scholarly journals Using Barcoded Zika Virus to Assess Virus Population Structure in vitro and in Aedes aegypti Mosquitoes

2017 ◽  
Author(s):  
James Weger-Lucarelli ◽  
Selene M. Garcia ◽  
Claudia Rückert ◽  
Alex Byas ◽  
Shelby L. O’Connor ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Next Generation Sequencing ◽  
Aedes Aegypti ◽  
Zika Virus ◽  
Virus Transmission ◽  
Viral Diversity ◽  
Virus Population ◽  
Generation Sequencing

ABSTRACTArboviruses such as Zika virus (ZIKV, Flaviviridae; Flavivirus) replicate in both mammalian and insect hosts where they encounter a variety of distinct host defenses. To overcome these pressures, arboviruses exist as diverse populations of distinct genomes. However, transmission between hosts and replication within hosts can involve genetic bottlenecks, during which population size and viral diversity may be significantly reduced, potentially resulting in large fitness losses. Understanding the points at which bottlenecks exist during arbovirus transmission is critical to identifying targets for preventing transmission. To study these bottleneck effects, we constructed 4 “barcoded” ZIKV clones - 2 with an 8-base-pair degenerate insertion in the 3’ UTR and 2 with 8 or 9 degenerate synonymous changes in the coding sequence, theoretically containing thousands of variants each. We passaged these viruses 3 times each in 2 mammalian and 2 mosquito cell lines and characterized selection of the “barcode” populations using deep sequencing. Additionally, the viruses were used to feed three recently field-caught populations of Aedes aegypti mosquitoes to assess bottlenecks in a natural host. The barcoded viruses replicated well in multiple cell lines in vitro and in vivo in mosquitoes and could be characterized using next-generation sequencing. The stochastic nature of mosquito transmission was clearly shown by tracking individual barcodes in Ae. aegypti mosquitoes. Barcoded viruses provide an efficient method to examine bottlenecks during virus infection.AUTHOR SUMMARYIn general, mosquito-borne viruses like ZIKV must replicate in two very different host environments: an insect and a mammalian host. RNA viruses such as ZIKV must maintain genetic diversity in order to adapt to these changing conditions. During this transmission cycle, several barriers exist which can severely restrict viral genetic diversity, causing bottlenecks in the virus population. It is critical to understand these bottlenecks during virus transmission as this will provide important insights into the selective forces shaping arbovirus evolution within and between hots. Here, we employ a set of barcoded ZIKV constructs containing a degenerate stretch of nucleotides that can be tracked using next-generation sequencing. We found that the insertion site in the genome was an important determinant of the resulting diversity of the genetic barcode. We also found that bottlenecks varied between different mosquito populations and patterns of genetic diversity were distinct among individual mosquitoes within a single population, highlighting the randomness of virus dissemination in mosquitoes. Our study characterizes a new tool for tracking bottlenecks during virus transmission in vivo and highlights the importance of both viral and host factors on the maintenance of viral diversity.

scholarly journals Next-Generation Sequencing in a Direct Model of HIV Infection Reveals Important Parallels to and Differences from In Vivo Reservoir Dynamics

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Marilia Rita Pinzone ◽  
Maria Paola Bertuccio ◽  
D. Jake VanBelzen ◽  
Ryan Zurakowski ◽  
Una O’Doherty
Keyword(s):  
T Cells ◽  
Next Generation Sequencing ◽  
Cd4 T Cells ◽  
Next Generation ◽  
Activated T Cells ◽  
Selection Pressures ◽  
Hiv Dna ◽  
Generation Sequencing

ABSTRACT Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir, but limited data exist on its use in vitro. Moreover, most NGS studies do not separate integrated from unintegrated DNA, even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare the infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions, we obtained HIV sequences 2, 4, and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/ml interleukin-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection, the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo. Massively deleted viral sequences formed more frequently in resting cells, likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures. IMPORTANCE The major implication of our work is that the decay of intact proviruses in vitro is extremely rapid, perhaps as a result of enhanced expression. Gaining a better understanding of why intact proviruses decay faster in vitro might help the field identify strategies to purge the reservoir in vivo. When used wisely, in vitro models are a powerful tool to study the selective pressures shaping the viral landscape. Our finding that massively deleted sequences rarely succeed in integrating has several ramifications. It demonstrates that the total HIV DNA can differ substantially in character from the integrated HIV DNA under certain circumstances. The presence of unintegrated HIV DNA has the potential to obscure selection pressures and confound the interpretation of clinical studies, especially in the case of trials involving treatment interruptions.

