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NAalytics / Assemblies Of Putative SARS CoV2 Spike Encoding MRNA Sequences For Vaccines BNT 162b2 And MRNA 1273RNA vaccines have become a key tool in moving forward through the challenges raised both in the current pandemic and in numerous other public health and medical challenges. With the rollout of vaccines for COVID-19, these synthetic mRNAs have become broadly distributed RNA species in numerous human populations. Despite their ubiquity, sequences are not always available for such RNAs. Standard methods facilitate such sequencing. In this note, we provide experimental sequence information for the RNA components of the initial Moderna (https://pubmed.ncbi.nlm.nih.gov/32756549/) and Pfizer/BioNTech (https://pubmed.ncbi.nlm.nih.gov/33301246/) COVID-19 vaccines, allowing a working assembly of the former and a confirmation of previously reported sequence information for the latter RNA. Sharing of sequence information for broadly used therapeutics has the benefit of allowing any researchers or clinicians using sequencing approaches to rapidly identify such sequences as therapeutic-derived rather than host or infectious in origin. For this work, RNAs were obtained as discards from the small portions of vaccine doses that remained in vials after immunization; such portions would have been required to be otherwise discarded and were analyzed under FDA authorization for research use. To obtain the small amounts of RNA needed for characterization, vaccine remnants were phenol-chloroform extracted using TRIzol Reagent (Invitrogen), with intactness assessed by Agilent 2100 Bioanalyzer before and after extraction. Although our analysis mainly focused on RNAs obtained as soon as possible following discard, we also analyzed samples which had been refrigerated (~4 ℃) for up to 42 days with and without the addition of EDTA. Interestingly a substantial fraction of the RNA remained intact in these preparations. We note that the formulation of the vaccines includes numerous key chemical components which are quite possibly unstable under these conditions-- so these data certainly do not suggest that the vaccine as a biological agent is stable. But it is of interest that chemical stability of RNA itself is not sufficient to preclude eventual development of vaccines with a much less involved cold-chain storage and transportation. For further analysis, the initial RNAs were fragmented by heating to 94℃, primed with a random hexamer-tailed adaptor, amplified through a template-switch protocol (Takara SMARTerer Stranded RNA-seq kit), and sequenced using a MiSeq instrument (Illumina) with paired end 78-per end sequencing. As a reference material in specific assays, we included RNA of known concentration and sequence (from bacteriophage MS2). From these data, we obtained partial information on strandedness and a set of segments that could be used for assembly. This was particularly useful for the Moderna vaccine, for which the original vaccine RNA sequence was not available at the time our study was carried out. Contigs encoding full-length spikes were assembled from the Moderna and Pfizer datasets. The Pfizer/BioNTech data [Figure 1] verified the reported sequence for that vaccine (https://berthub.eu/articles/posts/reverse-engineering-source-code-of-the-biontech-pfizer-vaccine/), while the Moderna sequence [Figure 2] could not be checked against a published reference. RNA preparations lacking dsRNA are desirable in generating vaccine formulations as these will minimize an otherwise dramatic biological (and nonspecific) response that vertebrates have to double stranded character in RNA (https://www.nature.com/articles/nrd.2017.243). In the sequence data that we analyzed, we found that the vast majority of reads were from the expected sense strand. In addition, the minority of antisense reads appeared different from sense reads in lacking the characteristic extensions expected from the template switching protocol. Examining only the reads with an evident template switch (as an indicator for strand-of-origin), we observed that both vaccines overwhelmingly yielded sense reads (>99.99%). Independent sequencing assays and other experimental measurements are ongoing and will be needed to determine whether this template-switched sense read fraction in the SmarterSeq protocol indeed represents the actual dsRNA content in the original material. This work provides an initial assessment of two RNAs that are now a part of the human ecosystem and that are likely to appear in numerous other high throughput RNA-seq studies in which a fraction of the individuals may have previously been vaccinated. ProtoAcknowledgements: Thanks to our colleagues for help and suggestions (Nimit Jain, Emily Greenwald, Lamia Wahba, William Wang, Amisha Kumar, Sameer Sundrani, David Lipman, Bijoyita Roy). Figure 1: Spike-encoding contig assembled from BioNTech/Pfizer BNT-162b2 vaccine. Although the full coding region is included, the nature of the methodology used for sequencing and assembly is such that the assembled contig could lack some sequence from the ends of the RNA. Within the assembled sequence, this hypothetical sequence shows a perfect match to the corresponding sequence from documents available online derived from manufacturer communications with the World Health Organization [as reported by https://berthub.eu/articles/posts/reverse-engineering-source-code-of-the-biontech-pfizer-vaccine/]. The 5’ end for the assembly matches the start site noted in these documents, while the read-based assembly lacks an interrupted polyA tail (A30(GCATATGACT)A70) that is expected to be present in the mRNA.
