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Asset Tag: Pacific Biosciences

April 21, 2020

Genome sequence of the Chinese white wax scale insect Ericerus pela: the first draft genome for the Coccidae family of scale insects.

The Chinese white wax scale insect, Ericerus pela, is best known for producing wax, which has been widely used in candle production, casting, Chinese medicine, and wax printing products for thousands of years. The secretion of wax, and other unusual features of scale insects, is thought to be an adaptation to their change from an ancestral ground-dwelling lifestyle to a sedentary lifestyle on the higher parts of plants. As well as helping to improve its economic value, studies of E. pela might also help to explain the adaptation of scale insects. However, no genomic data are currently available for E. pela.To assemble the E. pela genome, 303.92 Gb of data were generated using Illumina and Pacific Biosciences sequencing, producing 277.22 Gb of clean data for assembly. The assembled genome size was 0.66 Gb, with 1,979 scaffolds and a scaffold N50 of 735 kb. The guanine + cytosine content was 33.80%. A total of 12,022 protein-coding genes were predicted, with a mean coding sequence length of 1,370 bp. Twenty-six fatty acyl-CoA reductase genes and 35 acyltransferase genes were identified. Evolutionary analysis revealed that E. pela and aphids formed a sister group and split ~241.1 million years ago. There were 214 expanded gene families and 2,219 contracted gene families in E. pela.We present the first genome sequence from the Coccidae family. These results will help to increase our understanding of the evolution of unique features in scale insects, and provide important genetic information for further research. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Whole Genome Sequencing of the Giant Grouper (Epinephelus lanceolatus) and High-Throughput Screening of Putative Antimicrobial Peptide Genes.

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (gapdh1 and gapdh2) were involved in glycolysis, which had extremely high expression levels in giant grouper muscle. As it has been reported that AMPs inhibit the growth of a broad spectrum of microbes and participate in regulating innate and adaptive immune responses, genome sequencing of this study provides a comprehensive cataloging of putative AMPs of groupers, supporting antimicrobial research and aquaculture therapy. These genomic resources will be beneficial to further molecular breeding of this economically important fish.

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April 21, 2020

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

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 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genes pheV or pheR The length of the target site was 52 or 55?bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Tn6674 Is a Novel Enterococcal optrA-Carrying Multiresistance Transposon of the Tn554 Family.

The novel 12,932-bp nonconjugative multiresistance transposon Tn6674 was identified in the chromosomal DNA of a porcine Enterococcus faecalis strain. Tn6674 belongs to the Tn554 family of transposons. It shares the same arrangement of the transposase genes tnpA, tnpB, and tnpC with Tn554 However, in addition to the Tn554-associated resistance genes spc and erm(A), Tn6674 harbored the resistance genes fexA and optrA Circular forms of Tn6674 were detected and suggest the functional activity of this transposon.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus.

CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair pathways in bacteria, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4-400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems. © FEMS 2019.

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April 21, 2020

Complete Genome Sequence of the Silicimonas algicola Type Strain, a Representative of the Marine Roseobacter Group Isolated from the Cell Surface of the Marine Diatom Thalassiosira delicatula.

Silicimonas algicola strain KC90BT is an alphaproteobacterium of the Roseobacter clade that was isolated from a culture of the marine diatom Thalassiosira delicatula. Here, we report the complete genome sequence of this type strain, which is 4,351,658?bp in size with 4,272 coding sequences and an average G+C content of 65.2%.

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April 21, 2020

Mitochondrial DNA Variants in Patients with Liver Injury Due to Anti-Tuberculosis Drugs.

Hepatotoxicity is the most severe adverse effect of anti-tuberculosis therapy. Isoniazid’s metabolite hydrazine is a mitochondrial complex II inhibitor. We hypothesized that mitochondrial DNA variants are risk factors for drug-induced liver injury (DILI) due to isoniazid, rifampicin or pyrazinamide.We obtained peripheral blood from tuberculosis (TB) patients before anti-TB therapy. A total of 38 patients developed DILI due to anti-TB drugs. We selected 38 patients with TB but without DILI as controls. Next-generation sequencing detected point mutations in the mitochondrial DNA genome. DILI was defined as ALT =5 times the upper limit of normal (ULN), or ALT =3 times the ULN with total bilirubin =2 times the ULN.In 38 patients with DILI, the causative drug was isoniazid in eight, rifampicin in 14 and pyrazinamide in 16. Patients with isoniazid-induced liver injury had more variants in complex I’s NADH subunit 5 and 1 genes, more nonsynonymous mutations in NADH subunit 5, and a higher ratio of nonsynonymous to total substitutions. Patients with rifampicin- or pyrazinamide-induced liver injury had no association with mitochondrial DNA variants.Variants in complex I’s subunit 1 and 5 genes might affect respiratory chain function and predispose isoniazid-induced liver injury when exposed to hydrazine, a metabolite of isoniazid and a complex II inhibitor.

