgMLST, was the most frequently used annotation approach for typing bacterial genomes suggesting potential bioinformatics pipeline compatibility. Further capacity development for WGS-based typing is ongoing in many countries and upon consolidation and harmonization of methods should enable pan-EU data exchange for genomic surveillance in the medium-term subject to the development of suitable data management systems and appropriate agreements for data sharing.

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July 19, 2019

Accurate detection of complex structural variations using single-molecule sequencing.

Structural variations are the greatest source of genetic variation, but they remain poorly understood because of technological limitations. Single-molecule long-read sequencing has the potential to dramatically advance the field, although high error rates are a challenge with existing methods. Addressing this need, we introduce open-source methods for long-read alignment (NGMLR; https://github.com/philres/ngmlr ) and structural variant identification (Sniffles; https://github.com/fritzsedlazeck/Sniffles ) that provide unprecedented sensitivity and precision for variant detection, even in repeat-rich regions and for complex nested events that can have substantial effects on human health. In several long-read datasets, including healthy and cancerous human genomes, we discovered thousands of novel variants and categorized systematic errors in short-read approaches. NGMLR and Sniffles can automatically filter false events and operate on low-coverage data, thereby reducing the high costs that have hindered the application of long reads in clinical and research settings.

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July 19, 2019

Advances in Sequencing and Resequencing in Crop Plants.

DNA sequencing technologies have changed the face of biological research over the last 20 years. From reference genomes to population level resequencing studies, these technologies have made significant contributions to our understanding of plant biology and evolution. As the technologies have increased in power, the breadth and complexity of the questions that can be asked has increased. Along with this, the challenges of managing unprecedented quantities of sequence data are mounting. This chapter describes a few aspects of the journey so far and looks forward to what may lie ahead.

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July 19, 2019

A near-complete haplotype-phased genome of the dikaryotic wheat stripe rust fungus Puccinia striiformis f. sp. tritici reveals high interhaplotype diversity.

A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.IMPORTANCE Current representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organism’s evolutionary potential and ability to adapt to stress conditions. Yet, it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which lead to allelic variation and the presence of genes lacking allelic counterparts. Nonallelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis. Copyright © 2018 Schwessinger et al.

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July 19, 2019

Utility of DNA, RNA, protein, and functional approaches to solve cryptic immunodeficiencies.

We report a female infant identified by newborn screening for severe combined immunodeficiencies (NBS SCID) with T cell lymphopenia (TCL). The patient had persistently elevated alpha-fetoprotein (AFP) with IgA deficiency, and elevated IgM. Gene sequencing for a SCID panel was uninformative. We sought to determine the cause of the immunodeficiency in this infant.We performed whole-exome sequencing (WES) on the patient and parents to identify a genetic diagnosis. Based on the WES result, we developed a novel flow cytometric panel for rapid assessment of DNA repair defects using blood samples. We also performed whole transcriptome sequencing (WTS) on fibroblast RNA from the patient and father for abnormal transcript analysis.WES revealed a pathogenic paternally inherited indel in ATM. We used the flow panel to assess several proteins in the DNA repair pathway in lymphocyte subsets. The patient had absent phosphorylation of ATM, resulting in absent or aberrant phosphorylation of downstream proteins, including ?H2AX. However, ataxia-telangiectasia (AT) is an autosomal recessive condition, and the abnormal functional data did not correspond with a single ATM variant. WTS revealed in-frame reciprocal fusion transcripts involving ATM and SLC35F2 indicating a chromosome 11 inversion within 11q22.3, of maternal origin. Inversion breakpoints were identified within ATM intron 16 and SLC35F2 intron 7.We identified a novel ATM-breaking chromosome 11 inversion in trans with a pathogenic indel (compound heterozygote) resulting in non-functional ATM protein, consistent with a diagnosis of AT. Utilization of several molecular and functional assays allowed successful resolution of this case.

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July 19, 2019

Genome sequence of the progenitor of wheat A subgenome Triticum urartu.

Triticum urartu (diploid, AA) is the progenitor of the A subgenome of tetraploid (Triticum turgidum, AABB) and hexaploid (Triticum aestivum, AABBDD) wheat1,2. Genomic studies of T. urartu have been useful for investigating the structure, function and evolution of polyploid wheat genomes. Here we report the generation of a high-quality genome sequence of T. urartu by combining bacterial artificial chromosome (BAC)-by-BAC sequencing, single molecule real-time whole-genome shotgun sequencing 3 , linked reads and optical mapping4,5. We assembled seven chromosome-scale pseudomolecules and identified protein-coding genes, and we suggest a model for the evolution of T. urartu chromosomes. Comparative analyses with genomes of other grasses showed gene loss and amplification in the numbers of transposable elements in the T. urartu genome. Population genomics analysis of 147 T. urartu accessions from across the Fertile Crescent showed clustering of three groups, with differences in altitude and biostress, such as powdery mildew disease. The T. urartu genome assembly provides a valuable resource for studying genetic variation in wheat and related grasses, and promises to facilitate the discovery of genes that could be useful for wheat improvement.

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July 19, 2019

Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma.

To understand how genomic heterogeneity of glioblastoma (GBM) contributes to poor therapy response, we performed DNA and RNA sequencing on GBM samples and the neurospheres and orthotopic xenograft models derived from them. We used the resulting dataset to show that somatic driver alterations including single-nucleotide variants, focal DNA alterations and oncogene amplification on extrachromosomal DNA (ecDNA) elements were in majority propagated from tumor to model systems. In several instances, ecDNAs and chromosomal alterations demonstrated divergent inheritance patterns and clonal selection dynamics during cell culture and xenografting. We infer that ecDNA was unevenly inherited by offspring cells, a characteristic that affects the oncogenic potential of cells with more or fewer ecDNAs. Longitudinal patient tumor profiling found that oncogenic ecDNAs are frequently retained throughout the course of disease. Our analysis shows that extrachromosomal elements allow rapid increase of genomic heterogeneity during GBM evolution, independently of chromosomal DNA alterations.

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July 19, 2019

Resequencing of 243 diploid cotton accessions based on an updated A genome identifies the genetic basis of key agronomic traits.

The ancestors of Gossypium arboreum and Gossypium herbaceum provided the A subgenome for the modern cultivated allotetraploid cotton. Here, we upgraded the G. arboreum genome assembly by integrating different technologies. We resequenced 243?G. arboreum and G. herbaceum accessions to generate a map of genome variations and found that they are equally diverged from Gossypium raimondii. Independent analysis suggested that Chinese G. arboreum originated in South China and was subsequently introduced to the Yangtze and Yellow River regions. Most accessions with domestication-related traits experienced geographic isolation. Genome-wide association study (GWAS) identified 98 significant peak associations for 11 agronomically important traits in G. arboreum. A nonsynonymous substitution (cysteine-to-arginine substitution) of GaKASIII seems to confer substantial fatty acid composition (C16:0 and C16:1) changes in cotton seeds. Resistance to fusarium wilt disease is associated with activation of GaGSTF9 expression. Our work represents a major step toward understanding the evolution of the A genome of cotton.

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July 19, 2019

Genomic variation in 3,010 diverse accessions of Asian cultivated rice.

Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence-absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding.

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