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April 21, 2020

The evaluation of RNA-Seq de novo assembly by PacBio long read sequencing

RNA-Seq de novo assembly is an important method to generate transcriptomes for non-model organisms before any downstream analysis. Given many great de novo assembly methods developed by now, one critical issue is that there is no consensus on the evaluation of de novo assembly methods yet. Therefore, to set up a benchmark for evaluating the quality of de novo assemblies is very critical. Addressing this challenge will help us deepen the insights on the properties of different de novo assemblers and their evaluation methods, and provide hints on choosing the best assembly sets as transcriptomes of non-model organisms for the further functional analysis. In this article, we generate a textquotedblleftreal timetextquotedblright transcriptome using PacBio long reads as a benchmark for evaluating five de novo assemblers and two model-based de novo assembly evaluation methods. By comparing the de novo assmblies generated by RNA-Seq short reads with the textquotedblleftreal timetextquotedblright transcriptome from the same biological sample, we find that Trinity is best at the completeness by generating more assemblies than the alternative assemblers, but less continuous and having more misassemblies; Oases is best at the continuity and specificity, but less complete; The performance of SOAPdenovo-Trans, Trans-AByss and IDBA-Tran are in between of five assemblers. For evaluation methods, DETONATE leverages multiple aspects of the assembly set and ranks the assembly set with an average performance as the best, meanwhile the contig score can serve as a good metric to select assemblies with high completeness, specificity, continuity but not sensitive to misassemblies; TransRate contig score is useful for removing misassemblies, yet often the assemblies in the optimal set is too few to be used as a transcriptome.

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April 21, 2020

Biochemical characterization of a novel cold-adapted agarotetraose-producing a-agarase, AgaWS5, from Catenovulum sediminis WS1-A.

Although many ß-agarases that hydrolyze the ß-1,4 linkages of agarose have been biochemically characterized, only three a-agarases that hydrolyze the a-1,3 linkages are reported to date. In this study, a new a-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an a-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 °C, respectively. AgaWS5 showed a cold-tolerance, and it retained more than 40% of its maximum enzymatic activity at 10 °C. AgaWS5 is predicted to have several calcium-binding sites. Thus, its activity was slightly enhanced in the presence of Ca2+, and was strongly inhibited by EDTA. The Km and Vmax of AgaWS5 for agarose were 10.6 mg/mL and 714.3 U/mg, respectively. Agarose-liquefication, thin layer chromatography, and mass and NMR spectroscopic analyses demonstrated that AgaWS5 is an endo-type a-agarase that degrades agarose and mainly produces agarotetraose. Thus, in this study, a novel cold-adapted GH96 agarotetraose-producing a-agarase was identified.

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April 21, 2020

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

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.

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April 21, 2020

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

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

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April 21, 2020

A robust benchmark for germline structural variant detection

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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April 21, 2020

RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.

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April 21, 2020

Large Fragment Deletions Induced by Cas9 Cleavage While Not in BEs System in Rabbit

CRISPR-Cas9 and BEs system are poised to become the gene editing tool of choice in clinical contexts, however large fragment deletion was found in Cas9-mediated mutation cells without animal level validation. By analyzing 16 gene-edited rabbit lines (including 112 rabbits) generated using SpCas9, BEs, xCas9 and xCas9-BEs with long-range PCR genotyping and long-read sequencing by PacBio platform, we show that extending thousands of bases fragment deletions in single-guide RNA/Cas9 and xCas9 system mutation rabbit, but few large deletions were found in BEs-induced mutation rabbits. We firstly validated that no large fragment deletion induced by BEs system at animal level, suggesting that BE systems can be beneficial tools for the further development of highly accurate and secure gene therapy for the clinical treatment of human genetic disorders

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April 21, 2020

Comparison of mitochondrial DNA variants detection using short- and long-read sequencing.

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.

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April 21, 2020

Genomic analysis of Marinobacter sp. NP-4 and NP-6 isolated from the deep-sea oceanic crust on the western flank of the Mid-Atlantic Ridge

Two Marinobacter sp. NP-4 and NP-6 were isolated from a deep oceanic basaltic crust at North Pond, located at the western flank of the Mid-Atlantic Ridge. These two strains are capable of using multiple carbon sources such as acetate, succinate, glucose and sucrose while take oxygen as a primary electron acceptor. The strain NP-4 is also able to grow anaerobically under 20?MPa, with nitrate as the electron acceptor, thus represents a piezotolerant. To explore the metabolic potentials of Marinobacter sp. NP-4 and NP-6, the complete genome of NP-4 and close-to-complete genome of NP-6 were sequenced. The genome of NP-4 contains one chromosome and two plasmids with the size of 4.6?Mb in total, and with average GC content of 57.0%. The genome of NP-6 is 4.5?Mb and consists of 6 scaffolds, with an average GC content of 57.1%. Complete glycolysis, citrate cycle and aromatics compounds degradation pathways are identified in genomes of these two strains, suggesting that they possess a heterotrophic life style. Additionally, one plasmid of NP-4 contains genes for alkane degradation, phosphonate ABC transporter and cation efflux system, enabling NP-4 extra surviving abilities. In total, genomic information of these two strains provide insights into the physiological features and adaptation strategies of Marinobacter spp. in the deep oceanic crust biosphere.