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April 21, 2020

A chromosomal-level genome assembly for the insect vector for Chagas disease, Triatoma rubrofasciata.

Triatoma rubrofasciata is a widespread pathogen vector for Chagas disease, an illness that affects approximately 7 million people worldwide. Despite its importance to human health, its evolutionary origin has not been conclusively determined. A reference genome for T. rubrofasciata is not yet available.We have sequenced the genome of a female individual with T. rubrofasciatausing a single molecular DNA sequencing technology (i.e., PacBio Sequel platform) and have successfully reconstructed a whole-genome (680-Mb) assembly that covers 90% of the nuclear genome (757 Mb). Through Hi-C analysis, we have reconstructed full-length chromosomes of this female individual that has 13 unique chromosomes (2n = 24 = 22 + X1 + X2) with a contig N50 of 2.72 Mb and a scaffold N50 of 50.7 Mb. This genome has achieved a high base-level accuracy of 99.99%. This platinum-grade genome assembly has 12,691 annotated protein-coding genes. More than 95.1% of BUSCO genes were single-copy completed, indicating a high level of completeness of the genome.The platinum-grade genome assembly and its annotation provide valuable information for future in-depth comparative genomics studies, including sexual determination analysis in T. rubrofasciata and the pathogenesis of Chagas disease. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

A chromosome-level draft genome of the grain aphid Sitobion miscanthi.

Sitobion miscanthi is an ideal model for studying host plant specificity, parthenogenesis-based phenotypic plasticity, and interactions between insects and other species of various trophic levels, such as viruses, bacteria, plants, and natural enemies. However, the genome information for this species has not yet to be sequenced and published. Here, we analyzed the entire genome of a parthenogenetic female aphid colony using Pacific Biosciences long-read sequencing and Hi-C data to generate chromosome-length scaffolds and a highly contiguous genome assembly.The final draft genome assembly from 33.88 Gb of raw data was ~397.90 Mb in size, with a 2.05 Mb contig N50. Nine chromosomes were further assembled based on Hi-C data to a 377.19 Mb final size with a 36.26 Mb scaffold N50. The identified repeat sequences accounted for 26.41% of the genome, and 16,006 protein-coding genes were annotated. According to the phylogenetic analysis, S. miscanthi is closely related to Acyrthosiphon pisum, with S. miscanthi diverging from their common ancestor ~25.0-44.9 million years ago.We generated a high-quality draft of the S. miscanthi genome. This genome assembly should help promote research on the lifestyle and feeding specificity of aphids and their interactions with each other and species at other trophic levels. It can serve as a resource for accelerating genome-assisted improvements in insecticide-resistant management and environmentally safe aphid management. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Intercellular Transfer of Chromosomal Antimicrobial Resistance Genes between Acinetobacter baumannii Strains Mediated by Prophages.

The spread of antimicrobial resistance genes (ARGs) among Gram-negative pathogens, including Acinetobacter baumannii, is primarily mediated by transferable plasmids; however, ARGs are frequently integrated into its chromosome. How ARG gets horizontally incorporated into the chromosome of A. baumannii, and whether it functions as a cause for further spread of ARG, remains unknown. Here, we demonstrated intercellular prophage-mediated transfer of chromosomal ARGs without direct cell-cell interaction in A. baumannii We prepared ARG-harboring extracellular DNA (eDNA) components from the culture supernatant of a multidrug-resistant (MDR) A. baumannii NU-60 strain and exposed an antimicrobial-susceptible (AS) A. baumannii ATCC 17978 strain to the eDNA components. The antimicrobial-resistant (AR) A. baumannii ATCC 17978 derivatives appeared to acquire various ARGs, originating from dispersed loci of the MDR A. baumannii chromosome, along with their surrounding regions, by homologous recombination, with the ARGs including armA (aminoglycoside resistance), blaTEM-1 (ß-lactam resistance), tet(B) (tetracycline resistance), and gyrA-81L (nalidixic acid resistance) genes. Notably, the eDNAs conferring antimicrobial resistance were enveloped in specific capsid proteins consisting of phage particles, thereby protecting the eDNAs from detergent and DNase treatments. The phages containing ARGs were likely released into the extracellular space from MDR A. baumannii, thereby transducing ARGs into AS A. baumannii, resulting in the acquisition of AR properties by the recipient. We concluded that the generalized transduction, in which phages were capable of carrying random pieces of A. baumannii genomic DNAs, enabled efficacious intercellular transfer of chromosomal ARGs between A. baumannii strains without direct cell-cell interaction. Copyright © 2019 American Society for Microbiology.

