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Auto Tag: Aneuploidy

April 21, 2020  |  

Updated assembly resource of Phytophthora ramorum Pr102 isolate incorporating long reads from PacBio sequencing.

The NA1 clonal lineage of Phytophthora ramorum is responsible for Sudden Oak Death, an epidemic that has devastated California’s coastal forest ecosystems. An NA1 isolate Pr102 derived from coast live oak in California was previously sequenced and reported with 65 Mb assembly containing 12 Mb gaps in 2576 scaffolds. Here we report an improved 70 Mb genome in 1512 scaffolds with 6752 bp gaps after incorporating PacBio P5-C3 longreads. This assembly contains 19494 gene models (average gene length 2515 bp) compared to 16134 genes (average gene length of 1673 bp) in the previous version. We predicted 29 new RXLRs and 76 new paralogs of a total 392 RXLRs from this assembly. We predicted 35 CRNs compared to 19 in earlier version with six paralogs. Our lncRNAs prediction identified 255 candidates. This new resource will be invaluable for future evolution studies on the invasive plant pathogen.

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April 21, 2020  |  

Finding the needle in a haystack: Mapping antifungal drug resistance in fungal pathogen by genomic approaches.

Fungi are ubiquitous on earth and are essential for the maintenance of the global ecological equilibrium. Despite providing benefits to living organisms, they can also target specific hosts and inflict damage. These fungal pathogens are known to affect, for example, plants and mam- mals and thus reduce crop production necessary to sustain food supply and cause mortality in humans and animals. Designing defenses against these fungi is essential for the control of food resources and human health. As far as fungal pathogens are concerned, the principal option has been the use of antifungal agents, also called fungicides when they are used in the environment.

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April 21, 2020  |  

Enrichment of fetal and maternal long cell-free DNA fragments from maternal plasma following DNA repair.

Cell-free DNA (cfDNA) fragments in maternal plasma contain DNA damage and may negatively impact the sensitivity of noninvasive prenatal testing (NIPT). However, some of these DNA damages are potentially reparable. We aimed to recover these damaged cfDNA molecules using PreCR DNA repair mix.cfDNA was extracted from 20 maternal plasma samples and was repaired and sequenced by the Illumina platform. Size profiles and fetal DNA fraction changes of repaired samples were characterized. Targeted sequencing of chromosome Y sequences was used to enrich fetal cfDNA molecules following repair. Single-molecule real-time (SMRT) sequencing platform was employed to characterize long (>250 bp) cfDNA molecules. NIPT of five trisomy 21 samples was performed.Size profiles of repaired libraries were altered, with significantly increased long (>250 bp) cfDNA molecules. Single nucleotide polymorphism (SNP)-based analyses showed that both fetal- and maternal-derived cfDNA molecules were enriched by the repair. Fetal DNA fractions in maternal plasma showed a small but consistent (4.8%) increase, which were contributed by a higher increment of long fetal cfDNA molecules. z-score values were improved in NIPT of all trisomy 21 samples.Plasma DNA repair recovers and enriches long cfDNA molecules of both fetal and maternal origins in maternal plasma. © 2018 John Wiley & Sons, Ltd.

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April 21, 2020  |  

Assembly of allele-aware, chromosomal-scale autopolyploid genomes based on Hi-C data.

Construction of chromosome-level assembly is a vital step in achieving the goal of a ‘Platinum’ genome, but it remains a major challenge to assemble and anchor sequences to chromosomes in autopolyploid or highly heterozygous genomes. High-throughput chromosome conformation capture (Hi-C) technology serves as a robust tool to dramatically advance chromosome scaffolding; however, existing approaches are mostly designed for diploid genomes and often with the aim of reconstructing a haploid representation, thereby having limited power to reconstruct chromosomes for autopolyploid genomes. We developed a novel algorithm (ALLHiC) that is capable of building allele-aware, chromosomal-scale assembly for autopolyploid genomes using Hi-C paired-end reads with innovative ‘prune’ and ‘optimize’ steps. Application on simulated data showed that ALLHiC can phase allelic contigs and substantially improve ordering and orientation when compared to other mainstream Hi-C assemblers. We applied ALLHiC on an autotetraploid and an autooctoploid sugar-cane genome and successfully constructed the phased chromosomal-level assemblies, revealing allelic variations present in these two genomes. The ALLHiC pipeline enables de novo chromosome-level assembly of autopolyploid genomes, separating each allele. Haplotype chromosome-level assembly of allopolyploid and heterozygous diploid genomes can be achieved using ALLHiC, overcoming obstacles in assembling complex genomes.

