PacBio 2014 User Group Meeting Presentation Slides: Alisha Holloway of the Gladstone Institutes presented on the use of isoform sequencing (Iso-Seq) to improve the annotation of the chicken genome as a model reference for cardiovascular research.
While genome assembly projects have been successful in many haploid and inbred species, the assembly of non-inbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short-…
SMRT-Cappable-seq combines the isolation of full-length prokaryotic primary transcripts with long read sequencing technology. It is the first experimental methodology to sequence entire prokaryotic transcripts. It identifies the transcription start site and termination site, thereby directly defines the operon structures genome-wide in prokaryotes. Applied to E.coli, SMRT-Cappable-seq identifies a total of ~2300 operons, among which ~900 are novel. Importantly, our result reveals a pervasive read-through of previous experimentally validated transcription termination sites. Termination read-through represents a powerful strategy to control gene expression. Taken together this data provides a first glance at the complexity of the ‘operome’ in bacteria and presents…
With PacBio single-cell RNA sequencing using the Iso-Seq method, you can now distinguish between alternative transcript isoforms at the single-cell level. The highly accurate long reads (HiFi reads) can span the entire 5′ to 3′ end of a transcript, allowing a high-resolution view of isoform diversity and revealing cell-to-cell heterogeneity without the need for assembly.
In this AGBT presentation from AGBT 2019, Jason Underwood, shares information about single-cell isoform sequencing (scIso-Seq), focusing on a collaborative project with the labs of Evan Eichler and Alex Pollen. For this effort, scientists used Drop-seq sample prep and then loaded cDNA products onto the Sequel System. Results from a barnyard experiment using mouse and human cells as well as from cerebral organoids demonstrated that this approach could deliver cell type-specific gene expression data. Underwood also presents data from the Sequel II System comparing chimp and human organoids, resulting in information about 14,000 unique genes with important insights for post-transcriptional…
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…
Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made towards understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57862 high-quality full-length mRNA sequences derived from 18362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length…
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…
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…
Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81?kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3…
More than 3,000 species of octocorals (Cnidaria, Anthozoa) inhabit an expansive range of environments, from shallow tropical seas to the deep-ocean floor. They are important foundation species that create coral “forests,” which provide unique niches and 3-dimensional living space for other organisms. The octocoral genus Renilla inhabits sandy, continental shelves in the subtropical and tropical Atlantic and eastern Pacific Oceans. Renilla is especially interesting because it produces secondary metabolites for defense, exhibits bioluminescence, and produces a luciferase that is widely used in dual-reporter assays in molecular biology. Although several anthozoan genomes are currently available, the majority of these are hexacorals.…
Supernumerary B chromosomes (Bs) are extra karyotype units in addition to A chromosomes, and are found in some fungi and thousands of animals and plant species. Bs are uniquely characterized due to their non-Mendelian inheritance, and represent one of the best examples of genomic conflict. Over the last decades, their genetic composition, function and evolution have remained an unresolved query, although a few successful attempts have been made to address these phenomena. A classical concept based on cytogenetics and genetics is that Bs are selfish and abundant with DNA repeats and transposons, and in most cases, they do not carry…
Lacerta viridis and Lacerta bilineata are sister species of European green lizards (eastern and western clades, respectively) that, until recently, were grouped together as the L. viridis complex. Genetic incompatibilities were observed between lacertid populations through crossing experiments, which led to the delineation of two separate species within the L. viridis complex. The population history of these sister species and processes driving divergence are unknown. We constructed the first high-quality de novo genome assemblies for both L. viridis and L. bilineata through Illumina and PacBio sequencing, with annotation support provided from transcriptome sequencing of several tissues. To estimate gene flow…
Trichoplusiani derived cell lines are commonly used to enable recombinant protein expression via baculovirus infection to generate materials approved for clinical use and in clinical trials. In order to develop systems biology and genome engineering tools to improve protein expression in this host, we performed de novo genome assembly of the Trichoplusiani-derived cell line Tni-FNL.By integration of PacBio single-molecule sequencing, Bionano optical mapping, and 10X Genomics linked-reads data, we have produced a draft genome assembly of Tni-FNL.Our assembly contains 280 scaffolds, with a N50 scaffold size of 2.3 Mb and a total length of 359 Mb. Annotation of the Tni-FNL…
Algal polysaccharides are an important bacterial nutrient source and central component of marine food webs. However, cellular and ecological aspects concerning the bacterial degradation of polysaccharide mixtures, as presumably abundant in natural habitats, are poorly understood. Here, we contextualize marine polysaccharide mixtures and their bacterial utilization in several ways using the model bacterium Alteromonas macleodii 83-1, which can degrade multiple algal polysaccharides and contributes to polysaccharide degradation in the oceans. Transcriptomic, proteomic and exometabolomic profiling revealed cellular adaptations of A. macleodii 83-1 when degrading a mix of laminarin, alginate and pectin. Strain 83-1 exhibited substrate prioritization driven by catabolite repression,…