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April 21, 2020  |  

A full-length transcriptome of Sepia esculenta using a combination of single-molecule long-read (SMRT) and Illumina sequencing

As an economically important cephalopods species, wild-caught Sepia esculenta fishery has suffered a server decline due to over-fishing and ocean environmental damage. To restore this seriously declining fishery resource, we should understand the genetic foundation and molecular mechanism of spawning, reproduction and mortal of golden cuttlefish. In this study, we generated the full-length transcriptome of S. esculenta based on the total RNA of tissue samples (brain, optic gland, nidamental gland, ovary and muscle at different developmental stages) using a combination of single-molecule real-time (SMRT) and Illumina RNA-seq technology. A total of 14.16 Gb SMRT sequencing data were assembled into 94,635 transcripts. Meanwhile, 35.15 Gb Illumina HiSeq data were assembled into 177,226 non-redundant transcripts. Then, we merged SMRT and Illumina assembled data to generate a more complete/full-length S. esculenta transcriptome with 177,951 high-quality transcripts. Based on the obtained transcriptome data, total 81,459 transcripts were annotated in at least one of seven functional databases and 49,189 nucleotide sequences of coding regions were identified. Additionally, 161,327 SSRs distributed in 64,933 transcripts were identified based on SSR analysis. This full-length and high-quality transcriptome of S. esculenta can provide an important foundation for future genomic research on growth and development, reproduction and mortal of cephalopod and further recovery of this recessionary fisheries resources.

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April 21, 2020  |  

The role of long-term mineral and organic fertilisation treatment in changing pathogen and symbiont community composition in soil

Application of organic fertilisers to soil prevents erosion, improves fertility and may suppress certain soil-borne plant pathogens, but it is still unclear how different trophic groups of fungi and oomycetes respond to long-term fertilisation treatment. The objective of the study was to examine the effect of different fertilisation regimes on fungal and oomycete pathogen- and mycorrhizal symbiont diversity and community structure in both soil and roots, using PacBio SMRT sequencing. The field experiment included three fertilisation treatments that have been applied since 1989: nitrogen fertilisation (WOM), nitrogen fertilisation with manure amendment (FYM) and alternative organic fertilisation (AOF), each applied at five different rates. Soil samples were collected three times during the growing season, while root samples were collected during the flowering stage. There was no influence of the studied variables on soil and root pathogen richness. Contrary to our hypothesis, pathogen relative abundance in both soil and roots was significantly higher in plots with the AOF treatment. Furthermore, richness and relative abundance of arbuscular mycorrhizal (AM) fungi decreased significantly in the AOF treatment. Permutational analysis of variance (PERMANOVA) demonstrated the effect of fertilisation treatment on pathogen community composition in both soil and roots. Our findings indicate that organic fertilisers may not always benefit soil microbial community composition. Therefore, further studies are needed to understand how fertilisation affects mycorrhizal mutualists and pathogens.

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April 21, 2020  |  

Investigating the bacterial microbiota of traditional fermented dairy products using propidium monoazide with single-molecule real-time sequencing.

Traditional fermented dairy foods have been the major components of the Mongolian diet for millennia. In this study, we used propidium monoazide (PMA; binds to DNA of nonviable cells so that only viable cells are enumerated) and single-molecule real-time sequencing (SMRT) technology to investigate the total and viable bacterial compositions of 19 traditional fermented dairy foods, including koumiss from Inner Mongolia (KIM), koumiss from Mongolia (KM), and fermented cow milk from Mongolia (CM); sample groups treated with PMA were designated PKIM, PKM, and PCM. Full-length 16S rRNA sequencing identified 195 bacterial species in 121 genera and 13 phyla in PMA-treated and untreated samples. The PMA-treated and untreated samples differed significantly in their bacterial community composition and a-diversity values. The predominant species in KM, KIM, and CM were Lactobacillus helveticus, Streptococcus parauberis, and Lactobacillus delbrueckii, whereas the predominant species in PKM, PKIM, and PCM were Enterobacter xiangfangensis, Lactobacillus helveticus, and E. xiangfangensis, respectively. Weighted and unweighted principal coordinate analyses showed a clear clustering pattern with good separation and only minor overlapping. In addition, a pure culture method was performed to obtain lactic acid bacteria resources in dairy samples according to the results of SMRT sequencing. A total of 102 LAB strains were identified and Lb. helveticus (68.63%) was the most abundant, in agreement with SMRT sequencing results. Our results revealed that the bacterial communities of traditional dairy foods are complex and vary by type of fermented dairy product. The PMA treatment induced significant changes in bacterial community structure.Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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April 21, 2020  |  

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale.