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April 21, 2020

Complete genome sequence of Paenisporosarcina antarctica CGMCC 1.6503 T, a marine psychrophilic bacterium isolated from Antarctica

A marine psychrophilic bacterium _Paenisporosarcina antarctica_ CGMCC 1.6503T (= JCM 14646T) was isolated off King George Island, Antarctica (62°13’31? S 58°57’08? W). In this study, we report the complete genome sequence of _Paenisporosarcina antarctica_, which is comprised of 3,972,524?bp with a mean G?+?C content of 37.0%. By gene function and metabolic pathway analyses, studies showed that strain CGMCC 1.6503T encodes a series of genes related to cold adaptation, including encoding fatty acid desaturases, dioxygenases, antifreeze proteins and cold shock proteins, and possesses several two-component regulatory systems, which could assist this strain in responding to the cold stress, the oxygen stress and the osmotic stress in Antarctica. The complete genome sequence of _P. antarctica_ may provide further insights into the genetic mechanism of cold adaptation for Antarctic marine bacteria.

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April 21, 2020

Genome sequence resource for Ilyonectria mors-panacis, causing rusty root rot of Panax notoginseng.

Ilyonectria mors-panacis is a serious disease hampering the production of Panax notoginseng, an important Chinese medicinal herb, widely used for its anti-inflammatory, anti-fatigue, hepato-protective, and coronary heart disease prevention effects. Here, we report the first Illumina-Pacbio hybrid sequenced draft genome assembly of I. mors-panacis strain G3B and its annotation. The availability of this genome sequence not only represents an important tool toward understanding the genetics behind the infection mechanism of I. mors-panacis strain G3B but also will help illuminate the complexities of the taxonomy of this species.

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April 21, 2020

Decoding and analysis of organelle genomes of Indian tea (Camellia assamica) for phylogenetic confirmation.

The NCBI database has >15 chloroplast (cp) genome sequences available for different Camellia species but none for C. assamica. There is no report of any mitochondrial (mt) genome in the Camellia genus or Theaceae family. With the strong believes that these organelle genomes can play a great tool for taxonomic and phylogenetic analysis, we successfully assembled and analyzed cp and mt genome of C. assamica. We assembled the complete mt genome of C. assamica in a single circular contig of 707,441?bp length comprising of a total of 66 annotated genes, including 35 protein-coding genes, 29 tRNAs and two rRNAs. The first ever cp genome of C. assamica resulted in a circular contig of 157,353?bp length with a typical quadripartite structure. Phylogenetic analysis based on these organelle genomes showed that C. assamica was closely related to C. sinensis and C. leptophylla. It also supports Caryophyllales as Superasterids. Copyright © 2019. Published by Elsevier Inc.

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April 21, 2020

Complete genome of a marine bacterium Vibrio chagasii ECSMB14107 with the ability to infect mussels

Vibrio strains are pervasive in the aquatic environment and may form pathogenic and symbiotic relationships with the host. Vibrio chagasii ECSMB14107 was isolated from natural biofilms and is used as a model to elucidate the role of Vibrio in hard-shelled mussel (Mytilus coruscus) settlement, health and disease. The genome of the Vibrio strain ECSMB14107, comprised of two circular chromosomes that together encompass 5,549,357?bp with a mean GC content of 44.39% was determined. Knowledge about the genome of V. chagasii ECSMB14107 will provide insight into its contribution to mussel development and health.

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April 21, 2020

Integrating multiple genomic technologies to investigate an outbreak of carbapenemase-producing Enterobacter hormaechei

Carbapenem-resistant Enterobacteriaceae (CRE) represent one of the most urgent threats to human health posed by antibiotic resistant bacteria. Enterobacter hormaechei and other members of the Enterobacter cloacae complex are the most commonly encountered Enterobacter spp. within clinical settings, responsible for numerous outbreaks and ultimately poorer patient outcomes. Here we applied three complementary whole genome sequencing (WGS) technologies to characterise a hospital cluster of blaIMP-4 carbapenemase-producing E. hormaechei.In response to a suspected CRE outbreak in 2015 within an Intensive Care Unit (ICU)/Burns Unit in a Brisbane tertiary referral hospital we used Illumina sequencing to determine that all outbreak isolates were sequence type (ST)90 and near-identical at the core genome level. Comparison to publicly available data unequivocally linked all 10 isolates to a 2013 isolate from the same ward, confirming the hospital environment as the most likely original source of infection in the 2015 cases. No clonal relationship was found to IMP-4-producing isolates identified from other local hospitals. However, using Pacific Biosciences long-read sequencing we were able to resolve the complete context of the blaIMP-4 gene, which was found to be on a large IncHI2 plasmid carried by all IMP-4-producing isolates. Continued surveillance of the hospital environment was carried out using Oxford Nanopore long-read sequencing, which was able to rapidly resolve the true relationship of subsequent isolates to the initial outbreak. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing.Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak, including the transmission dynamics of a carbapenemase-producing E. hormaechei cluster, identification of possible hospital reservoirs and the full context of blaIMP-4 on a multidrug resistant IncHI2 plasmid that appears to be widely distributed in Australia.

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