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April 21, 2020

The blaNDM-1-Carrying IncA/C2 Plasmid Underlies Structural Alterations and Cointegrate Formation In Vivo.

In 2012, a carbapenemase-producing Salmonella enterica serovar Corvallis isolate carrying a blaNDM-1 multiresistance IncA/C2 plasmid, apart from IncHI2 and ColE-like plasmids, was detected in a wild bird in Germany. In a recent broiler chicken infection study, we observed transfer of this blaNDM-1-carrying IncA/C2 plasmid to other Enterobacteriaceae Here, we focused on the stability of this plasmid and gained insight into the type and frequency of its structural alterations after an in vivo passage in a broiler chicken infection study.Copyright © 2019 Hadziabdic et al.

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April 21, 2020

Medusavirus, a Novel Large DNA Virus Discovered from Hot Spring Water.

Recent discoveries of new large DNA viruses reveal high diversity in their morphologies, genetic repertoires, and replication strategies. Here, we report the novel features of medusavirus, a large DNA virus newly isolated from hot spring water in Japan. Medusavirus, with a diameter of 260?nm, shows a T=277 icosahedral capsid with unique spherical-headed spikes on its surface. It has a 381-kb genome encoding 461 putative proteins, 86 of which have their closest homologs in Acanthamoeba, whereas 279 (61%) are orphan genes. The virus lacks the genes encoding DNA topoisomerase II and RNA polymerase, showing that DNA replication takes place in the host nucleus, whereas the progeny virions are assembled in the cytoplasm. Furthermore, the medusavirus genome harbored genes for all five types of histones (H1, H2A, H2B, H3, and H4) and one DNA polymerase, which are phylogenetically placed at the root of the eukaryotic clades. In contrast, the host amoeba encoded many medusavirus homologs, including the major capsid protein. These facts strongly suggested that amoebae are indeed the most promising natural hosts of medusavirus, and that lateral gene transfers have taken place repeatedly and bidirectionally between the virus and its host since the early stage of their coevolution. Medusavirus reflects the traces of direct evolutionary interactions between the virus and eukaryotic hosts, which may be caused by sharing the DNA replication compartment and by evolutionarily long lasting virus-host relationships. Based on its unique morphological characteristics and phylogenomic relationships with other known large DNA viruses, we propose that medusavirus represents a new family, MedusaviridaeIMPORTANCE We have isolated a new nucleocytoplasmic large DNA virus (NCLDV) from hot spring water in Japan, named medusavirus. This new NCLDV is phylogenetically placed at the root of the eukaryotic clades based on the phylogenies of several key genes, including that encoding DNA polymerase, and its genome surprisingly encodes the full set of histone homologs. Furthermore, its laboratory host, Acanthamoeba castellanii, encodes many medusavirus homologs in its genome, including the major capsid protein, suggesting that the amoeba is the genuine natural host from ancient times of this newly described virus and that lateral gene transfers have repeatedly occurred between the virus and amoeba. These results suggest that medusavirus is a unique NCLDV preserving ancient footprints of evolutionary interactions with its hosts, thus providing clues to elucidate the evolution of NCLDVs, eukaryotes, and virus-host interaction. Based on the dissimilarities with other known NCLDVs, we propose that medusavirus represents a new viral family, Medusaviridae.Copyright © 2019 Yoshikawa et al.

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April 21, 2020

Combining Viral Genetics and Statistical Modeling to Improve HIV-1 Time-of-infection Estimation towards Enhanced Vaccine Efficacy Assessment.