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April 21, 2020  |  

Single-Molecule Sequencing: Towards Clinical Applications.

In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that are being applied to pathogenic microorganisms and viruses, constitutional disorders, pharmacogenomics, cancer, and more.Copyright © 2018 Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

Genetic Variation, Comparative Genomics, and the Diagnosis of Disease.

The discovery of mutations associated with human genetic dis- ease is an exercise in comparative genomics (see Glossary). Although there are many different strategies and approaches, the central premise is that affected persons harbor a significant excess of pathogenic DNA variants as com- pared with a group of unaffected persons (controls) that is either clinically defined1 or established by surveying large swaths of the general population.2 The more exclu- sive the variant is to the disease, the greater its penetrance, the larger its effect size, and the more relevant it becomes to both disease diagnosis and future therapeutic investigation. The most popular approach used by researchers in human genetics is the case–control design, but there are others that can be used to track variants and disease in a family context or that consider the probability of different classes of mutations based on evolutionary patterns of divergence or de novo mutational change.3,4 Although the approaches may be straightforward, the discovery of patho- genic variation and its mechanism of action often is less trivial, and decades of research can be required in order to identify the variants underlying both mendelian and complex genetic traits.

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April 21, 2020  |  

Dnase1l3 deletion causes aberrations in length and end-motif frequencies in plasma DNA.

Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3-/- mice carrying Dnase1l3+/- fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3 Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity. Copyright © 2019 the Author(s). Published by PNAS.

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April 21, 2020  |  

Multiple modes of convergent adaptation in the spread of glyphosate-resistant Amaranthus tuberculatus.

The selection pressure exerted by herbicides has led to the repeated evolution of herbicide resistance in weeds. The evolution of herbicide resistance on contemporary timescales in turn provides an outstanding opportunity to investigate key questions about the genetics of adaptation, in particular the relative importance of adaptation from new mutations, standing genetic variation, or geographic spread of adaptive alleles through gene flow. Glyphosate-resistant Amaranthus tuberculatus poses one of the most significant threats to crop yields in the Midwestern United States, with both agricultural populations and herbicide resistance only recently emerging in Canada. To understand the evolutionary mechanisms driving the spread of resistance, we sequenced and assembled the A. tuberculatus genome and investigated the origins and population genomics of 163 resequenced glyphosate-resistant and susceptible individuals from Canada and the United States. In Canada, we discovered multiple modes of convergent evolution: in one locality, resistance appears to have evolved through introductions of preadapted US genotypes, while in another, there is evidence for the independent evolution of resistance on genomic backgrounds that are historically nonagricultural. Moreover, resistance on these local, nonagricultural backgrounds appears to have occurred predominantly through the partial sweep of a single haplotype. In contrast, resistant haplotypes arising from the Midwestern United States show multiple amplification haplotypes segregating both between and within populations. Therefore, while the remarkable species-wide diversity of A. tuberculatus has facilitated geographic parallel adaptation of glyphosate resistance, more recently established agricultural populations are limited to adaptation in a more mutation-limited framework.Copyright © 2019 the Author(s). Published by PNAS.

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April 21, 2020  |  

Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease.

Current diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test.We performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases.We found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs.Robust identification of CNVs by GS is possible within a clinical testing environment.

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April 21, 2020  |  

Meiotic sex in Chagas disease parasite Trypanosoma cruzi.