Genome study of PDGFRA D842V mutant GIST using next generation sequencing approach.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 10540-10540
Author(s):  
Maria A. Pantaleo ◽  
Annalisa Astolfi ◽  
Milena Urbini ◽  
Valentina Indio ◽  
Margherita Nannini ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Tyrosine Kinases ◽  
Therapeutic Targets ◽  
Mapk Signaling ◽  
Gastric Gist ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Generation Sequencing

10540 Background: Mutations of the receptors KIT and PDGFRA in GIST are the oncogenetic events of disease as well as the targets of molecular therapies. Within the PDGFRA mutations, the D842V mutation in exon 18 confers in vitro and in vivo resistance to imatinib. Next generation sequencing techniques may completely dissect all the possible somatic mutations and genomic rearrangements in order to identify novel therapeutic targets in this patients population. Methods: Five patients with gastric GIST were analyased (3 M, 2 F; mean age 65,5 years, range 51-77). The tumor dimension ranged between 3 and 15 cm, MI < 2 and 8 /50 HPF. No metastases were present in all cases. The KIT and PDGFRA anlysis showed a D842V mutation in exon 18 of PDGFRA in all cases. Whole transcriptome sequencing was performed with Illumina HiScanSQ platform with a paired-end strategy (75x2). After performing quality controls, the short reads were mapped with Tophat-Botie pipeline against the human reference genome (HG19). The variations, such as Single nucleotide variants (SNVs) and InDels, were called by SNVMix2 (a software suited for SNVs discovery in tumor samples) implementing an accurate filtering procedures developed in our laboratory. Two predictors of mutations effect at protein level (SNPs&GO and Provean) were employed in order to prioritize the emerging variation. Results: An average of 206 coding non-synonymous variants were highlighted in the five GIST samples, of which ~ 30% were predicted as deleterious with at least one predictor. In addition to PDGFRA D842V mutation, in all five patients were found mutations on different receptor tyrosine kinases, such as FGFR4 and DDR2. Moreover three out of five patients harboured mutations on members of the MDR/TAP subfamily that are involved in multidrug resistance, in particular on ABCB1, ABCB4 and on ABCB6 genes. Other mutations were found on the hedgehog and MAPK signaling pathway and on SNF/SWI chromatin remodeling complex. Conclusions: New molecular events have been identified in PDGFRA D842V mutant GIST. These data should be validated in larger series and the role of these mutations as therapeutic targets should be further investigated.

scholarly journals Insertion Specificity of the hATx-6 Transposase of Hydra magnipapillata

2021 ◽  
Vol 8 ◽  
Author(s):  
Paul Riggs ◽  
George Blundell-Hunter ◽  
Joanna Hagelberger ◽  
Guoping Ren ◽  
Laurence Ettwiller ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Bioinformatic Analysis ◽  
Next Generation ◽  
Hydra Vulgaris ◽  
Domains Of Life ◽  
Hydra Magnipapillata ◽  
Sequence Bias ◽  
Generation Sequencing

Transposable elements (TE) are mobile genetic elements, present in all domains of life. They commonly encode a single transposase enzyme, that performs the excision and reintegration reactions, and these enzymes have been used in mutagenesis and creation of next-generation sequencing libraries. All transposases have some bias in the DNA sequence they bind to when reintegrating the TE DNA. We sought to identify a transposase that showed minimal sequence bias and could be produced recombinantly, using information from the literature and a novel bioinformatic analysis, resulting in the selection of the hATx-6 transposase from Hydra vulgaris (aka Hydra magnipapillata) for further study. This transposase was tested and shown to be active both in vitro and in vivo, and we were able to demonstrate very low sequence bias in its integration preference. This transposase could be an excellent candidate for use in biotechnology, such as the creation of next-generation sequencing libraries.

