This work was supported by Wellcome [207569/Z/17/Z, 224670/Z/21/Z], Newton funding from the Medical Research Council [MR/R025649/1] and the Philippines Department of Science and Technology (DOST), the UK Research and Innovation Global effort on COVID-19 [MR/V035444/1], the University of Glasgow Institutional Strategic Support Fund [204820], Medical Research Council New Investigator Award (KB) [MR/X002047/1], and International Partnership Development Fund, a DOST British Council-Philippines studentship (CB), a National Institute for Health Research [17/63/82] GemVi scholarship (GJ), and University of Glasgow studentships from the MVLS DTP (KC) [125638-06], the EPSRC DTP (RD) [EP/T517896/1], and the Wellcome IIB DTP (HF) [218518/Z/19/Z]. We are grateful to colleagues and collaborators who have supported this work: Daniel Streicker, Alice Broos, Elizabeth Miranda, DVM, Daria Manalo, DVM, Thumbi Mwangi, Kennedy Lushasi, Charles Kayuki, Jude Karlo Bolivar, Jeromir Bondoc, Esteven&#160;Balbin, Ronnel Tongohan, Agatha Ukande, Davis Kuchaka, Mumbua Mutunga, Lwitiko Sikana, and Anna Czupryna.

A Nanopore-Based Genomic Approach to Control Rabies in Low-Income Countries

The authors have nothing to disclose.

An accessible RABV, nanopore-based, whole genome sequencing workflow was developed by Brunker et al.61, using resources from the ARTIC network46. Here, we present an updated workflow, with complete sample-to-sequence-to-interpretation steps. The workflow details the preparation of brain tissue samples for whole genome sequencing, presents a bioinformatics pipeline to process reads and generate consensus sequences, and highlights two rabies-specific tools to automate lineage assignment and determine phylogenetic context. The updated workflow also provides comprehensive instructions for the setup of appropriate computational and laboratory workspaces, with considerations for implementation in different contexts (including low-resource settings). We have demonstrated the successful implementation of the workflow in both academic and research institute settings in four RABV endemic LMICs with no or limited genomic surveillance capacity. The workflow has proven resilient to application across diverse settings, and comprehensible by users with varying expertise.
This workflow for RABV sequencing is the most comprehensive publicly available protocol (covering sample-to-sequence-to-interpretation steps) and specifically adapted to reduce both startup and running costs. The time and cost required for library preparation and sequencing with nanopore technology is greatly reduced relative to other platforms, such as Illumina61, and continual technology developments are improving sequence quality and accuracy to be comparable with Illumina62.
This protocol is designed to be resilient in diverse low-resource contexts. By referring to the troubleshooting and modifications guidance provided alongside the core protocol, users are supported to adapt the workflow to their needs. The addition of user-friendly bioinformatic tools to the workflow constitutes a major development to the original protocol, providing rapid and standardized methods that can be applied by users with minimal prior bioinformatics experience to interpret sequence data in local contexts. The capacity to do this in situ is often limited by the need to have specific programming and phylogenetic skills, which require an intensive and long-term skills training investment. While this skillset is important to thoroughly interpret sequence data, basic and accessible interpretation tools are equally desirable in order to capacitate local &#34;sequencing champions&#34;, whose core expertise may be wet lab based, enabling them to interpret and take ownership over their data.
As the protocol has been undertaken for a number of years in several countries, we now can provide guidance on how to optimize multiplex primer schemes to improve coverage and deal with accumulated diversity. Efforts have also been made to help users improve the cost-effectiveness or to allow for ease of procurement in a given region, which is typically a challenge for the sustainability of molecular approaches63. For example, in Africa (Tanzania, Kenya, and Nigeria), we opted for blunt/TA ligase master mix at the adapter ligation step, which was more readily available from local suppliers and a cheaper alternative to other ligation reagents.
From experience, there are several ways of reducing the cost per sample and per run. Reducing the number of samples per run (e.g., from 24 down to 12 samples) can extend the life of flow cells over multiple runs, whereas increasing the number of samples per run maximizes the time and reagents. In our hands, we were able to wash and reuse flow cells for one in every three sequencing runs, enabling an additional 55 samples to be sequenced. Washing the flow cell immediately after use, or if not possible, removing the waste fluid from the waste channel after every run, seemed to preserve the number of pores available for a second run. Taking into consideration the initial number of pores available in a flow cell, one run can also be optimized to plan how many samples to run in a particular flow cell.
