A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae

Mycoviruses infect a large number of diverse fungal species, but considering their prevalence, relatively few high-quality genome sequences have been determined. Many mycoviruses have linear double-stranded RNA genomes, which makes it technically challenging to ascertain their nucleotide sequence using conventional sequencing methods. Different specialist methodologies have been developed for the extraction of double-stranded RNAs from fungi and the subsequent synthesis of cDNAs for cloning and sequencing. However, these methods are often labor-intensive, time-consuming, and can require several days to produce cDNAs from double-stranded RNAs. Here, we describe a comprehensive method for the rapid extraction and sequencing of dsRNAs derived from yeasts, using short-read next generation sequencing. This method optimizes the extraction of high-quality double-stranded RNAs from yeasts and 3′ polyadenylation for the initiation of cDNA synthesis for next-generation sequencing. We have used this method to determine the sequence of two mycoviruses and a double-stranded RNA satellite present within a single strain of the model yeast Saccharomyces cerevisiae. The quality and depth of coverage was sufficient to detect fixed and polymorphic mutations within viral populations extracted from a clonal yeast population. This method was also able to identify two fixed mutations within the alpha-domain of a variant K1 killer toxin encoded on a satellite double-stranded RNA. Relative to the canonical K1 toxin, these newly reported mutations increased the cytotoxicity of the K1 toxin against a specific species of yeast.

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Optimizing preservation protocols to extract high‐quality RNA from different tissues of echinoderms for next‐generation sequencing

Molecular Ecology Resources ◽

10.1111/1755-0998.12122 ◽

2013 ◽

Vol 13 (5) ◽

pp. 884-889 ◽

Cited By ~ 12

Author(s):

Rocío Pérez‐Portela ◽

Ana Riesgo

Keyword(s):

Next Generation Sequencing ◽

Next Generation ◽

High Quality ◽

Generation Sequencing ◽

Different Tissues

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Draft Genome Sequence of Saccharomyces cerevisiae Strain Awamori Number 101, Commonly Used to Make Awamori, a Traditional Spirit, in Okinawa, Japan

Microbiology Resource Announcements ◽

10.1128/mra.01414-20 ◽

2021 ◽

Vol 10 (25) ◽

Author(s):

Masatoshi Tsukahara ◽

Kotaro Ise ◽

Maiko Nezuo ◽

Haruna Azuma ◽

Takeshi Akao ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Next Generation Sequencing ◽

Genome Sequence ◽

Draft Genome ◽

Draft Genome Sequence ◽

Next Generation ◽

Industrial Strain ◽

Content Type ◽

Short Reads ◽

Generation Sequencing

We report here the draft genome sequence for Saccharomyces cerevisiae strain Awamori number 101, an industrial strain used for producing awamori, a distilled alcohol beverage. It was constructed by assembling the short reads obtained by next-generation sequencing. The 315 contigs constitute an 11.5-Mbp genome sequence coding 6,185 predicted proteins.

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A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases

Circulation Research ◽

10.1161/circresaha.115.306723 ◽

2015 ◽

Vol 117 (7) ◽

pp. 603-611 ◽

Cited By ~ 20

Author(s):

Kitchener D. Wilson ◽

Peidong Shen ◽

Eula Fung ◽

Ioannis Karakikes ◽

Angela Zhang ◽

...

Keyword(s):

Next Generation Sequencing ◽

Heart Diseases ◽

Cost Effective ◽

Next Generation ◽

High Quality ◽

Quality Cost ◽

Targeted Next Generation Sequencing ◽

Generation Sequencing

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Use of Next Generation Sequencing to study two cowpox virus outbreaks

PeerJ ◽

10.7717/peerj.6561 ◽

2019 ◽

Vol 7 ◽

pp. e6561 ◽

Cited By ~ 4

Author(s):

Markus H. Antwerpen ◽

Enrico Georgi ◽

Alexandra Nikolic ◽

Gudrun Zoeller ◽

Peter Wohlsein ◽

...

Keyword(s):

