In this webinar, Emily Hatas of PacBio shares information about the applications and benefits of SMRT Sequencing in plant and animal biology, agriculture, and industrial research fields. This session contains…
In this PacBio User Group Meeting presentation, Mount Sinai’s Ethan Ellis presents results from the HLS-CATCH method, which involves the use of the SageHLS instrument with CRISPR design methods to…
In this webinar, Jenny Ekholm and Paul Kotturi provide an overview of the PacBio No-Amp targeted sequencing application and its uses for targeting hard-to-amplify genes. This approach couples CRISPR-Cas9 with…
In this presentation, Emily Hatas of PacBio offers a look a how SMRT Sequencing has changed over the years as well as the most common applications in human genome analysis:…
In this talk at PAG 2020, PacBio Plant and Animal Sciences Marketing Manager Michelle Vierra discusses recent updates to Single Molecule, Real-Time (SMRT) Sequencing technology, including the Sequel II System,…
In this webinar, Adam Ameur of SciLifeLab at Uppsala University shares how he uses Single Molecule, Real-Time (SMRT) Sequencing applications for medical diagnostics and human genetics research, including sequencing of…
In this ASHG 2020 PacBio Workshop Jonas Korlach, CSO, shares how the new PacBio Sequel IIe System makes highly accurate long-read sequencing easy and affordable so?all scientists can gain comprehensive…
The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with ‘ready-to-use’ deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and that may proliferate in public database repositories affecting all downstream analyses. As a case study, we provide examples of the Atlantic cod genome, whose sequencing and assembly were hindered by a particularly high prevalence of tandem repeats. We complement this case study with examples from other species, where mis-annotations and sequencing errors have propagated into protein databases. With this review, we aim to raise the awareness level within the community of database users, and alert scientists working in the underlying workflow of database creation that the data they omit or improperly assemble may well contain important biological information valuable to others. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
The recent advent of long-read sequencing technologies is expected to provide reasonable answers to genetic challenges unresolvable by short-read sequencing, primarily the inability to accurately study structural variations, copy number variations, and homologous repeats in complex parts of the genome. However, long-read sequencing comes along with higher rates of random short deletions and insertions, and single nucleotide errors. The relatively higher sequencing accuracy of short-read sequencing has kept it as the first choice of screening for single nucleotide variants and short deletions and insertions. Albeit, short-read sequencing still suffers from systematic errors that tend to occur at specific positions where a high depth of reads is not always capable to correct for these errors. In this study, we compared the genotyping of mitochondrial DNA variants in three samples using PacBio’s Sequel (Pacific Biosciences Inc., Menlo Park, CA, USA) long-read sequencing and illumina’s HiSeqX10 (illumine Inc., San Diego, CA, USA) short-read sequencing data. We concluded that, despite the differences in the type and frequency of errors in the long-reads sequencing, its accuracy is still comparable to that of short-reads for genotyping short nuclear variants; due to the randomness of errors in long reads, a lower coverage, around 37 reads, can be sufficient to correct for these random errors.
Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome with chromosome-scale sequences for Chinese chestnut (C. mollissima), the disease-resistance donor for American chestnut restoration. We also demonstrate the value of the genome as a platform for research and species restoration, including new insights into the evolution of blight resistance in Asian chestnut species, the locations in the genome of ecologically important signatures of selection differentiating American chestnut from Chinese chestnut, the identification of candidate genes for disease resistance, and preliminary comparisons of genome organization with related species.
Sequencing technology and assembly algorithms have matured to the point that high-quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse transposable elements (TEs) and allow for annotation of TEs. There are numerous methods for each class of elements with unknown relative performance metrics. We benchmarked existing programs based on a curated library of rice TEs. Using the most robust programs, we created a comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a condensed TE library for annotations of structurally intact and fragmented elements. EDTA is open-source and freely available: https://github.com/oushujun/EDTA.List of abbreviationsTETransposable ElementsLTRLong Terminal RepeatLINELong Interspersed Nuclear ElementSINEShort Interspersed Nuclear ElementMITEMiniature Inverted Transposable ElementTIRTerminal Inverted RepeatTSDTarget Site DuplicationTPTrue PositivesFPFalse PositivesTNTrue NegativeFNFalse NegativesGRFGeneric Repeat FinderEDTAExtensive de-novo TE Annotator
DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spreads to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5′ ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for twelve different gene targets indicated that neighbors within 500-nt were often co-silenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, cis-spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not co-silenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in Phytophthora but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.
Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinsontextquoterights Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here we apply the first study of using long read sequencing with targeted capture of both the gDNA and cDNA of the SNCA gene in brain tissues of PD, DLB, and control samples using the PacBio Sequel system. The targeted full-length cDNA (Iso-Seq) data confirmed complex usage of known alternative start sites and variable 3textquoteright UTR lengths, as well as novel 5textquoteright starts and 3textquoteright ends not previously described. The targeted gDNA data allowed phasing of up to 81% of the ~114kb SNCA region, with the longest phased block excedding 54 kb. We demonstrate that long gDNA and cDNA reads have the potential to reveal long-range information not previously accessible using traditional sequencing methods. This approach has a potential impact in studying disease risk genes such as SNCA, providing new insights into the genetic etiologies, including perturbations to the landscape the gene transcripts, of human complex diseases such as synucleinopathies.
Suppressed recombination allows divergence between homologous sex chromosomes and the functionality of their genes. Here, we reveal patterns of the earliest stages of sex-chromosome evolution in the diploid dioecious herb Mercurialis annua on the basis of cytological analysis, de novo genome assembly and annotation, genetic mapping, exome resequencing of natural populations, and transcriptome analysis. The genome assembly contained 34,105 expressed genes, of which 10,076 were assigned to linkage groups. Genetic mapping and exome resequencing of individuals across the species range both identified the largest linkage group, LG1, as the sex chromosome. Although the sex chromosomes of M. annua are karyotypically homomorphic, we estimate that about a third of the Y chromosome has ceased recombining, containing 568 transcripts and spanning 22.3 cM in the corresponding female map. Nevertheless, we found limited evidence for Y-chromosome degeneration in terms of gene loss and pseudogenization, and most X- and Y-linked genes appear to have diverged in the period subsequent to speciation between M. annua and its sister species M. huetii which shares the same sex-determining region. Taken together, our results suggest that the M. annua Y chromosome has at least two evolutionary strata: a small old stratum shared with M. huetii, and a more recent larger stratum that is probably unique to M. annua and that stopped recombining about one million years ago. Patterns of gene expression within the non-recombining region are consistent with the idea that sexually antagonistic selection may have played a role in favoring suppressed recombination.Copyright © 2019, Genetics.
Acoels are primitive bilaterians with very simple soft bodies, in which many organs, including the gut, are not developed. They provide platforms for studying molecular and developmental mechanisms involved in the formation of the basic bilaterian body plan, whole-body regeneration, and symbiosis with photosynthetic microalgae. Because genomic information is essential for future research on acoel biology, we sequenced and assembled the nuclear genome of an acoel, Praesagittifera naikaiensis.To avoid sequence contamination derived from symbiotic microalgae, DNA was extracted from embryos that were free of algae. More than 290x sequencing coverage was achieved using a combination of Illumina (paired-end and mate-pair libraries) and PacBio sequencing. RNA sequencing and Iso-Seq data from embryos, larvae, and adults were also obtained. First, a preliminary ~17-kilobase pair (kb) mitochondrial genome was assembled, which was deleted from the nuclear sequence assembly. As a result, a draft nuclear genome assembly was ~656 Mb in length, with a scaffold N50 of 117 kb and a contig N50 of 57 kb. Although ~70% of the assembled sequences were likely composed of repetitive sequences that include DNA transposons and retrotransposons, the draft genome was estimated to contain 22,143 protein-coding genes, ~99% of which were substantiated by corresponding transcripts. We could not find horizontally transferred microalgal genes in the acoel genome. Benchmarking Universal Single-Copy Orthologs analyses indicated that 77% of the conserved single-copy genes were complete. Pfam domain analyses provided a basic set of gene families for transcription factors and signaling molecules.Our present sequencing and assembly of the P. naikaiensis nuclear genome are comparable to those of other metazoan genomes, providing basic information for future studies of genic and genomic attributes of this animal group. Such studies may shed light on the origins and evolution of simple bilaterians. © The Author(s) 2019. Published by Oxford University Press.
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