Though the workflow aims to be as comprehensive as possible, with the addition of detailed guidance and signposted resources, the procedure is still complex and can be daunting for a new user. The user is encouraged to seek in-person training and support, ideally locally, or alternatively through external collaborators. In the Philippines for example, a project on capacity building within regional laboratories for SARS-CoV-2 genomic surveillance using ONT has developed core competencies among health care diagnosticians that are readily transferrable to RABV sequencing. Important steps, such as SPRI bead clean up, can be difficult to master without hands-on training, and ineffective clean up can damage the flow cell and compromise the run. Sample contamination is always a major concern when amplicons are being processed in the lab and can be difficult to eliminate. In particular, cross-contamination between samples is extremely difficult to detect during post-run bioinformatics. Good laboratory technique and practices, such as maintaining clean work surfaces, separating pre- and post-PCR areas, and incorporating negative controls, are imperative to ensure quality control. The fast pace of nanopore sequencing developments is both an advantage and disadvantage for routine RABV genomic surveillance. Continuing improvements to nanopore&#39;s accuracy, accessibility, and protocol repertoire widen and improve the scope for its application. However, the same developments make it challenging to maintain standard operating procedures and bioinformatic pipelines. In this protocol, we provide a document assisting the transition from older to current nanopore library preparation kits (Table of Materials).
A common roadblock to sequencing in LMICs is accessibility, including not only the cost but also the ability to procure consumables in a timely manner (in particular sequencing reagents, which are relatively new to procurement teams and suppliers) and computational resources, as well as simply having access to stable power and the internet. Using portable nanopore sequencing technology as the foundation of this workflow helps with many of these accessibility issues, and we have demonstrated the use of our protocol across a range of settings, conducting the full protocol and analysis in-country. Admittedly, procuring equipment and sequencing consumables in a timely manner remains a challenge and, in many instances, we were forced to carry or ship reagents from the UK. However, in some areas, we were able to rely entirely on local supply routes for reagents, benefiting from investment in SARS-CoV-2 sequencing (e.g., the Philippines) that has streamlined procurement processes and begun to normalize the application of pathogen genomics.
The need for a stable internet connection is minimized by one-time-only installs; for instance, GitHub repositories, software download, and nanopore sequencing itself only require internet access to start the run (not throughout) or can be performed completely offline with agreement from the company. If mobile data is available, a phone can be used as a hotspot to the laptop to begin the sequencing run, before disconnecting for the run duration. When routinely processing samples, data storage requirements can grow rapidly, and ideally data would be stored on a server. Otherwise, solid state drive (SSD) hard drives are relatively cheap to source.
While we recognize that there are still barriers to genomic surveillance in LMICs, increasing investment in building genomics accessibility and expertise (e.g., Africa Pathogen Genomics Initiative [Africa PGI])64 suggests that this situation will improve. Genomic surveillance is critical for pandemic preparedness6, and capacity can be established through routinizing the genomic surveillance of endemic pathogens such as RABV. Global disparities in sequencing capacities highlighted during the SARS-CoV-2 pandemic should be a driver of catalytic change to address these structural inequities.
This sample-to-sequence-to-interpretation workflow for RABV, including accessible bioinformatics tools, has the potential to be used to guide control measures targeting the goal of zero human deaths from dog-mediated rabies by 2030, and ultimately for the elimination of RABV variants. Combined with relevant metadata, genomic data generated from this protocol facilitates rapid RABV characterization during outbreak investigations and in the identification of circulating lineages in a country or region60,61,65. We illustrate our pipeline mostly using examples from dog-mediated rabies; however, the workflow is directly applicable to wildlife rabies. This transferability and low cost minimize the challenges in making routine sequencing easily available, not only for rabies but also for other pathogens46,66,67, to improve disease management and control.

