Recent advances in sequencing chemistry and software in the Sequel II System enable generating highly accurate long reads that are up to 25 kb in length with >99% accuracy. The…
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…
Complete, high-quality microbial genomes are very valuable across a broad array of fields, from environmental studies, to human microbiome health, food pathogen surveillance, etc. Long-read sequencing enables accurate resolution of…
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.
Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a ‘one-step operation’. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513?Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars ‘Smooth Cayenne’ and ‘Queen’ exhibited ancient and recent admixture, while ‘Singapore Spanish’ supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated bromelain inhibitors. Four candidate genes for self-incompatibility were linked in F153, but were not functional in self-compatible CB5. Our findings support the coexistence of sexual recombination and a one-step operation in the domestication of clonally propagated crops. This work guides the exploration of sexual and asexual domestication trajectories in other clonally propagated crops.
A Gram-stain-negative bacterial strain, designated CA10T, was isolated from bovine raw milk sampled in Anseong, Republic of Korea. Cells were yellow-pigmented, aerobic, non-motile bacilli and grew optimally at 30?°C and pH 7.0 on tryptic soy agar without supplementation of NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain CA10T belonged to the genus Chryseobacterium, family Flavobacteriaceae, and was most closely related to Chryseobacterium indoltheticum ATCC 27950T (98.75?% similarity). The average nucleotide identity and digital DNA-DNA hybridization values of strain CA10T were 94.4 and 56.9?%, respectively, relative to Chryseobacterium scophthalmum DSM 16779T, being lower than the cut-off values of 95-96?and 70?%, respectively. The predominant respiratory quinone was menaquinone-6; major polar lipid, phosphatidylethanolamine; major fatty acids, iso-C15?:?0, summed feature 9 (iso-C17?:?1?9c and/or C16?:?0 10-methyl), summed feature 3 (iso-C15?:?0 2-OH and/or C16?:?1?7c) and iso-C17?:?0 3-OH. The results of physiological, chemotaxonomic and biochemical analyses suggested that strain CA10T is a novel species of genus Chryseobacterium, for which the name Chryseobacterium mulctrae sp. nov. is proposed. The type strain is CA10T (=KACC 21234T=JCM 33443T).
A Gram-stain-negative, rod-shaped and red-pigmented strain, HME7025T, was isolated from freshwater sampled in the Republic of Korea. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain HME7025T formed a lineage within the family Cytophagaceae of the phylum Bacteroidetes. Strain HME7025T was closely related to the genera Pseudarcicella, Arcicella and Flectobacillus. The 16S rRNA gene sequence similarity values of strain HME7025T were under 94.5?% to its closest phylogenetic neighbours. The major fatty acids of strain HME7025T were iso-C15?:?0 (41.9?%), summed feature 3 (comprising C16?:?1?7c and/or C16?:?1?6c; 12.2?%) and anteiso-C15?:?0 (10.8?%). The major respiratory quinone was menaquinone-7. The major polar lipids were phosphatidylethanolamine, two unidentified aminophospholipids and one unidentified polar lipid. The DNA G+C content of strain HME7025T was 37.9?mol%. On the basis of the evidence presented in this study, strain HME7025T represents a novel species of a novel genus within the family Cytophagaceae, for which the name Allopseudarcicella aquatilis gen. nov., sp. nov. is proposed. The type strain is HME7025T (=KCTC 23617T=CECT 7957T).
Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing.Published by Elsevier Inc.
Bacillus cereus is an opportunistic human pathogen causing food-borne gastrointestinal infections and non-gastrointestinal infections worldwide. The strain B. cereus FORC_013 was isolated from fried eel. Its genome was completely sequenced by PacBio technology, analyzed and compared with other complete genome sequences of Bacillus to elucidate the distinct pathogenic features of the strain isolated in South Korea. Genomic analysis revealed pathogenesis and host immune evasion-associated genes encoding tissue-destructive exoenzymes, and pore-forming toxins. In particular, tissue-destructive (hemolysin BL, nonhaemolytic enterotoxins) and cytolytic proteins (cytolysin) were observed in the genome, which damage the plasma membrane of the epithelial cells of the small intestine causing diarrhea in humans. Capsule biosynthesis gene found in both chromosome and plasmid, which might be responsible for protecting the pathogen from the host cell immune defense system after host cell invasion. Additionally, multidrug resistance operon and efflux pumps were identified in the genome, which play a prominent role in multi-antibiotic resistance. Comparative phylogenetic tree analysis of the strain FORC_013 and other B. cereus strains revealed that the closest strains are ATCC 14579 and B4264. This genome data can be used to identify virulence factors that can be applied for the development of novel biomarkers for the rapid detection of this pathogen in foods.Copyright © 2018. Published by Elsevier Inc.
