Characterization and Comparative Genomics Analysis of two Bacillus megaterium lytic bacteriophages

Next Generation Sequencing (NGS) technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. Several Bacillus bacteriophages have been isolated, but very few Bacillus megaterium bacteriophages have been characterized. In this study, we describe the biological characteristics, whole genome sequences, and their annotations for two new isolates of the B. megaterium bacteriophages (BM5 and BM10), which were isolated from Egyptian soil samples. Growth analyses indicated that the phages BM5 and BM10 have a shorter latent period (25 and 30 minutes respectively) and a smaller burst size (103 and 117 PFU respectively), in comparison to that which is typical for Bacillus phages. The genome sizes of the phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with a modular organization. Bioinformatic analyses of these genomes enabled the assignment of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of the BM5 and BM10 genome structures, in conjunction with other B. megaterium bacteriophages, revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7 respectively), within the L-cluster, and display different lifestyles (lysogenic and lytic respectively). Moreover, sequenced phages encode proteins associated with Bacillus pathogenesis. In addition, BM5 does not contain any tRNA sequences, whereas BM10 genome codes for 17 tRNAs.

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Characterization and comparative genomic analysis of virulent and temperateBacillus megateriumbacteriophages

PeerJ ◽

10.7717/peerj.5687 ◽

2018 ◽

Vol 6 ◽

pp. e5687 ◽

Cited By ~ 2

Author(s):

Abdoallah Sharaf ◽

Miroslav Oborník ◽

Adel Hammad ◽

Sohair El-Afifi ◽

Eman Marei

Keyword(s):

Burst Size ◽

Phylogenetic Analyses ◽

Genomic Analysis ◽

Modular Organization ◽

Comparative Genomic ◽

Environmental Diversity ◽

Egyptian Soil ◽

Next Generation Sequencing Ngs ◽

Growth Analyses ◽

Generation Sequencing

Next-Generation Sequencing (NGS) technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. SeveralBacillusbacteriophages have been isolated, but very fewBacillus megateriumbacteriophages have been characterized. In this study, we describe the biological characteristics, whole genome sequences, and annotations for two new isolates of theB. megateriumbacteriophages (BM5 and BM10), which were isolated from Egyptian soil samples. Growth analyses indicated that the phages BM5 and BM10 have a shorter latent period (25 and 30 min, respectively) and a smaller burst size (103 and 117 PFU, respectively), in comparison to what is typical forBacillusphages. The genome sizes of the phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with modular organization. Bioinformatic analyses of these genomes enabled the assignment of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of the BM5 and BM10 genome structures, in conjunction with otherB. megateriumbacteriophages, revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7, respectively), within the L-cluster, and display different lifestyles (lysogenic and lytic, respectively). Moreover, sequenced phages encode proteins associated withBacilluspathogenesis. In addition, BM5 does not contain any tRNA sequences, whereas BM10 genome codes for 17 tRNAs.

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A68 Characterization and comparative genomic analysis of two Bacillus megaterium lytic bacteriophages

Virus Evolution ◽

10.1093/ve/vez002.067 ◽

2019 ◽

Vol 5 (Supplement_1) ◽

Author(s):

A Sharaf ◽

M Oborník ◽

A Hammad ◽

S El-Afifi ◽

E Marei

Keyword(s):