The rabies virus (RABV) is a lyssavirus in the Rhabdoviridae family that causes a fatal neurological disease in mammals1. Although rabies is 100% preventable by vaccination, it remains a major public health and economic concern in endemic countries. Of the 60,000 human rabies deaths estimated to occur each year, over 95% are in Africa and Asia where dogs are the primary reservoir2. In contrast, dog vaccination has led to the elimination of dog-mediated rabies across Western Europe, North America, and much of Latin America. In these regions, reservoirs of rabies are now restricted to wildlife, such as bats, raccoons, skunks, and wild canids3. Across Latin America, the common vampire bat is a problematic source of rabies due to regular spillover transmission from bats to both humans and livestock during nightly blood feeding4. The annual global economic impact of rabies is estimated to be $8.6 billion, with livestock losses accounting for 6%5.
Sequence data from viral pathogens combined with metadata on the timing and source of infections can provide robust epidemiological insights6. For RABV, sequencing has been used to investigate the origin of outbreaks7,8, identify host associations with wildlife or domestic dogs8,9,10,11,12, and trace sources of human cases13,14. Outbreak investigations using phylogenetic analysis have indicated that rabies emerged in the formerly rabies-free province of Bali, Indonesia, through a single introduction from the nearby endemic areas of Kalimantan or Sulawesi15. Meanwhile, in the Philippines, an outbreak on Tablas Island, Romblon Province was proven to be introduced from the main island of Luzon16. Viral genomic data have also been used to better understand pathogen transmission dynamics required for targeting control measures geographically. For example, genomic characterization of RABV illustrates the geographic clustering of clades17,18,19, co-circulation of lineages20,21,22, human-mediated viral movement17,23,24, and metapopulation dynamics25,26.
Disease monitoring is one important function of genomic surveillance that has been enhanced with the global increase in sequencing capacity in response to the SARS-CoV-2 pandemic. Genomic surveillance has supported the real-time tracking of SARS-COV-2 variants of concern27,28 and associated countermeasures6. Advances in accessible sequencing technology, such as nanopore technology, have led to improved and more affordable protocols for the rapid sequencing of both human29,30,31,32 and animal33,34,35 pathogens. However, in many rabies endemic countries, there are still barriers to operationalizing pathogen genomic surveillance, as shown by global disparities in SARS-CoV-2 sequencing capacity36. Limitations in laboratory infrastructure, supply chains, and technical knowledge make the establishment and routinization of genomic surveillance challenging. In this paper, we demonstrate how an optimized, rapid, and affordable whole genome sequencing workflow can be deployed for RABV surveillance in resource-limited settings.

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Brand name&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sequencing software&lt;br/&gt; (&lt;em&gt;MinKnow&lt;/em&gt;)</td><td>Oxford Nanopore Technologies</td><td>https://community.nanoporetech.com/downloads</td><td></td></tr><tr><td>Bioinformatics tool kit&lt;br/&gt; (&lt;em&gt;Guppy&lt;/em&gt;)</td><td>Oxford Nanopore Technologies</td><td>https://community.nanoporetech.com/docs/prepare/library_prep_protocols/Guppy-protocol/v/gpb_2003_v1_revao_14dec2018</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thermal cycler&lt;br/&gt; &lt;em&gt;(miniPCR&amp;trade; mini16 thermal cycler)&lt;/em&gt;</td><td>Cambio</td><td>MP-QP-1016-01</td><td></td></tr><tr><td>Homogenizer&lt;br/&gt; (&lt;em&gt;Precellys Evolution Touch Homogenizer&lt;/em&gt;)</td><td>Bertin Instruments</td><td>EQ02520-300</td><td></td></tr><tr><td>Cold Racks (0.2-0.5mL)&lt;br/&gt; (&lt;em&gt;PCR Mini-cooler with transparent lid&lt;/em&gt;)</td><td>BRAND&amp;nbsp;</td><td>781260</td><td></td></tr><tr><td>Pipettor</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;(Pipetman L Fixed F1000L, 1000 uL)&lt;/em&gt;</td><td>Gilson&amp;nbsp;</td><td>SKU: FA10030</td><td></td></tr><tr><td>&lt;em&gt;(Pipetman L Fixed F100L, 100 uL)&lt;/em&gt;</td><td>Gilson&amp;nbsp;</td><td>SKU: FA10024</td><td></td></tr><tr><td>&lt;em&gt;(Pipetman L Fixed F10L, 10 uL)&lt;/em&gt;</td><td>Gilson&amp;nbsp;</td><td>SKU: FA10020</td><td></td></tr><tr><td>&lt;em&gt;(Pipetman L Fixed F1L, 1 uL)&lt;/em&gt;</td><td>Gilson&amp;nbsp;</td><td>SKU: FA10025</td><td></td></tr><tr><td>&lt;em&gt;(Pipetman L Fixed F20L, 20 uL)&lt;/em&gt;</td><td>Gilson&amp;nbsp;</td><td>SKU: FA10021</td><td></td></tr><tr><td>&lt;em&gt;(Pipetman L Fixed F250L, 250 uL)&lt;/em&gt;</td><td>Gilson&amp;nbsp;</td><td>SKU: FA10026</td><td></td></tr><tr><td>Fluorometer&lt;br/&gt; (&lt;em&gt;Qubit&amp;nbsp; 4 Fluorometer&lt;/em&gt;)&amp;nbsp;</td><td>Thermofisher scientific/Fisher scientific</td><td>Q33238</td><td></td></tr><tr><td>Laptop&amp;nbsp;&lt;br/&gt; (&lt;em&gt;Any brand with ~2 GB of drive space, minimum of 512 GB storage space, msi installer [GPU]&lt;/em&gt;)</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge&lt;br/&gt; (&lt;em&gt;Refrigerated centrifuge&lt;/em&gt;)</td><td>Thermofisher scientific/Fisher scientific</td><td>75004081</td><td></td></tr><tr><td>Vortex mixer&lt;br/&gt; (&lt;em&gt;Basic vortex mixer&lt;/em&gt;)&amp;nbsp;</td><td>Thermofisher scientific/Fisher scientific</td><td>88882011</td><td></td></tr><tr><td>Magnetic rack&lt;br/&gt; (&lt;em&gt;DynaMag -2 Magnet&lt;/em&gt;)</td><td>Thermofisher scientific/Fisher scientific</td><td>12321D</td><td></td></tr><tr><td>Sequencing device&lt;br/&gt; (&lt;em&gt;MinION&lt;/em&gt;)</td><td>Oxford Nanopore Technologies</td><td>MinION Mk1B</td><td></td></tr><tr><td>&lt;strong&gt;RNA Extraction&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RNA extraction kit&lt;br/&gt; (&lt;em&gt;Qiagen RNEasy Mini Kit 250&lt;/em&gt;)</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>RNA stabilizing reagents&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;(RNA later)&amp;nbsp;&lt;/em&gt;</td><td>Invitrogen</td><td>AM7020</td><td></td></tr><tr><td>&lt;em&gt;(DNA/RNA Shield)&lt;/em&gt;</td><td>Zymo Research</td><td>R1100-50</td><td></td></tr><tr><td>&lt;strong&gt;PCR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nuclease-free Water&lt;br/&gt; (&lt;em&gt;Nuclease-free Water [not&amp;nbsp; DEPC-treated]&lt;/em&gt;)</td><td>Thermofisher scientific/Fisher scientific</td><td>AM9937</td><td></td></tr><tr><td>Master mix for first strand cDNA synthesis&lt;br/&gt; (&lt;em&gt;LunaScript RT SuperMix Kit&lt;/em&gt;)</td><td>New England Biolabs</td><td>E3010S</td><td></td></tr><tr><td>DNA amplification master mix&lt;br/&gt; (&lt;em&gt;Q5&amp;reg; Hot Start High-Fidelity 2X Master Mix [NEB]&lt;/em&gt;)</td><td>New England Biolabs</td><td>M0494L</td><td></td></tr><tr><td>Primer (Scheme)&lt;br/&gt; (&lt;em&gt;Custom DNA oligos&lt;/em&gt;)</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>&lt;strong&gt;SPRI Bead Clean-up&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SPRI beads&lt;br/&gt; (&lt;em&gt;Aline Biosciences PCR Clean DX&lt;/em&gt; )</td><td>Cambio</td><td>AL-AC1003-50</td><td></td></tr><tr><td>Ethanol, Pure Absolute, &gt;99.8% (GC) [Riedel-De Haen]</td><td>Merck</td><td>818760</td><td></td></tr><tr><td>Short Fragment buffer&lt;br/&gt; (&lt;em&gt;SFB expansion pack&lt;/em&gt;)</td><td>Oxford Nanopore Technologies</td><td>EXP-SFB001</td><td></td></tr><tr><td>&lt;strong&gt;DNA Quantification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DNA quantification kit&lt;br/&gt; (&lt;em&gt;Qubit&amp;reg; dsDNA HS Assay Kit&lt;/em&gt;)</td><td>Thermofisher scientific/Fisher scientific</td><td>Q32854</td><td></td></tr><tr><td>DNA