scholarly journals Profiling a single-stranded DNA region within an rDNA segment that is a loading site for bacterial condensin

2021 ◽  
Author(s):  
Koichi Yano ◽  
Hideki Noguchi ◽  
Hironori Niki
Keyword(s):  
Next Generation Sequencing ◽  
Conversion Rate ◽  
Bacterial Genome ◽  
Single Stranded Dna ◽  
Deletion Mutations ◽  
Bisulfite Treatment ◽  
Generation Sequencing

Bacterial condensin preferentially loads to single-stranded DNA (ssDNA) in vitro and loads onto rDNA in vivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the topological loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines via uracils (CT-conversion) at locally melted DNA of a bacterial genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the PCR-amplified rDNA segment to obtain the actual CT-conversion rate. In principle, the CT-conversion rate is an accurate guide to detect the formation of the ssDNA segment. We expected that an increment of the CT-conversion rate would reflect a trend toward ssDNA accumulation at a given site within the rDNA. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA that affect the bacterial-condensin loading hindered the ssDNA formation only at the 100-500 bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the bacterial condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

scholarly journals Using Next Generation Sequencing to Study the Genetic Diversity of Candidate Live Attenuated Zika Vaccines

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 161
Author(s):  
Natalie D. Collins ◽  
Chao Shan ◽  
Bruno T.D. Nunes ◽  
Steven G. Widen ◽  
Pei-Yong Shi ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Next Generation Sequencing ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Sequencing Analysis ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Live Attenuated Vaccine ◽  
Generation Sequencing

Zika virus (ZIKV) is a mosquito-transmitted positive-sense RNA virus in the family Flaviviridae. Candidate live-attenuated vaccine (LAV) viruses with engineered deletions in the 3’ untranslated region (UTR) provide immunity and protection in animal models of ZIKV infection, and phenotypic studies show that LAVs retain protective abilities following in vitro passage. The present study investigated the genetic diversity of wild-type (WT) parent ZIKV and its candidate LAVs using next generation sequencing analysis of five sequential in vitro passages. The results show that genomic entropy of WT ZIKV steadily increases during in vitro passage, whereas that of LAVs also increased by passage number five but was variable throughout passaging. Additionally, clusters of single nucleotide variants (SNVs) were found to be present in the pre-membrane/membrane (prM), envelope (E), nonstructural protein NS1 (NS1), and other nonstructural protein genes, depending on the specific deletion, whereas in the parent WT ZIKV, they are more abundant in prM and NS1. Ultimately, both the parental WT and LAV derivatives increase in genetic diversity, with evidence of adaptation following passage.

scholarly journals LncRNA LEF1-AS1 promotes metastasis of prostatic carcinoma via the Wnt/β-catenin pathway

2020 ◽  
Author(s):  
Weiyuan Li ◽  
Ganggang Yang ◽  
Dengke Yang ◽  
Dong Li ◽  
Qian Sun
Keyword(s):  
Next Generation Sequencing ◽  
Rna Binding ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Normal Tissues ◽  
Factor C ◽  
Generation Sequencing

Abstract Background: Long noncoding RNAs (lncRNAs) are important functional regulators of many biological processes of cancers. However, the mechanisms by which lncRNAs modulate androgen-independent prostate cancer (AIPC) development remain largely unknown.Methods: Next-generation sequencing technology and RT-qPCR were used to assess LEF1-AS1 expression level in AIPC tissues and adjacent normal tissues. Functional in vitro experiments, including colony formation, EDU and transwell assays were performed to assess the role of LEF1-AS1 in AIPC. Xenograft assays were conducted to assess the effect of LEF1-AS1 on cell proliferation in vivo. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) assays were performed to elucidate the regulatory network of LEF1-AS1.Results: The next-generation sequencing results showed that LEF1-AS1 is significantly overexpressed in AIPC. Furthermore, our RT-qPCR assay data showed that LEF1-AS1 is overexpressed in AIPC tissues. Functional experiments showed that LEF1-AS1 promotes the proliferation, migration, invasion and angiogenic ability of AIPC cells in vitro and tumour growth in vivo by recruiting the transcription factor C-myb to the promoter of FZD2, inducing its transcription. Furthermore, LEF1-AS1 was shown to function as a competing endogenous RNA (ceRNA) that sponges miR-328 to activate CD44.Conclusion: In summary, the results of our present study revealed that LEF1-AS1 acts as a tumour promoter in the progression of AIPC. Furthermore, the results revealed that LEF1-AS1 functions as a ceRNA and regulates Wnt/β-catenin pathway activity via FZD2 and CD44. Our results provide new insights into the mechanism that links the function of LEF1-AS1 with AIPC and suggests that LEF1-AS1 may serve as a novel potential target for the improvement of AIPC therapy.