Next Generation Sequencing ◽

Group Method ◽

Common Source ◽

Cowpox Virus ◽

Next Generation ◽

Single Strain ◽

Hemagglutinin Gene ◽

Hospital Acquired ◽

Virus Isolates ◽

Generation Sequencing

BackgroundBetween 2008 and 2011 about 40 cases of human cowpox were reported from Germany and France. Infections had been acquired via close contact to infected, young pet rats. An identical and unique sequence of the hemagglutinin gene was found in various cowpox virus (CPXV) isolates pointing to a common source of infection. In a second CPXV outbreak in cats in a small animal clinic in Germany in 2015, four out of five hospitalized cats showed identical hemagglutinin sequences and thus, a hospital-acquired transmission had been assumed. Next-Generation Sequencing was performed in order to re-investigate the outbreaks, as epidemiological data could not confirm all cases.MethodsHomogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of four virus isolates, nine CPXVs from our strain collections and from DNA of three paraffin-embedded lesion materials were determined by Next Generation Sequencing (NGS). For phylogenetic analyses a MAFFT-alignment was generated. A distance matrix based on concatenated SNPs was calculated and plotted as dendrogram using Unweighted Pair Group Method with Arithmetic mean (UPGMA) for visualization.ResultsAligning of about 200.000 nucleotides of 8 virus isolates associated with the pet rat outbreak revealed complete identity of six genomes, the remainder two genomes differed in as little as 3 SNPs. When comparing this dataset with four already published CPXV genomes also associated with the pet rat outbreak, again a maximum difference of 3 SNPs was found. The outbreak which lasted from 2008 till 2011 was indeed caused by a single strain which has maintained an extremely high level of clonality over 4 years. Aligning genomic sequences from four cases of feline cowpox revealed 3 identical sequences and one sequence which differed in 65 nucleotides. Although identical hemagglutinin sequences had been obtained from four hospitalized cats, genomic sequencing proved that a hospital-acquired transmission had occurred in only three cats.ConclusionAnalyzing the rather short sequence of the hemagglutinin gene is not sufficient to conduct molecular trace back analyses. Instead, whole genome sequencing is the method of choice which can even be applied to paraffin-embedded specimens.

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Next-generation sequencing of double stranded RNA is greatly improved by treatment with the inexpensive denaturing reagent DMSO

10.1101/644591 ◽

2019 ◽

Author(s):

Alexander H. Wilcox ◽

Eric Delwart ◽

Samuel L. Díaz Muñoz

Keyword(s):

Next Generation Sequencing ◽

Limit Of Detection ◽

Genetic Material ◽

Dsrna Virus ◽

Next Generation ◽

Short Read ◽

Double Stranded Rna ◽

Ncbi Short Read Archive ◽

Dmso Treatment ◽

Generation Sequencing

AbstractDouble stranded RNA (dsRNA) is the genetic material of important viruses and a key component of RNA interference-based immunity in eukaryotes. Previous studies have noted difficulties in determining the sequence of dsRNA molecules that have affected studies of immune function and estimates of viral diversity in nature. Dimethyl sulfoxide (DMSO) has been used to denature dsRNA prior to the reverse transcription stage to improve RT-PCR and Sanger sequencing. We systematically tested the utility of DMSO to improve sequencing yield of a dsRNA virus (Φ6) in a short-read next generation sequencing platform. DMSO treatment improved sequencing read recovery by over two orders of magnitude, even when RNA and cDNA concentrations were below the limit of detection. We also tested the effects of DMSO on a mock eukaryotic viral community and found that dsRNA virus reads increased with DMSO treatment. Furthermore, we provide evidence that DMSO treatment does not adversely affect recovery of reads from a single-stranded RNA viral genome (Influenza A/California/07/2009). We suggest that up to 50% DMSO treatment be used prior to cDNA synthesis when samples of interest are composed of or may contain dsRNA.Data SummarySequence data was deposited in the NCBI Short Read Archive (accession numbers: PRJNA527100, PRJNA527101, PRJNA527098). Data and code for analysis is available on GitHub (https://github.com/awilcox83/dsRNA-sequencing/, doi:10.5281/zenodo.1453423). Protocol for dsRNA sequencing is posted on protocols.io (doi:10.17504/protocols.io.ugnetve).

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Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics

Cancers ◽

10.3390/cancers11111691 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1691

Author(s):

Muscarella ◽

Fabrizio ◽

De Bonis ◽

Mancini ◽

Balsamo ◽

...

Keyword(s):

Next Generation Sequencing ◽

Scale Up ◽

Cancer Diagnostics ◽

Sequencing Analysis ◽

Library Preparation ◽

Next Generation ◽

High Quality ◽

High Coverage ◽

Dna Recovery ◽

Generation Sequencing

Thanks to personalized medicine trends and collaborations between industry, clinical research groups and regulatory agencies, next generation sequencing (NGS) is turning into a common practice faster than one could have originally expected. When considering clinical applications of NGS in oncology, a rapid workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, as well as producing high quality library preparation, can be real challenges. Here we consider these targets and how applying effective automation technology to NGS workflows may help improve yield, timing and quality-control. We firstly evaluated DNA recovery from archived FFPE blocks from three different manual extraction methods and two automated extraction workstations. The workflow was then implemented to somatic (lung/colon panel) and germline (BRCA1/2) library preparation for NGS analysis exploiting two automated workstations. All commercial kits gave good results in terms of DNA yield and quality. On the other hand, the automated workstation workflow has been proven to be a valid automatic extraction system to obtain high quality DNA suitable for NGS analysis (lung/colon Ampli-seq panel). Moreover, it can be efficiently integrated with an open liquid handling platform to provide high-quality libraries from germline DNA with more reproducibility and high coverage for targeted sequences in less time (BRCA1/2). The introduction of automation in routine workflow leads to an improvement of NGS standardization and increased scale up of sample preparations, reducing labor and timing, with optimization of reagents and management.

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