quantification assay tubes&lt;br/&gt; &lt;em&gt;(Qubit&amp;trade; Assay Tubes)&lt;/em&gt;</td><td>Thermofisher scientific/Fisher scientific</td><td>Q32856</td><td></td></tr><tr><td>&lt;strong&gt;End Prep and barcoding&lt;/strong&gt;&lt;br/&gt; &lt;strong&gt;(Qubit&amp;trade; Assay Tubes)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>End Prep master mix&lt;br/&gt; &lt;em&gt;(NEBNext Ultra End Repair/dA-Tailing Module)&lt;/em&gt;</td><td>New England Biolabs</td><td>E7546L</td><td></td></tr><tr><td>Barcoding kit</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;*Chemistry 9&amp;nbsp;&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 9&amp;nbsp;&lt;/em&gt;</td><td></td></tr><tr><td>&lt;em&gt;(Native Barcoding Expansion 1-12)&lt;/em&gt;</td><td></td><td>EXP-NBD104&amp;nbsp;</td><td></td></tr><tr><td>&lt;em&gt;(Native Barcoding Expansion 13-24)&amp;nbsp;&lt;/em&gt;</td><td></td><td>EXP-NBD114&amp;nbsp;</td><td></td></tr><tr><td>&lt;em&gt;(Native Barcoding Expansion 96)&lt;/em&gt;</td><td></td><td>EXP-NBD196</td><td></td></tr><tr><td>&lt;em&gt;*Chemistry 14&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 14&lt;/em&gt;</td><td></td></tr><tr><td>&lt;em&gt;(not compatible)&amp;nbsp;&lt;/em&gt;</td><td></td><td>&lt;em&gt;(not compatible)&amp;nbsp;&lt;/em&gt;</td><td></td></tr><tr><td>&lt;em&gt;(Native Barcoding Kit 24 V14)&lt;/em&gt;</td><td></td><td>SQK-NBD114.24</td><td></td></tr><tr><td>&lt;em&gt;(Native Barcoding Kit 96 V14)&lt;/em&gt;</td><td></td><td>SQK-NBD114.96</td><td></td></tr><tr><td>Ligation mastermix&lt;br/&gt; &lt;em&gt;(Blunt/TA Ligase Master Mix)&lt;/em&gt;</td><td>New England Biolabs</td><td>M0367S</td><td></td></tr><tr><td>&lt;strong&gt;Adapter Ligation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adapter ligation master mix</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;(NEBNext&amp;nbsp; Quick Ligation Module)&lt;/em&gt;</td><td>New England Biolabs</td><td>E6056S&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>&lt;em&gt;(NEBNext Ultra II Ligation Module)&amp;nbsp;&lt;/em&gt;</td><td>New England Biolabs</td><td>E7595S</td><td></td></tr><tr><td>&lt;em&gt;(Blunt/TA Ligase Master Mix)&lt;/em&gt;</td><td>New England Biolabs</td><td>M0367S</td><td></td></tr><tr><td>Adapter mix</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;*Chemistry 9&amp;nbsp;&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 9&lt;/em&gt;&lt;br/&gt; EXP-AMII001</td><td></td></tr><tr><td>&lt;em&gt;(Adapter Mix II [AMII])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;*Chemistry 14&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 14&lt;/em&gt;&lt;br/&gt; EXP-NBA114</td><td></td></tr><tr><td>&lt;em&gt;(Native adapter [NA])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Sequencing&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Flowcell priming kit&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;*Chemistry 9&amp;nbsp;&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 9&lt;/em&gt;&lt;br/&gt; EXP-FLP002</td><td></td></tr><tr><td>&lt;em&gt;(Flush Buffer [FB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;(Flush Tether [FT])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;*Chemistry 14&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 14&lt;/em&gt;&lt;br/&gt; EXP-FLP004</td><td></td></tr><tr><td>&lt;em&gt;(Flow Cell Flush [FCF])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;(Flow Cell Tether [FCT])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Ligation Sequencing Kit&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;*Chemistry 9&amp;nbsp;&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 9&lt;/em&gt;&lt;br/&gt; SQK-LSK109</td><td></td></tr><tr><td>Adapter Mix&lt;br/&gt; &lt;em&gt;(Adapter Mix [AMX])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Ligation Buffer&lt;br/&gt; &lt;em&gt;(Ligation buffer [LNB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Short Fragment Buffer&lt;br/&gt; &lt;em&gt;(Short