To track stepwise changes in genetic diversity and antimicrobial resistance in rapidly evolving OXA-232-producing Klebsiella pneumoniae ST14, an emerging carbapenem-resistant high-risk clone, in clinical settings.Twenty-six K. pneumoniae ST14 isolates were collected by the Korean Nationwide Surveillance of Antimicrobial Resistance system over the course of 1 year. Isolates were subjected to whole-genome sequencing and MIC determinations using 33 antibiotics from 14 classes.Single-nucleotide polymorphism (SNP) typing identified 72 unique SNP sites spanning the chromosomes of the isolates, dividing them into three clusters (I, II and III). The initial isolate possessed two plasmids with 18 antibiotic-resistance genes, including blaOXA-232, and exhibited resistance to 11 antibiotic classes. Four other plasmids containing 12 different resistance genes, including blaCTX-M-15 and strA/B, were introduced over time, providing additional resistance to aztreonam and streptomycin. Moreover, chromosomal integration of insertion sequence Ecp1-blaCTX-M-15 mediated the inactivation of mgrB responsible for colistin resistance in four isolates from cluster III. To the best of our knowledge, this is the first description of K. pneumoniae ST14 resistant to both carbapenem and colistin in South Korea. Furthermore, although some acquired genes were lost over time, the retention of 12 resistance genes and inactivation of mgrB provided resistance to 13 classes of antibiotics.We describe stepwise changes in OXA-232-producing K. pneumoniae ST14 in vivo over time in terms of antimicrobial resistance. Our findings contribute to our understanding of the evolution of emerging high-risk K. pneumoniae clones and provide reference data for future outbreaks.Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Chlorella vulgaris is a fast-growing fresh-water microalga cultivated at the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light-HL versus low light -LL) enabled to identify 10,724 nuclear genes, coding for 11,082 transcripts. Moreover 121 and 48 genes were respectively found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed peculiar features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL vs HL provide insights into the molecular basis for metabolic rearrangement in HL vs. LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway can be predicted and its upregulation upon HL exposure is observed, consistent with increased lipid amount under HL. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.This article is protected by copyright. All rights reserved.
Pacific Biosciences’ SMRT sequencing method was used to extend the sequence of HLA-A*02:13. © 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions in their entirety with higher continuity and accuracy than is possible with other methods.Results We used trio binning to assemble reference genomes for two species from a single individual using an interspecies cross of yak (Bos grunniens) and cattle (Bos taurus). The high heterozygosity inherent to interspecies hybrids allowed us to confidently assign >99% of long reads from the F1 offspring to parental bins using unique k-mers from parental short reads. Both the maternal (yak) and paternal (cattle) assemblies contain over one third of the acrocentric chromosomes, including the two largest chromosomes, in single haplotigs.Conclusions These haplotigs are the first vertebrate chromosome arms to be assembled gap-free and fully phased, and the first time assemblies for two species have been created from a single individual. Both assemblies are the most continuous currently available for non-model vertebrates.MbmegabaseskbkilobasesMYAmillions of years agoMHCmajor histocompatibility complexSMRTsingle molecule real time
Horizontal transfer of plasmids encoding antimicrobial-resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s the first CA-MRSA isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline and penicillin-resistance genes on plasmid pWBG753 (~30 kb). WA-5 and pWBG753 appeared only briefly in WA, however, fusidic-acid-resistance plasmids related to pWBG753 were also present in the first European CA-MRSA at the time. Here we characterized a 72-kb conjugative plasmid pWBG731 present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749-family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium and penicillin-resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS257, IS257-dependent miniature inverted-repeat transposable elements (MITEs) and the BinL resolution system of the ß-lactamase transposon Tn552 An evolutionary intermediate ~42-kb non-conjugative plasmid pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline-resistance plasmid pT181. IS257 likely facilitated replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized non-conjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous CA-MSSA. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA.Copyright © 2019 American Society for Microbiology.
New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls =50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.
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