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

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April 21, 2020  |  

A 12-kb structural variation in progressive myoclonic epilepsy was newly identified by long-read whole-genome sequencing.

We report a family with progressive myoclonic epilepsy who underwent whole-exome sequencing but was negative for pathogenic variants. Similar clinical courses of a devastating neurodegenerative phenotype of two affected siblings were highly suggestive of a genetic etiology, which indicates that the survey of genetic variation by whole-exome sequencing was not comprehensive. To investigate the presence of a variant that remained unrecognized by standard genetic testing, PacBio long-read sequencing was performed. Structural variant (SV) detection using low-coverage (6×) whole-genome sequencing called 17,165 SVs (7,216 deletions and 9,949 insertions). Our SV selection narrowed down potential candidates to only five SVs (two deletions and three insertions) on the genes tagged with autosomal recessive phenotypes. Among them, a 12.4-kb deletion involving the CLN6 gene was the top candidate because its homozygous abnormalities cause neuronal ceroid lipofuscinosis. This deletion included the initiation codon and was found in a GC-rich region containing multiple repetitive elements. These results indicate the presence of a causal variant in a difficult-to-sequence region and suggest that such variants that remain enigmatic after the application of current whole-exome sequencing technology could be uncovered by unbiased application of long-read whole-genome sequencing.

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April 21, 2020  |  

The CF Canada-Sick Kids Program in individual CF therapy: A resource for the advancement of personalized medicine in CF.

Therapies targeting certain CFTR mutants have been approved, yet variations in clinical response highlight the need for in-vitro and genetic tools that predict patient-specific clinical outcomes. Toward this goal, the CF Canada-Sick Kids Program in Individual CF Therapy (CFIT) is generating a “first of its kind”, comprehensive resource containing patient-specific cell cultures and data from 100 CF individuals that will enable modeling of therapeutic responses.The CFIT program is generating: 1) nasal cells from drug naïve patients suitable for culture and the study of drug responses in vitro, 2) matched gene expression data obtained by sequencing the RNA from the primary nasal tissue, 3) whole genome sequencing of blood derived DNA from each of the 100 participants, 4) induced pluripotent stem cells (iPSCs) generated from each participant’s blood sample, 5) CRISPR-edited isogenic control iPSC lines and 6) prospective clinical data from patients treated with CF modulators.To date, we have recruited 57 of 100 individuals to CFIT, most of whom are homozygous for F508del (to assess in-vitro: in-vivo correlations with respect to ORKAMBI response) or heterozygous for F508del and a minimal function mutation. In addition, several donors are homozygous for rare nonsense and missense mutations. Nasal epithelial cell cultures and matched iPSC lines are available for many of these donors.This accessible resource will enable development of tools that predict individual outcomes to current and emerging modulators targeting F508del-CFTR and facilitate therapy discovery for rare CF causing mutations.Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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April 21, 2020  |  

Vaccine-induced protection from homologous tier 2 SHIV challenge in nonhuman primates depends on serum-neutralizing antibody titers.

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ~1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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April 21, 2020  |  

De Novo Genome Sequence Assembly of Dwarf Coconut (Cocos nucifera L. ‘Catigan Green Dwarf’) Provides Insights into Genomic Variation Between Coconut Types and Related Palm Species.