Bacillus Megaterium ◽

Thermal Inactivation ◽

Burst Size ◽

Phylogenetic Analyses ◽

Genomic Analysis ◽

Modular Organization ◽

Comparative Genomic ◽

Environmental Diversity ◽

Ph Tolerance

Abstract Next-generation sequencing technologies provide unique possibilities for the comprehensive assessment of the environmental diversity of bacteriophages. Many Bacillus bacteriophages have been isolated, but very few Bacillus megaterium bacteriophages have been characterized. Here, we describe biological characteristics and whole-genome sequences and their annotations for two new isolates of the B. megaterium bacteriophages (BM5 and BM10), isolated from Egyptian soil samples and representing two different groups according to their host range and amplified fragment length polymorphism profiles. Both phages have been displaying different thermal inactivation points (82 and 78 °C) and pH tolerance range (5–9.2 and 5–8.4 pH) while having the same longevity in vitro (192 h). Electron microscopy observation has proved that both phages belonged to the Myoviridiae family. Furthermore, growth analyses indicated that phages BM5 and BM10 have a shorter latent period (20 and 25 min) and smaller burst size (103 and 117 PFU) than is typical for Bacillus phages. The genome sizes of phages BM5 and BM10 were 165,031 bp and 165,213 bp, respectively, with a modular organization. Bioinformatic analyses of BM5 and BM10 genomes enabled assignments of putative functions to 97 and 65 putative ORFs, respectively. Comparative analysis of BM5 and BM10 genome structures with other B. megaterium bacteriophages revealed relatively high levels of sequence and organizational identity. Both genomic comparisons and phylogenetic analyses support the conclusion that the sequenced phages (BM5 and BM10) belong to different sub-clusters (L5 and L7) within L cluster and display different lifestyles (lysogenic and lytic). Sequenced phages encode proteins associated with Bacillus pathogenesis. BM5 does not contain any tRNA sequences, while BM10 genome codes for 17 tRNAs.

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Molecular epidemiology and clinical characteristics of hepatitis delta virus (HDV) infected patients with elevated transaminases in Shanghai, China

10.21203/rs.2.22573/v1 ◽

2020 ◽

Author(s):

Shanshan Wu ◽

Yi Zhang ◽

Yuyan Tang ◽

Ting Yao ◽

Mengjiao Lv ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hepatitis Delta Virus ◽

Clinical Characteristics ◽

Phylogenetic Analyses ◽

Serum Samples ◽

Next Generation Sequencing Ngs ◽

Elevated Transaminases ◽

Generation Sequencing ◽

Hdv Genotype ◽

Delta Virus

Abstract Background: Patients coinfected with HBV and hepatitis D virus (HDV) have a greater risk of HCC and cirrhosis. The current study was undertaken to assess HDV genotype distribution and determine clinical characteristics of hepatitis delta virus (HDV) among HBsAg positive individuals in Shanghai.Method: This retrospective study involved 225 serum samples from HBsAg positive hospitalized patients from October 2010 to April 2013. HDV-specific RT-nested PCR was used to amplify HDV RNA. HDV genotypes were characterized by Next-generation sequencing (NGS), followed by phylogenetic analyses. HDV/HBV co-infected patients and HBV mono-infected patients were compared clinically and virologically.Results: Out of the 225 HBsAg-positive serum samples with elevated transaminases, HDV-RNA was identified in 11 (4.9%) HBsAg positive patients. The HBV loads in the HDV positive group were significantly lower than the HDV negative HBV-infected patients. The aminotransferase enzymes were significantly higher in HDV/HBV co-infected compared to HDV negative patients (P<0.05). Phylogenetic analyses indicated that HDV-2 genotype being the predominant genotype, other HDV genotypes were not observed. HDV/HBV patients were significantly associated with a rather unfavourable clinical outcomeConclusion： In summary, our study showed that the prevalence of HDV infection in patients with elevated transaminases is not low and the predominance of HDV genotype 2 infection in Shanghai. This finding helps us to better understand the correlation of HDV/HBV co-infection. Moreover, Next-generation sequencing (NGS) technologies provide a rapid, precise method for generating HDV genomes to define infecting genotypes.