scholarly journals LncRNA LEF1-AS1 promotes metastasis of prostatic carcinoma via the Wnt/β-catenin pathway

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Weiyuan Li ◽  
Ganggang Yang ◽  
Dengke Yang ◽  
Dong Li ◽  
Qian Sun
Keyword(s):  
Next Generation Sequencing ◽  
Rna Binding ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Normal Tissues ◽  
Factor C ◽  
Generation Sequencing

Abstract Background Long noncoding RNAs (lncRNAs) are important functional regulators of many biological processes of cancers. However, the mechanisms by which lncRNAs modulate androgen-independent prostate cancer (AIPC) development remain largely unknown. Methods Next-generation sequencing technology and RT-qPCR were used to assess LEF1-AS1 expression level in AIPC tissues and adjacent normal tissues. Functional in vitro experiments, including colony formation, EDU and transwell assays were performed to assess the role of LEF1-AS1 in AIPC. Xenograft assays were conducted to assess the effect of LEF1-AS1 on cell proliferation in vivo. Chromatin immunoprecipitation (ChIP) and RNA binding protein immunoprecipitation (RIP) assays were performed to elucidate the regulatory network of LEF1-AS1. Results The next-generation sequencing results showed that LEF1-AS1 is significantly overexpressed in AIPC. Furthermore, our RT-qPCR assay data showed that LEF1-AS1 is overexpressed in AIPC tissues. Functional experiments showed that LEF1-AS1 promotes the proliferation, migration, invasion and angiogenic ability of AIPC cells in vitro and tumour growth in vivo by recruiting the transcription factor C-myb to the promoter of FZD2, inducing its transcription. Furthermore, LEF1-AS1 was shown to function as a competing endogenous RNA (ceRNA) that sponges miR-328 to activate CD44. Conclusion In summary, the results of our present study revealed that LEF1-AS1 acts as a tumour promoter in the progression of AIPC. Furthermore, the results revealed that LEF1-AS1 functions as a ceRNA and regulates Wnt/β-catenin pathway activity via FZD2 and CD44. Our results provide new insights into the mechanism that links the function of LEF1-AS1 with AIPC and suggests that LEF1-AS1 may serve as a novel potential target for the improvement of AIPC therapy.

scholarly journals Novel CYP2A6 diplotypes identified through next-generation sequencing are associated with in-vitro and in-vivo nicotine metabolism

2018 ◽  
Vol 28 (1) ◽  
pp. 7-16 ◽  
Author(s):  
Julie-Anne Tanner ◽  
Andy Z. Zhu ◽  
Katrina G. Claw ◽  
Bhagwat Prasad ◽  
Viktoriya Korchina ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Nicotine Metabolism ◽  
Generation Sequencing

scholarly journals Using barcoded Zika virus to assess virus population structure in vitro and in Aedes aegypti mosquitoes

Virology ◽  
2018 ◽  
Vol 521 ◽  
pp. 138-148 ◽  
Author(s):  
James Weger-Lucarelli ◽  
Selene M. Garcia ◽  
Claudia Rückert ◽  
Alex Byas ◽  
Shelby L. O’Connor ◽  
...  
Keyword(s):  
Population Structure ◽  
Aedes Aegypti ◽  
Zika Virus ◽  
Virus Population
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