Fragment buffer [SFB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Sequencing Buffer&lt;br/&gt; &lt;em&gt;(Sequencing Buffer [SQB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Elution Buffer&lt;br/&gt; &lt;em&gt;(Elution buffer [EB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Loading Beads&lt;br/&gt; &lt;em&gt;(Loading Beads [LB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Sequencing Tether&lt;br/&gt; &lt;em&gt;(Sequencing Tether [SQT])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;*Chemistry 14&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 14&lt;/em&gt;&lt;br/&gt; SQK-LSK114</td><td></td></tr><tr><td>Adapter Mix&lt;br/&gt; &lt;em&gt;(Ligation Adapter [LA])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Ligation Buffer&lt;br/&gt; &lt;em&gt;(Ligation buffer [LNB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Short Fragment Buffer&lt;br/&gt; &lt;em&gt;(Short Fragment buffer [SFB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Sequencing Buffer&lt;br/&gt; &lt;em&gt;(Sequencing Buffer [SB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Elution Buffer&lt;br/&gt; &lt;em&gt;(Elution buffer [EB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Loading Beads&lt;br/&gt; &lt;em&gt;(Loading Beads [LIB])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Sequencing Tether&lt;br/&gt; &lt;em&gt;(Flow Cell Tether [FCT])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Library solution&lt;br/&gt; &lt;em&gt;(Library solution [LIS])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Flush buffer&lt;br/&gt; &lt;em&gt;(Flow Cell Flush [FCF])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Flow Cell&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;*Chemistry 9&amp;nbsp;&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 9&lt;/em&gt;&lt;br/&gt; FLO-MIN106D</td><td></td></tr><tr><td>&lt;em&gt;(Flow Cell&amp;nbsp; [R9.4.1])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>*Chemistry 14</td><td>Oxford Nanopore Technologies</td><td>&lt;em&gt;*Chemistry 14&lt;/em&gt;&lt;br/&gt; FLO-MIN114</td><td></td></tr><tr><td>&lt;em&gt;(Flow Cell&amp;nbsp; [R10.4.1])&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Flow Cell wash&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Flowcell wash kit&lt;br/&gt; &lt;em&gt;(Flow cell wash kit)&lt;/em&gt;</td><td>Oxford Nanopore Technologies</td><td>EXP-WSH004</td><td></td></tr><tr><td>&lt;strong&gt;Consummables&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Surface decontaminant&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;(DNA Away Surface Decontaminant, Squeeze Bottle [Molecular Bio])&lt;/em&gt;</td><td>Thermofisher scientific/Fisher scientific</td><td>7010PK</td><td></td></tr><tr><td>&lt;em&gt;(RNase Away Surface Decontaminant, Bottle [Molecular Bio])&lt;/em&gt;</td><td>Thermofisher scientific/Fisher scientific</td><td>7002PK</td><td></td></tr><tr><td>PCR 8-Tube Strip 0.2ml, individual cap&lt;br/&gt; &lt;em&gt;(PCR 8-Tube Strip 0.2ml, with Individual attached Flat Caps, Sterile, DNAse/RNAse, Pyrogen Free,Natural [Greiner])&lt;/em&gt;</td><td>Greiner</td><td>608281</td><td></td></tr><tr><td>PCR Tube 0.2ml&lt;br/&gt; &lt;em&gt;(PCR Tube 0.2ml, Natural [Domed Cap] Bagged in 500s, Non-Sterile [Greiner])&lt;/em&gt;</td><td>Greiner</td><td>671201</td><td></td></tr><tr><td>1000&amp;micro;L Filter Tips (500)&lt;br/&gt; &lt;em&gt;(Stacked 1000&amp;micro;L Filter Tips [500])&lt;/em&gt;</td><td>Thermofisher scientific/Fisher scientific</td><td>11977724</td><td></td></tr><tr><td>100&amp;micro;L Filter Tips (1000)</td><td>Thermofisher scientific/Fisher scientific</td><td>11947724</td><td></td></tr><tr><td>10&amp;micro;L Filter Tips (1000)&lt;br/&gt; &lt;em&gt;(Stacked 100&amp;micro;L Filter Tips [1000])&lt;/em&gt;</td><td>Thermofisher scientific/Fisher scientific</td><td>11907724</td><td></td></tr><tr><td>Reinforced tubes&amp;nbsp; tubes (2ml)&amp;nbsp; with screw caps and