UVVM / UVVMUVVM (Universal VHDL Verification Methodology) is a free and Open Source Methodology and Library for very efficient VHDL verification of FPGA and ASIC – resulting also in significant quality improvement. Community forum: https://forum.uvvm.org/ UVVM.org: https://uvvm.org/
OSVVM / OSVVMOSVVM Utility Library: AlertLogPkg, CoveragePkg, RandomPkg, ScoreboardGenericPkg, MemoryPkg, TbUtilPkg, TranscriptPkg, ...
alihussainzada / BugHunterMethodologyA comprehensive bug bounty methodology compiled from extensive research, covering web application reconnaissance, checklists, and methods for identifying various bugs. This guide aims to help bug hunters improve their skills in finding, verifying, and responsibly reporting security vulnerabilities.
Siddhi-95 / AHB To APB Bridge VerificationMaven Silicon project - AHB-to-APB Bridge Verification using UVM Methodology.
AlchemyTweaks / Verified TweaksThe “Verified Tweaks” section includes empirically validated system configurations, derived from controlled benchmarking scenarios and frame-level telemetry analysis. Each modification has been replicated, documented, and verified through captured test sessions,ensuring methodological rigor and reliable performance impact.
dpretet / SvutSVUT is a simple framework to create Verilog/SystemVerilog unit tests. Just focus on your tests!
GianfrancoBazzani / Awesome Advanced Smart Contracts Testing And VerificationA curated collection of tools, frameworks, and methodologies for testing and verifying Solidity smart contracts.
Ghonimo / Pre Silicon AHB To APB VerificationComprehensive verification suite for the AHB2APB Bridge design, featuring SystemVerilog and UVM-based methodologies. 🌉🚀
amamory-verification / Uvm Basicsmy UVM training projects
chiggs / UVMMirror of the Universal Verification Methodology from sourceforge
4get / Uvm Book ExamplesUVM Book Examples - A Practical Guide to Adopting the Universal Verification Methodology (UVM) Second Edition
kowsyap / Physical Design And Verification Of DPRAM Using SV UVM And Semi Custom DesignMemory Level Verification of Dual Port RAM using SystemVerilog and Universal Verification Methodology Environments with assertions,functional coverage and code coverage report
A-T-Kristensen / Simple Alu UvmThis is full tutorial of UVM (Universal Verification Methodology) for a simple ALU unit
universal-verification-methodology / Learn Uvm PyuvmA comprehensive, modular learning path for mastering UVM (Universal Verification Methodology) and pyuvm (Python UVM implementation) with progressive complexity levels. This project provides a complete educational resource with examples, testbenches, and documentation covering all aspects of UVM verification.
tahandy / ToroExactPython implementation of the exact Riemann solver of Toro (1999).