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Molecular epidemiology and clinical characteristics of hepatitis delta virus (HDV) infected patients with elevated transaminases in Shanghai, China

10.21203/rs.2.22573/v2 ◽

2020 ◽

Author(s):

Shanshan Wu ◽

Yi Zhang ◽

Yuyan Tang ◽

Ting Yao ◽

Mengjiao Lv ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hepatitis Delta Virus ◽

Clinical Characteristics ◽

Phylogenetic Analyses ◽

Serum Samples ◽

Next Generation Sequencing Ngs ◽

Elevated Transaminases ◽

Generation Sequencing ◽

Hdv Genotype ◽

Delta Virus

Abstract Background: Patients coinfected with HBV and hepatitis D virus (HDV) have a greater risk of HCC and cirrhosis. The current study was undertaken to assess HDV genotype distribution and determine clinical characteristics of hepatitis delta virus (HDV) among HBsAg positive individuals in Shanghai.Method: This retrospective study involved 225 serum samples from HBsAg positive hospitalized patients from October 2010 to April 2013. HDV-specific RT-nested PCR was used to amplify HDV RNA. HDV genotypes were characterized by Next-generation sequencing (NGS), followed by phylogenetic analyses. HDV/HBV co-infected patients and HBV mono-infected patients were compared clinically and virologically.Results: Out of the 225 HBsAg-positive serum samples with elevated transaminases, HDV-RNA was identified in 11 (4.9%) HBsAg positive patients. The HBV loads in the HDV positive group were significantly lower than the HDV negative HBV-infected patients. The aminotransferase enzymes were significantly higher in HDV/HBV co-infected compared to HDV negative patients (P<0.05). Phylogenetic analyses indicated that HDV-2 genotype being the predominant genotype, other HDV genotypes were not observed. HDV/HBV patients were significantly associated with a rather unfavourable clinical outcomeConclusion： In summary, our study showed that the prevalence of HDV infection in patients with elevated transaminases is not low and the predominance of HDV genotype 2 infection in Shanghai. This finding helps us to better understand the correlation of HDV/HBV co-infection. Moreover, Next-generation sequencing (NGS) technologies provide a rapid, precise method for generating HDV genomes to define infecting genotypes.

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A20 Sample preparation for whole-genome next-generation sequencing (NGS) of hepatitis C virus (HCV) routine RNA samples

Virus Evolution ◽

10.1093/ve/vez002.019 ◽

2019 ◽

Vol 5 (Supplement_1) ◽

Author(s):

Julia Hillung ◽

María Alma Bracho ◽

Javier Pons Tamarit ◽

Fernando González-Candelas

Keyword(s):

Sample Preparation ◽

Nucleic Acids ◽

Blood Plasma ◽

Reference Genome ◽

Phylogenetic Analyses ◽

Whole Genome ◽

Library Preparation ◽

Genome Sequences ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Next-generation sequencing (NGS) is a technique that can capture the variability of viral populations in transmission studies. The conventional sample preparation for NGS, based on amplicons, is a potential source of errors, derived from the variable affinity of specific primers for different viral variants and from irregular DNA polymerase efficiency. In this context, we propose a more reliable method for viral whole genome sample preparation, starting from nucleic acids obtained and stored with conventional procedures. Our goal was to obtain complete hepatitis C virus (HCV) genome sequences to subsequently perform extensive phylogenetic analyses. Additionally, we aimed to test the effectiveness of nuclease treatment used to remove contaminating host DNA. Nucleic acids were obtained from almost cell-free blood plasma of HCV-infected patients. As a source for Illumina library preparation, double-stranded cDNA was generated using random primers. The HCV genome was not amplified before library preparation, avoiding possible biases derived from unequal copying. To get rid of possible host contaminants in the samples, a DNase treatment step was added. Libraries were paired-end sequenced on the Illumina platform using MiSeq reagent kit v3. After conservative filtering of contaminant human reads by alignment with the human reference genome using Burrows-Wheeler Aligner (BWA), the remaining reads were mapped to the HCV reference genome using BWA. Primary maximum likelihood phylogenetic analyses were performed using ClustalW and IQTREE to infer the phylogenetic relationships of the sequenced samples in the context of complete genome sequences of the same genotype. NGS sample preparation method of HCV from blood plasma was established. Complete genome sequences of HCV could be obtained with variable coverage depending on the viral load of plasma samples. No significant reduction of host DNA proportion in DNase treated samples in comparison to the controls was observed. The new sequences clustered within the Los Alamos National Laboratory database-deposited HCV subtype 4d samples. The method can be used to obtain full-length sequences of HCV from nucleic acid samples not previously planned for NGS. No improvement was observed when DNase pre-treatment of nucleic acids extracted from blood plasma was performed.