o-rings&lt;br/&gt; &lt;em&gt;(Fisherbrand&amp;trade;&amp;nbsp;Bulk tubes)&lt;/em&gt;</td><td>Thermofisher scientific/Fisher scientific</td><td>15545809</td><td></td></tr><tr><td>Microcentrifuge tube (1.5ml)&lt;br/&gt; &lt;em&gt;(1.5 ml Eppendorf Tubes [500])&lt;/em&gt;</td><td>Eppendorf</td><td>1229888</td><td></td></tr><tr><td>DNA LoBind Tubes&amp;nbsp; (1.5ml)&lt;br/&gt; &lt;em&gt;(DNA LoBind Tubes)&lt;/em&gt;</td><td>Thermofisher scientific/Fisher scientific</td><td>10051232</td><td></td></tr><tr><td>Cryobabies labels</td><td></td><td></td><td></td></tr><tr><td>Gloves (S/M/L)</td><td></td><td></td><td></td></tr><tr><td>Paper towel</td><td></td><td></td><td></td></tr></tbody></table>

whole genome sequencing for rapid characterization of rabies virus using nanopore technology

Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic surveillance remains limited in many low- and middle-income countries (LMICs), where dog-mediated rabies and/or rabies transmitted by wildlife such as vampire bats pose major public health and economic concerns. We present here a rapid and affordable sample-to-sequence-to-interpretation workflow using nanopore technology. Protocols for sample collection and the diagnosis of rabies are briefly described, followed by details of the optimized whole genome sequencing workflow, including primer design and optimization for multiplex polymerase chain reaction (PCR), a modified, low-cost sequencing library preparation, sequencing with live and offline base calling, genetic lineage designation, and phylogenetic analysis. Implementation of the workflow is demonstrated, and critical steps are highlighted for local deployment, such as pipeline validation, primer optimization, inclusion of negative controls, and the use of publicly available data and genomic tools (GLUE, MADDOG) for classification and placement within regional and global phylogenies. The turnaround time for the workflow is 2-3 days, and the cost ranges from $25 per sample for a 96 sample run to $80 per sample for a 12 sample run. We conclude that setting up rabies virus genomic surveillance in LMICs is feasible and can support progress toward the global goal of zero dog-mediated human rabies deaths by 2030, as well as enhanced monitoring of wildlife rabies spread. Moreover, the platform can be adapted for other pathogens, helping to build a versatile genomic capacity that contributes to epidemic and pandemic preparedness.

The sample-to-sequence-to-interpretation workflow for RABV described in this protocol has been used successfully in different laboratory conditions in endemic countries, such as Tanzania, Kenya, Nigeria, and the Philippines (Figure 4). The protocol was used on different sample types and conditions (Table 6): fresh and frozen brain tissue, cDNA and RNA extracts from brain tissue transported under cold chain for extended periods, and FTA cards with brain tissue smears.
Live basecalling using RAMPART (Figure 5) shows the almost real-time generation of reads and the percent coverage per sample. This is particularly useful in deciding when to stop the run and save the flow cell for reuse. Variation in run time was observed, with some finished in 2 h, while others could take more than 12 h for an adequate depth of coverage (x100) to be reached. We can also view regions with poor amplification; for example, Figure 6 shows a snapshot of one sequencing run where coverage profiles show some amplicons with very low amplification, indicating potentially problematic primers. By investigating these poorly amplifying regions more thoroughly, we have been able to identify primer mismatches, which will enable us to redesign and improve individual primers. Some primer schemes have shown more mismatches than others. This is observed in the East Africa primer scheme, as compared to the Philippines, in line with the targeted diversity, as the East Africa scheme aims to capture a much broader diversity.