fvmformal / FvmA Formal Verification Methodology to lower the adoption barriers for Formal Verification of ASIC and FPGA designs in the Space sector (this is a mirror of https://gitlab.com/fvmformal/fvm : you can open issues there)
relativitydev / Relativity Test HelpersOpen Source Community: Integration testing is a software testing methodology used to test individual software components or units of code to verify their interaction. These components are tested as a single group or organized in an iterative manner.That said, we have created Relativity Integration Test Helpers to assist you with writing good Integration Tests for your Relativity application. You can use this framework to test event handlers, agents or any workflow that combines agents and Eventhandlers. We will continue adding more helpers but in the mean time you should be able to create workspaces, create dbcontext, proxy and create documents with this framework. This framework is only compatible with Relativity 9.5
shrestha-bishal / BankoneerCompleteBankingSystemCoreBANKONEER (A Co-operative Banking System Software with Online Banking) [JAVA, PHP, HTML & CSS, JS, MS-SQL With ODBC-Connector Driver] BANKONEER, a Co-operative Banking System Software for handling transactions carried in co-operative banks daily in a computerized manner & to nurture the needs of an end banking by providing various ways to perform banking tasks with different departments as CEO, account, loan, e-banking, cashier and online banking. BANKONEER system comes in three platforms as Desktop Workstation software used in co-operative administrative and administration (JAVA platform & framework), Online Banking for members of co-operative banks (PHP, HTML, CSS & JS with ODBC connector driver) & MS-SQL (server). The system is designed with simple GUI for effective user interaction and increased effectiveness for handling the transactions and flow of the financials in a computerized manner with sophisticated algorithms. With Online Banking the members of the co-operative banks can be transparent. Also the features like Transactions, Loans Details (on-going & completed) & the installments, Third-Party Transfer, Wallet Concept etc. have made the software stand-out. The system deals with data entry, validation & confirmation, handling transactions and financials flow, updating etc. We have also developed our own Online Payment Portal ‘B-Payment’ and embedded it with BANKONEER for third-party transfer. Thus BANKONEER designed according to the specifications & requirements saves transaction time, increase efficiency & also makes the bond between co-operative banks and its members stronger. “BANKONEER, Co-operative Banking System Software with Online Banking” project is a model desktop application and internet banking site for complete cooperative banking transactions and to maintain all related issues in very efficient manner in computerized way. This project enables both bank staffs to handle day to day co-operative banking operations and costumers to perform the basic banking transactions through online too. This system provides banking operations handling to banking staff at one end whereas the access to the customer to create an account, deposit/withdraw the cash from account, third-party transfers, wallet and also to view reports of all accounts, transactions and loan details(on-going and completed) at the other end. The customers can access the banks website for viewing their account details and perform the transactions on account as per their requirements. The simple GUI (Graphical User Interface) in desktop application makes it user friendly and efficient to handle all the transactions for admins or staffs. Bank administrative have full control over the system as they can add, remove, manage transactions/financials and manage other utilities. Customers can also make account form their home with required paperwork and verifications from administrative. With this system the brick and mortar structure of traditional co-operative banking gets converted into a click and portal model, thereby giving a concept of virtual banking a real shape in our sole purpose. E-banking/Online Banking facilitates banking transactions by members round the clock globally. The primary aim of this project is to provide an improved design methodology which envisages the future expansions and modification necessary for the core sector like banking. This necessitates the design to be expandable and modifiable and so a modular approach in this bank can become a member of banking system.
SandhyaRani18 / ChatbotChat bots in health care services have the potential to provide patients access to immediate medical information. Health care chat bots could help patients better manage own health related issues. An AI based chat bot can provide an adequate and instant solution for human health related queries, so this proposed methodology provides a reliable solution that resolves users’ health care issues through a user-friendly environment. Chat bots could be useful for preliminary information; however, it’s vital that patients don’t try to use them to replace human doctors. They can answer fundamental questions, but patients should always verify the information with a medical professional before taking the prescribed prescription. Hence, the chat bot reaches each and every individual for not only answering his/her questions but also provides reliable suggestions. In the study AWS has been implemented for a trouble-free output, which provides sequences of services to provide effective and efficient usage of bot. The Simulations have been enhanced in the study and automated the bot to do the simulations by itself.
