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Auto Tag: Variant detection

July 19, 2019

The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution.

The domesticated sunflower, Helianthus annuus L., is a global oil crop that has promise for climate change adaptation, because it can maintain stable yields across a wide variety of environmental conditions, including drought. Even greater resilience is achievable through the mining of resistance alleles from compatible wild sunflower relatives, including numerous extremophile species. Here we report a high-quality reference for the sunflower genome (3.6 gigabases), together with extensive transcriptomic data from vegetative and floral organs. The genome mostly consists of highly similar, related sequences and required single-molecule real-time sequencing technologies for successful assembly. Genome analyses enabled the reconstruction of the evolutionary history of the Asterids, further establishing the existence of a whole-genome triplication at the base of the Asterids II clade and a sunflower-specific whole-genome duplication around 29 million years ago. An integrative approach combining quantitative genetics, expression and diversity data permitted development of comprehensive gene networks for two major breeding traits, flowering time and oil metabolism, and revealed new candidate genes in these networks. We found that the genomic architecture of flowering time has been shaped by the most recent whole-genome duplication, which suggests that ancient paralogues can remain in the same regulatory networks for dozens of millions of years. This genome represents a cornerstone for future research programs aiming to exploit genetic diversity to improve biotic and abiotic stress resistance and oil production, while also considering agricultural constraints and human nutritional needs.

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July 19, 2019

SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis.

The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication.Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome.Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.

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July 19, 2019

Dual redundant sequencing strategy: Full-length gene characterisation of 1056 novel and confirmatory HLA alleles.

The high-throughput department of DKMS Life Science Lab encounters novel human leukocyte antigen (HLA) alleles on a daily basis. To characterise these alleles, we have developed a system to sequence the whole gene from 5′- to 3′-UTR for the HLA loci A, B, C, DQB1 and DPB1 for submission to the European Molecular Biology Laboratory – European Nucleotide Archive (EMBL-ENA) and the IPD-IMGT/HLA Database. Our workflow is based on a dual redundant sequencing strategy. Using shotgun sequencing on an Illumina MiSeq instrument and single molecule real-time (SMRT) sequencing on a PacBio RS II instrument, we are able to achieve highly accurate HLA full-length consensus sequences. Remaining conflicts are resolved using the R package DR2S (Dual Redundant Reference Sequencing). Given the relatively high throughput of this strategy, we have developed the semi-automated web service TypeLoader, to aid in the submission of sequences to the EMBL-ENA and the IPD-IMGT/HLA Database. In the IPD-IMGT/HLA Database release 3.24.0 (April 2016; prior to the submission of the sequences described here), only 5.2% of all known HLA alleles have been fully characterised together with intronic and UTR sequences. So far, we have applied our strategy to characterise and submit 1056 HLA alleles, thereby more than doubling the number of fully characterised alleles. Given the increasing application of next generation sequencing (NGS) for full gene characterisation in clinical practice, extending the HLA database concomitantly is highly desirable. Therefore, we propose this dual redundant sequencing strategy as a workflow for submission of novel full-length alleles and characterisation of sequences that are as yet incomplete. This would help to mitigate the predominance of partially known alleles in the database.© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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July 19, 2019

Comparative genomics of two sequential Candida glabrata clinical isolates.

Candida glabrata is an important fungal pathogen which develops rapid antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator PDR1 result in upregulation of the transporters. In addition, we showed that the PDR1 mutations can contribute to enhance virulence in animal models. In this study, we were interested to compare genomes of two specific C. glabrata-related isolates, one of which was azole susceptible (DSY562) while the other was azole resistant (DSY565). DSY565 contained a PDR1 mutation (L280F) and was isolated after a time-lapse of 50 d of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacBio technology used here yielded 14 major contigs (sizes 0.18-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among the unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 each contained a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (63) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 nonsynonymous SNPs (among which the was the expected PDR1 mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes contained a DNA mismatch repair allele of MSH2 known to be involved in the so-called hyper-mutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a MSH2 defect. In conclusion, this study is the first to compare genomes of C. glabrata sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points exhibited limited variations, even if submitted to the host pressure. Copyright © 2017 Vale-Silva et al.

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July 19, 2019

Improved maize reference genome with single-molecule technologies.

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

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July 19, 2019

Contrasting evolutionary genome dynamics between domesticated and wild yeasts.

Structural rearrangements have long been recognized as an important source of genetic variation, with implications in phenotypic diversity and disease, yet their detailed evolutionary dynamics remain elusive. Here we use long-read sequencing to generate end-to-end genome assemblies for 12 strains representing major subpopulations of the partially domesticated yeast Saccharomyces cerevisiae and its wild relative Saccharomyces paradoxus. These population-level high-quality genomes with comprehensive annotation enable precise definition of chromosomal boundaries between cores and subtelomeres and a high-resolution view of evolutionary genome dynamics. In chromosomal cores, S. paradoxus shows faster accumulation of balanced rearrangements (inversions, reciprocal translocations and transpositions), whereas S. cerevisiae accumulates unbalanced rearrangements (novel insertions, deletions and duplications) more rapidly. In subtelomeres, both species show extensive interchromosomal reshuffling, with a higher tempo in S. cerevisiae. Such striking contrasts between wild and domesticated yeasts are likely to reflect the influence of human activities on structural genome evolution.

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July 19, 2019

SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms.

Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences’ Single Molecule, Real-Time (PacBio SMRT) sequencing technology.

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July 19, 2019

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

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July 19, 2019

A case study into microbial genome assembly gap sequences and finishing strategies.

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.