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Molecular epidemiology and clinical characteristics of hepatitis delta virus (HDV) infected patients with elevated transaminases in Shanghai, China

10.21203/rs.2.22573/v3 ◽

2020 ◽

Author(s):

Shanshan Wu ◽

Yi Zhang ◽

Yuyan Tang ◽

Ting Yao ◽

Mengjiao Lv ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hepatitis Delta Virus ◽

Clinical Characteristics ◽

Phylogenetic Analyses ◽

Serum Samples ◽

Next Generation Sequencing Ngs ◽

Elevated Transaminases ◽

Generation Sequencing ◽

Hdv Genotype ◽

Delta Virus

Abstract Background: Patients coinfected with HBV and hepatitis D virus (HDV) have a greater risk of HCC and cirrhosis. The current study was undertaken to assess HDV genotype distribution and determine clinical characteristics of hepatitis delta virus (HDV) among HBsAg positive individuals in Shanghai.Method: This retrospective study involved 225 serum samples from HBsAg positive hospitalized patients from October 2010 to April 2013. HDV-specific RT-nested PCR was used to amplify HDV RNA. HDV genotypes were characterized by Next-generation sequencing (NGS), followed by phylogenetic analyses. HDV/HBV co-infected patients and HBV mono-infected patients were compared clinically and virologically.Results: Out of the 225 HBsAg-positive serum samples with elevated transaminases, HDV-RNA was identified in 11 (4.9%) patients. The HBV loads in the HDV positive group were significantly lower than the HDV negative HBV-infected patients. The aminotransferase enzymes were significantly higher in HDV/HBV co-infected compared to HDV negative patients (P<0.05). Phylogenetic analyses indicated that HDV-2 genotype being the predominant genotype, other HDV genotypes were not observed. HDV/HBV patients were significantly associated with a rather unfavourable clinical outcome.Conclusion： In summary, our study showed that the prevalence of HDV infection in patients with elevated transaminases is not low and the predominance of HDV genotype 2 infection in Shanghai. This finding helps us to better understand the correlation of HDV/HBV co-infection. Moreover, Next-generation sequencing (NGS) technologies provide a rapid, precise method for generating HDV genomes to define infecting genotypes.

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Identification and phylogenetic analysis of voltage-gated sodium channel haplotypes in the malaria vector Anopheles sinensis using a high-throughput amplicon sequencing approach

Parasites & Vectors ◽

10.1186/s13071-021-05009-5 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Ruoyao Ni ◽

Nian Liu ◽

Mei Li ◽

Weiping Qian ◽

Xinghui Qiu

Keyword(s):