Knowledge of the time of HIV-1 infection and the multiplicity of viruses that establish HIV-1 infection is crucial for the in-depth analysis of clinical prevention efficacy trial outcomes. Better estimation methods would improve the ability to characterize immunological and genetic sequence correlates of efficacy within preventive efficacy trials of HIV-1 vaccines and monoclonal antibodies. We developed new methods for infection timing and multiplicity estimation using maximum likelihood estimators that shift and scale (calibrate) estimates by fitting true infection times and founder virus multiplicities to a linear regression model with independent variables defined by data on HIV-1 sequences, viral load, diagnostics, and sequence alignment statistics. Using Poisson models of measured mutation counts and phylogenetic trees, we analyzed longitudinal HIV-1 sequence data together with diagnostic and viral load data from the RV217 and CAPRISA 002 acute HIV-1 infection cohort studies. We used leave-one-out cross validation to evaluate the prediction error of these calibrated estimators versus that of existing estimators and found that both infection time and founder multiplicity can be estimated with improved accuracy and precision by calibration. Calibration considerably improved all estimators of time since HIV-1 infection, in terms of reducing bias to near zero and reducing root mean squared error (RMSE) to 5-10 days for sequences collected 1-2 months after infection. The calibration of multiplicity assessments yielded strong improvements with accurate predictions (ROC-AUC above 0.85) in all cases. These results have not yet been validated on external data, and the best-fitting models are likely to be less robust than simpler models to variation in sequencing conditions. For all evaluated models, these results demonstrate the value of calibration for improved estimation of founder multiplicity and of time since HIV-1 infection.

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April 21, 2020

Large Scale Profiling of Protein Isoforms Using Label-Free Quantitative Proteomics Revealed the Regulation of Nonsense-Mediated Decay in Moso Bamboo (Phyllostachys edulis).

Moso bamboo is an important forest species with a variety of ecological, economic, and cultural values. However, the gene annotation information of moso bamboo is only based on the transcriptome sequencing, lacking the evidence of proteome. The lignification and fiber in moso bamboo leads to a difficulty in the extraction of protein using conventional methods, which seriously hinders research on the proteomics of moso bamboo. The purpose of this study is to establish efficient methods for extracting the total proteins from moso bamboo for following mass spectrometry-based quantitative proteome identification. Here, we have successfully established a set of efficient methods for extracting total proteins of moso bamboo followed by mass spectrometry-based label-free quantitative proteome identification, which further improved the protein annotation of moso bamboo genes. In this study, 10,376 predicted coding genes were confirmed by quantitative proteomics, accounting for 35.8% of all annotated protein-coding genes. Proteome analysis also revealed the protein-coding potential of 1015 predicted long noncoding RNA (lncRNA), accounting for 51.03% of annotated lncRNAs. Thus, mass spectrometry-based proteomics provides a reliable method for gene annotation. Especially, quantitative proteomics revealed the translation patterns of proteins in moso bamboo. In addition, the 3284 transcript isoforms from 2663 genes identified by Pacific BioSciences (PacBio) single-molecule real-time long-read isoform sequencing (Iso-Seq) was confirmed on the protein level by mass spectrometry. Furthermore, domain analysis of mass spectrometry-identified proteins encoded in the same genomic locus revealed variations in domain composition pointing towards a functional diversification of protein isoform. Finally, we found that part transcripts targeted by nonsense-mediated mRNA decay (NMD) could also be translated into proteins. In summary, proteomic analysis in this study improves the proteomics-assisted genome annotation of moso bamboo and is valuable to the large-scale research of functional genomics in moso bamboo. In summary, this study provided a theoretical basis and technical support for directional gene function analysis at the proteomics level in moso bamboo.

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April 21, 2020

Nine Novel Phages from a Plateau Lake in Southwest China: Insights into Aeromonas Phage Diversity.

Aeromonas species are common pathogens of fish and some of them can opportunistically cause infectious diseases in humans. The overuse of antibiotics has led to the emergence of bacterial drug-resistance. To date, only 51 complete genome sequences of Aeromonas phages are available in GenBank. Here, we report the isolation of nine Aeromonas phages from a plateau lake in China. The protein cluster, dot plot and ANI analyses were performed on all 60 currently sequenced Aeromonas phage genomes and classified into nine clusters and thirteen singletons. Among the nine isolated phages, the DNA-packaging strategy of cluster 2L372D (including 2L372D, 2L372X, 4L372D, 4L372XY) is unknown, while the other five phages use the headful (P22/Sf6) DNA-packaging strategy. Notably, the isolated phages with larger genomes conservatively encode auxiliary metabolism genes, DNA replication and metabolism genes, while in smaller phage genomes, recombination-related genes were conserved. Finally, we propose a new classification scheme for Aeromonas phages.

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