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July 19, 2019

SplitThreader: Exploration and analysis of rearrangements in cancer genomes

Genomic rearrangements and associated copy number changes are important drivers in cancer as they can alter the expression of oncogenes and tumor suppressors, create gene fusions, and misregulate gene expression. Here we present SplitThreader (http://splitthreader.com), an open- source interactive web application for analysis and visualization of genomic rearrangements and copy number variation in cancer genomes. SplitThreader constructs a sequence graph of genomic rearrangements in the sample and uses a priority queue breadth-first search algorithm on the graph to search for novel interactions. This is applied to detect gene fusions and other novel sequences, as well as to evaluate distances in the rearranged genome between any genomic regions of interest, especially the repositioning of regulatory elements and their target genes. SplitThreader also analyzes each variant to categorize it by its relation to other variants and by its copy number concordance. This identifies balanced translocations, identifies simple and complex variants, and suggests likely false positives when copy number is not concordant across a candidate breakpoint. It also provides explanations when multiple variants affect the copy number state and obscure the contribution of a single variant, such as a deletion within a region that is overall amplified. Together, these categories triage the variants into groups and provide a starting point for further systematic analysis and manual curation. To demonstrate its utility, we apply SplitThreader to three cancer cell lines, MCF-7 and A549 with Illumina paired- end sequencing, and SK-BR-3, with long-read PacBio sequencing. Using SplitThreader, we examine the genomic rearrangements responsible for previously observed gene fusions in SK-BR-3 and MCF-7, and discover many of the fusions involved a complex series of multiple genomic rearrangements. We also find notable differences in the types of variants between the three cell lines, in particular a much higher proportion of reciprocal variants in SK-BR-3 and a distinct clustering of interchromosomal variants in SK-BR-3 and MCF-7 that is absent in A549.

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July 19, 2019

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications.The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

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July 19, 2019

Exonization of an intronic LINE-1 element causing Becker muscular dystrophy as a novel mutational mechanism in dystrophin gene.

A broad mutational spectrum in the dystrophin (DMD) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD, adding to the diversity of mutational events that give rise to D/BMD.

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July 19, 2019

The draft genome of tropical fruit durian (Durio zibethinus).

Durian (Durio zibethinus) is a Southeast Asian tropical plant known for its hefty, spine-covered fruit and sulfury and onion-like odor. Here we present a draft genome assembly of D. zibethinus, representing the third plant genus in the Malvales order and first in the Helicteroideae subfamily to be sequenced. Single-molecule sequencing and chromosome contact maps enabled assembly of the highly heterozygous durian genome at chromosome-scale resolution. Transcriptomic analysis showed upregulation of sulfur-, ethylene-, and lipid-related pathways in durian fruits. We observed paleopolyploidization events shared by durian and cotton and durian-specific gene expansions in MGL (methionine ?-lyase), associated with production of volatile sulfur compounds (VSCs). MGL and the ethylene-related gene ACS (aminocyclopropane-1-carboxylic acid synthase) were upregulated in fruits concomitantly with their downstream metabolites (VSCs and ethylene), suggesting a potential association between ethylene biosynthesis and methionine regeneration via the Yang cycle. The durian genome provides a resource for tropical fruit biology and agronomy.

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July 19, 2019

Long-read genome sequence assembly provides insight into ongoing retroviral invasion of the koala germline.

The koala retrovirus (KoRV) is implicated in several diseases affecting the koala (Phascolarctos cinereus). KoRV provirus can be present in the genome of koalas as an endogenous retrovirus (present in all cells via germline integration) or as exogenous retrovirus responsible for somatic integrations of proviral KoRV (present in a limited number of cells). This ongoing invasion of the koala germline by KoRV provides a powerful opportunity to assess the viral strategies used by KoRV in an individual. Analysis of a high-quality genome sequence of a single koala revealed 133 KoRV integration sites. Most integrations contain full-length, endogenous provirus; KoRV-A subtype. The second most frequent integrations contain an endogenous recombinant element (recKoRV) in which most of the KoRV protein-coding region has been replaced with an ancient, endogenous retroelement. A third set of integrations, with very low sequence coverage, may represent somatic cell integrations of KoRV-A, KoRV-B and two recently designated additional subgroups, KoRV-D and KoRV-E. KoRV-D and KoRV-E are missing several genes required for viral processing, suggesting they have been transmitted as defective viruses. Our results represent the first comprehensive analyses of KoRV integration and variation in a single animal and provide further insights into the process of retroviral-host species interactions.

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July 19, 2019

A comparative study on the characterization of hepatitis B virus quasispecies by clone-based sequencing and third-generation sequencing.

Hepatitis B virus (HBV) has a high mutation rate due to the extremely high replication rate and the proofreading deficiency during reverse transcription. The generated variants with genetic heterogeneity are described as viral quasispecies (QS). Clone-based sequencing (CBS) is thought to be the ‘gold standard’ for assessing QS complexity and diversity of HBV, but an important issue about CBS is cost-effectiveness and laborious. In this study, we investigated the utility of the third-generation sequencing (TGS) DNA sequencing to characterize genetic heterogeneity of HBV QS and assessed the possible contribution of TGS technology in HBV QS studies. Parallel experiments including 3 control samples, which consisted of HBV full gene genotype B and genotype C plasmids, and 10 patients samples were performed by using CBS and TGS to analyze HBV whole-genome QS. Characterization of QS heterogeneity was conducted by using comprehensive statistical analysis. The results showed that TGS had a high consistency with CBS when measuring the complexity and diversity of QS. In addition, to detect rare variants, there were strong advantages conferred by TGS. In summary, TGS was considered to be practicable in HBV QS studies and it might have a relevant role in the clinical management of HBV infection in the future.

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