Genetic exchange enables parasites to rapidly transform disease phenotypes and exploit new host populations. Trypanosoma cruzi, the parasitic agent of Chagas disease and a public health concern throughout Latin America, has for decades been presumed to exchange genetic material rarely and without classic meiotic sex. We present compelling evidence from 45 genomes sequenced from southern Ecuador that T. cruzi in fact maintains truly sexual, panmictic groups that can occur alongside others that remain highly clonal after past hybridization events. These groups with divergent reproductive strategies appear genetically isolated despite possible co-occurrence in vectors and hosts. We propose biological explanations for the fine-scale disconnectivity we observe and discuss the epidemiological consequences of flexible reproductive modes. Our study reinvigorates the hunt for the site of genetic exchange in the T. cruzi life cycle, provides tools to define the genetic determinants of parasite virulence, and reforms longstanding theory on clonality in trypanosomatid parasites.

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April 21, 2020  |  

CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.

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April 21, 2020  |  

The Impact of cDNA Normalization on Long-Read Sequencing of a Complex Transcriptome

Normalization of cDNA is widely used to improve the coverage of rare transcripts in analysis of transcriptomes employing next-generation sequencing. Recently, long-read technology has been emerging as a powerful tool for sequencing and construction of transcriptomes, especially for complex genomes containing highly similar transcripts and transcript-spliced isoforms. Here, we analyzed the transcriptome of sugarcane, with a highly polyploidy plant genome, by PacBio isoform sequencing (Iso-Seq) of two different cDNA library preparations, with and without a normalization step. The results demonstrated that, while the two libraries included many of the same transcripts, many longer transcripts were removed and many new generally shorter transcripts were detected by normalization. For the same input cDNA and the same data yield, the normalized library recovered more total transcript isoforms, number of predicted gene families and orthologous groups, resulting in a higher representation for the sugarcane transcriptome, compared to the non-normalized library. The non-normalized library, on the other hand, included a wider transcript length range with more longer transcripts above ~1.25 kb, more transcript isoforms per gene family and gene ontology terms per transcript. A large proportion of the unique transcripts comprising ~52% of the normalized library were expressed at a lower level than the unique transcripts from the non-normalized library, across three tissue types tested including leaf, stalk and root. About 83% of the total 5,348 predicted long noncoding transcripts was derived from the normalized library, of which ~80% was derived from the lowly expressed fraction. Functional annotation of the unique transcripts suggested that each library enriched different functional transcript fractions. This demonstrated the complementation of the two approaches in obtaining a complete transcriptome of a complex genome at the sequencing depth used in this study.

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April 21, 2020  |  

Comprehensive characterization of T-DNA integration induced chromosomal rearrangement in a birch T-DNA mutant.

Integration of T-DNA into plant genomes via Agrobacterium may interrupt gene structure and generate numerous mutants. The T-DNA caused mutants are valuable materials for understanding T-DNA integration model in plant research. T-DNA integration in plants is complex and still largely unknown. In this work, we reported that multiple T-DNA fragments caused chromosomal translocation and deletion in a birch (Betula platyphylla × B. pendula) T-DNA mutant yl.We performed PacBio genome resequencing for yl and the result revealed that two ends of a T-DNA can be integrated into plant genome independently because the two ends can be linked to different chromosomes and cause chromosomal translocation. We also found that these T-DNA were connected into tandem fragment regardless of direction before integrating into plant genome. In addition, the integration of T-DNA in yl genome also caused several chromosomal fragments deletion. We then summarized three cases for T-DNA integration model in the yl genome. (1) A T-DNA fragment is linked to the two ends of a double-stranded break (DSB); (2) Only one end of a T-DNA fragment is linked to a DSB; (3) A T-DNA fragment is linked to the ends of different DSBs. All the observations in the yl genome supported the DSB repair model.In this study, we showed a comprehensive genome analysis of a T-DNA mutant and provide a new insight into T-DNA integration in plants. These findings would be helpful for the analysis of T-DNA mutants with special phenotypes.

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September 22, 2019  |  

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

Parallel sequencing of a single cell’s genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ~3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

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