Next Generation Sequencing ◽

Sodium Channel ◽

Phylogenetic Analyses ◽

Cost Effective ◽

Amplicon Sequencing ◽

Anopheles Sinensis ◽

Next Generation ◽

Voltage Gated ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Background Anopheles sinensis is a dominant vector for malaria transmission in Asian countries. Voltage-gated sodium channel (VGSC) mutation-mediated knock-down resistance (kdr) has developed in many A. sinensis populations because of intensive and long-term use of pyrethroids. Our previous study showed that multiple mutations at position 1014 of the VGSC were heterogeneously distributed in A. sinensis populations across Sichuan, China. Methods To understand resistance genotypes at the haplotype level and reconstruct the phylogenetic relationship of VGSC haplotypes, a cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach was established to clarify haplotypes containing codon 1014 of the VGSC gene from a total of 446 adults collected in 12 locations of Sichuan, China. Results Nineteen (19) haplotypes were identified, including 11 wild 1014L, 6 resistance 1014F, and 2 resistance 1014C haplotypes. We found that resistance haplotypes of A. sinensis VGSC were widely distributed at frequencies ranging from 3.67 to 92.61%. The frequencies of the 1014C haplotype in the southeast of Sichuan (Luzhou, Guangan, and Suining) were relatively higher than those in other sampling locations. Phylogenetic analyses support that kdr-type mutation at position 1014 is not singly originated and resistance 1014C haplotypes evolve from TTT-encoding 1014F. Conclusions A cost-effective next-generation sequencing (NGS)-based amplicon sequencing approach has been established in this study. The data revealed the patchy distribution of VGSC resistance haplotypes with overall high frequencies in Sichuan, China. Phylogenetic analyses support multiple origins and sequential evolution (1014L → 1014F → 1014C) for kdr-type mutations in A. sinensis. Graphical abstract

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Sarcopeltis gen. nov. (Gigartinaceae, Rhodophyta), with S. skottsbergii comb. nov. from southern South America and S. antarctica sp. nov. from the Antarctic Peninsula

Phytotaxa ◽

10.11646/phytotaxa.468.1.4 ◽

2020 ◽

Vol 468 (1) ◽

pp. 75-88

Author(s):

JEFFERY R. HUGHEY ◽

GEOFFREY L. LEISTER ◽

PAUL W. GABRIELSON ◽

MAX H. HOMMERSAND

Keyword(s):

South America ◽

Antarctic Peninsula ◽

Dna Sequences ◽

Phylogenetic Analyses ◽

New Genus And Species ◽

Southern South America ◽

Complete Mitogenome ◽

Next Generation Sequencing Ngs ◽

The Antarctic ◽

Generation Sequencing

Gigartina skottsbergii is the currently accepted name for the large, peltate species of Gigartinaceae distributed in Argentina, Chile, and along the Antarctic Peninsula. However, phylogenetic analyses of DNA sequences for more than twenty years indicate that two species are passing under the name G. skottsbergii, and both DNA sequences and morpho-anatomy indicate that the species are assignable to an undescribed genus. To formally validate the new genus and species, we performed next generation sequencing (NGS) on the lectotype material of G. skottsbergii to document its genetic identity and obtained its complete mitogenome and partial plastid genome. Phylogenetic analysis of rbcL and cox1 sequences of the lectotype along with recent collections of G. skottsbergii from southern South America and Antarctica, confirmed the generic distinction and the presence of two morphologically similar taxa. We propose Sarcopeltis gen. nov., containing the generitype S. skottsbergii comb. nov. from southern South America, and S. antarctica sp. nov. from the Antarctic Peninsula. Sarcopeltis is characterized by the following suite of features: 1) peg-like secondary haptera that adhere to the substratum, 2) absence of an envelope surrounding each cystocarp, 3) presence of extensive, terminal, tubular gonimoblast filaments that fuse with surrounding vegetative cells, and 4) tetrasporangia formed from secondary filaments entirely in the medulla. This study demonstrates that genetic analysis of type material, together with recently obtained DNA sequences from field collected specimens, can provide clear and objective taxonomic conclusions.

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Saliva is more sensitive than nasopharyngeal or nasal swabs for diagnosis of asymptomatic and mild COVID-19 infection

Scientific Reports ◽

10.1038/s41598-021-82787-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alvin Kuo Jing Teo ◽

Yukti Choudhury ◽

Iain Beehuat Tan ◽

Chae Yin Cher ◽

Shi Hao Chew ◽

...

Keyword(s):

Migrant Workers ◽

Acute Respiratory Infection ◽

Community Care ◽

Phylogenetic Analyses ◽

Care Facility ◽

Rt Pcr ◽

Nasal Swabs ◽

Next Generation Sequencing Ngs ◽

Positive Cycle ◽

Generation Sequencing

AbstractWe aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples. We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.

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