We report the first whole genome sequence (WGS) assembly and annotation of a dwarf coconut variety, ‘Catigan Green Dwarf’ (CATD). The genome sequence was generated using the PacBio SMRT sequencing platform at 15X coverage of the expected genome size of 2.15 Gbp, which was corrected with assembled 50X Illumina paired-end MiSeq reads of the same genome. The draft genome was improved through Chicago sequencing to generate a scaffold assembly that results in a total genome size of 2.1 Gbp consisting of 7,998 scaffolds with N50 of 570,487 bp. The final assembly covers around 97.6% of the estimated genome size of coconut ‘CATD’ based on homozygous k-mer peak analysis. A total of 34,958 high-confidence gene models were predicted and functionally associated to various economically important traits, such as pest/disease resistance, drought tolerance, coconut oil biosynthesis, and putative transcription factors. The assembled genome was used to infer the evolutionary relationship within the palm family based on genomic variations and synteny of coding gene sequences. Data show that at least three (3) rounds of whole genome duplication occurred and are commonly shared by these members of the Arecaceae family. A total of 7,139 unique SSR markers were designed to be used as a resource in marker-based breeding. In addition, we discovered 58,503 variants in coconut by aligning the Hainan Tall (HAT) WGS reads to the non-repetitive regions of the assembled CATD genome. The gene markers and genome-wide SSR markers established here will facilitate the development of varieties with resilience to climate change, resistance to pests and diseases, and improved oil yield and quality.Copyright © 2019 Lantican et al.

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April 21, 2020  |  

Detecting a long insertion variant in SAMD12 by SMRT sequencing: implications of long-read whole-genome sequencing for repeat expansion diseases.

Long-read sequencing technology is now capable of reading single-molecule DNA with an average read length of more than 10?kb, fully enabling the coverage of large structural variations (SVs). This advantage may pave the way for the detection of unprecedented SVs as well as repeat expansions. Pathogenic SVs of only known genes used to be selectively analyzed based on prior knowledge of target DNA sequence. The unbiased application of long-read whole-genome sequencing (WGS) for the detection of pathogenic SVs has just begun. Here, we apply PacBio SMRT sequencing in a Japanese family with benign adult familial myoclonus epilepsy (BAFME). Our SV selection of low-coverage WGS data (7×) narrowed down the candidates to only six SVs in a 7.16-Mb region of the BAFME1 locus and correctly determined an approximately 4.6-kb SAMD12 intronic repeat insertion, which is causal of BAFME1. These results indicate that long-read WGS is potentially useful for evaluating all of the known SVs in a genome and identifying new disease-causing SVs in combination with other genetic methods to resolve the genetic causes of currently unexplained diseases.

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April 21, 2020  |  

Agricultural intensification reduces microbial network complexity and the abundance of keystone taxa in roots.

Root-associated microbes play a key role in plant performance and productivity, making them important players in agroecosystems. So far, very few studies have assessed the impact of different farming systems on the root microbiota and it is still unclear whether agricultural intensification influences the structure and complexity of microbial communities. We investigated the impact of conventional, no-till, and organic farming on wheat root fungal communities using PacBio SMRT sequencing on samples collected from 60 farmlands in Switzerland. Organic farming harbored a much more complex fungal network with significantly higher connectivity than conventional and no-till farming systems. The abundance of keystone taxa was the highest under organic farming where agricultural intensification was the lowest. We also found a strong negative association (R2?=?0.366; P?

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April 21, 2020  |  

High-Resolution Evolutionary Analysis of Within-Host Hepatitis C Virus Infection.

Despite recent breakthroughs in treatment of hepatitis C virus (HCV) infection, we have limited understanding of how virus diversity generated within individuals impacts the evolution and spread of HCV variants at the population scale. Addressing this gap is important for identifying the main sources of disease transmission and evaluating the risk of drug-resistance mutations emerging and disseminating in a population.We have undertaken a high-resolution analysis of HCV within-host evolution from 4 individuals coinfected with human immunodeficiency virus 1 (HIV-1). We used long-read, deep-sequenced data of full-length HCV envelope glycoprotein, longitudinally sampled from acute to chronic HCV infection to investigate the underlying viral population and evolutionary dynamics.We found statistical support for population structure maintaining the within-host HCV genetic diversity in 3 out of 4 individuals. We also report the first population genetic estimate of the within-host recombination rate for HCV (0.28 × 10-7 recombination/site/year), which is considerably lower than that estimated for HIV-1 and the overall nucleotide substitution rate estimated during HCV infection.Our findings indicate that population structure and strong genetic linkage shapes within-host HCV evolutionary dynamics. These results will guide the future investigation of potential HCV drug resistance adaptation during infection, and at the population scale. © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.

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April 21, 2020  |  

A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci.

Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain Purple Kush and the hemp variety “Finola.” The map reveals that cannabinoid biosynthesis genes are generally unlinked but that aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (CBCAS) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis. THCAS and CBDAS (which determine the drug vs. hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly nonhomologous between drug- and hemp-type alleles and are furthermore embedded within ~40 Mb of minimally recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant. © 2019 Laverty et al.; Published by Cold Spring Harbor Laboratory Press.

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April 21, 2020  |  

Rapid and Focused Maturation of a VRC01-Class HIV Broadly Neutralizing Antibody Lineage Involves Both Binding and Accommodation of the N276-Glycan.

The VH1-2 restricted VRC01-class of antibodies targeting the HIV envelope CD4 binding site are a major focus of HIV vaccine strategies. However, a detailed analysis of VRC01-class antibody development has been limited by the rare nature of these responses during natural infection and the lack of longitudinal sampling of such responses. To inform vaccine strategies, we mapped the development of a VRC01-class antibody lineage (PCIN63) in the subtype C infected IAVI Protocol C neutralizer PC063. PCIN63 monoclonal antibodies had the hallmark VRC01-class features and demonstrated neutralization breadth similar to the prototype VRC01 antibody, but were 2- to 3-fold less mutated. Maturation occurred rapidly within ~24 months of emergence of the lineage and somatic hypermutations accumulated at key contact residues. This longitudinal study of broadly neutralizing VRC01-class antibody lineage reveals early binding to the N276-glycan during affinity maturation, which may have implications for vaccine design.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020  |  

PacBio sequencing reveals bacterial community diversity in cheeses collected from different regions.

Cheese is a fermented dairy product that is popular for its unique flavor and nutritional value. Recent studies have shown that microorganisms in cheese play an important role in the fermentation process and determine the quality of the cheese. We collected 12 cheese samples from different regions and studied the composition of their bacterial communities using PacBio small-molecule real-time sequencing (Pacific Biosciences, Menlo Park, CA). Our data revealed 144 bacterial genera (including Lactobacillus, Streptococcus, Lactococcus, and Staphylococcus) and 217 bacterial species (including Lactococcus lactis, Streptococcus thermophilus, Staphylococcus equorum, and Streptococcus uberis). We investigated the flavor quality of the cheese samples using an electronic nose system and we found differences in flavor-quality indices among samples from different regions. We found a clustering tendency based on flavor quality using principal component analysis. We found correlations between lactic acid bacteria and the flavor quality of the cheese samples. Biodegradation and metabolism of xenobiotics, and lipid-metabolism-related pathways, were predicted to contribute to differences in cheese flavor using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). This preliminary study explored the bacterial communities in cheeses collected from different regions and their potential genome functions from the perspective of flavor quality.Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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April 21, 2020  |  

The complete genome sequence of Ethanoligenens harbinense reveals the metabolic pathway of acetate-ethanol fermentation: A novel understanding of the principles of anaerobic biotechnology.

Ethanol-type fermentation is one of three main fermentation types in the acidogenesis of anaerobic treatment systems. Non-spore-forming Ethanoligenens is as a typical genus capable of ethanol-type fermentation in mixed culture (i.e. acetate-ethanol fermentation). This genus can produce ethanol, acetate, CO2, and H2 using carbohydrates, and has application potential in anaerobic bioprocesses. Here, the complete genome sequences and methylome of Ethanoligenens harbinense strains with different autoaggregative and coaggregative abilities were obtained using the PacBio single-molecule real-time sequencing platform. The genome size of E. harbinense strains was about 2.97-3.10?Mb with 55.5% G+C content. 3020-3153 genes were annotated, most of which were methylated at specific sites or motifs. The methylation types included 6mA, 4mC, and unknown types. Comparative genomic analysis demonstrated low levels of genetic similarity between E. harbinense and other well-known hydrogen-producing bacteria (i.e., Clostridium and Thermoanaerobacter) in phylogenesis. Hydrogen production of E. harbinense was catalyzed by genes that encode [FeFe]-hydrogenases and that were synthesized by three maturases of [FeFe]-H2ase. The metabolic mechanism of H2-ethanol co-production fermentation, catalyzed by pyruvate ferredoxin oxidoreductase was proposed. This study provides genetic and evolutionary information of a model genus for the further investigation of the metabolic pathway and regulatory network of ethanol-type fermentation and anaerobic bioprocesses for waste or wastewater treatment.Copyright © 2019. Published by Elsevier Ltd.

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