RABV-GLUE42, a general-purpose resource for RABV genome data management, and MADDOG60, a lineage classification and nomenclature system, were used to compile and interpret resulting RABV sequences. Table 7 shows the major and minor clades circulating in each country assigned using RABV-GLUE. Also shown is a higher resolution classification of local lineages following the MADDOG assignment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65414/65414fig01.jpg" /> 
Figure 1: Sample-to-sequence-to-interpretation workflow for RABV. Summarized steps are shown for (A) sample preparation, (B) PCR and library preparation, and (C) sequencing and bioinformatics up to analysis and interpretation. <a href="https://www.jove.com/files/ftp_upload/65414/65414fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65414/65414fig02.jpg" /> 
Figure 2: Primer scheme schematic. Annealing positions along the &#39;index reference genome&#39; (dark purple) for pairs of forward and reverse primers (half arrows), which are assigned in two separate pools: A (red) and B (green). Primer pairs generate 400 bp overlapping amplicons (blue) which are numbered sequentially along the index reference genome in the format &#39;scheme_name_X_DIRECTION&#39;, where &#39;X&#39; is a number referring to the amplicon generated by the primer and &#39;DIRECTION&#39; is either &#39;LEFT&#39; or &#39;RIGHT&#39;, describing the forward or reverse respectively. Odd or even values of &#39;X&#39; determine the pool (A or B, respectively). <a href="https://www.jove.com/files/ftp_upload/65414/65414fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65414/65414fig04.jpg" /> 
Figure 3: Nanopore flow cell48. Blue labels illustrate the different parts of the flow cell, including the priming port cover which covers the priming port where the priming solution is added, the SpotON sample port cover covering the sample port where the sample is added in a dropwise fashion, the waste ports 1 and 2, and the flow cell ID. <a href="https://www.jove.com/files/ftp_upload/65414/65414fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65414/65414fig03.jpg" /> 
Figure 4: Map showing the location where RABV sequencing was conducted using the optimized workflow in 2021 and 2022. Bubble size and color correspond to the number of sequences per location, where smaller and darker is fewer, while larger and lighter is more. <a href="https://www.jove.com/files/ftp_upload/65414/65414fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65414/65414fig05v2.jpg" /> 
Figure 5: Screenshot of RAMPART visualization in web browser. Barcode names are replaced by sample names according to the bioinformatic setup. The top three panels show summary plots for the whole run: depth of coverage of mapped reads for each barcode per nucleotide position on the index reference genome (top left, colored by barcode), summed mapped reads from all barcodes over time (top middle), and mapped reads per barcode (top right, colored by barcode). Lower panels show rows of plots per barcoded. From left to right: the depth of coverage of mapped reads per nucleotide position on the index reference genome (left), length distribution of mapped reads (middle), and proportion of nucleotide positions on the index reference genome which have obtained a 10x, 100x and 1,000x coverage of mapped reads over time (right). <a href="https://www.jove.com/files/ftp_upload/65414/65414fig05largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65414/65414fig06v2.jpg" /> 
Figure 6: An example read coverage across the genome for a rabies virus sample from the Philippines sequenced using the protocol.&#160;Read coverage at each nucleotide position in the genome is shown, alongside the position of the overlapping amplicons (1-41) used to generate the library. Spikes in the depth of coverage correspond to areas of amplicon overlap. Amplicons with a low depth of coverage correspond to areas of amplicon overlap. Amplicons with a low depth of coverage are highlighted in red indicating problematic regions that may require&#160;optimization. <a href="https://www.jove.com/files/ftp_upload/65414/65414fig06largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Master mix and thermal cycler conditions for cDNA preparation. <a href="https://www.jove.com/files/ftp_upload/65414/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Master mix and thermal cycler conditions for multiplex PCR. <a href="https://www.jove.com/files/ftp_upload/65414/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Master mix and thermal cycler conditions for end-prep reaction. <a href="https://www.jove.com/files/ftp_upload/65414/Table 3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Master mix and thermal cycler conditions for barcoding. <a href="https://www.jove.com/files/ftp_upload/65414/Table 4.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Adapter ligation master mix and final library dilution for sequencing. <a href="https://www.jove.com/files/ftp_upload/65414/Table-5_revised.zip" target="_blank">Please click here to download this Table.</a>
Table 6: The number of rabies virus whole genome sequences generated and the type of samples used in different countries using the sample-to-sequence-to-interpretation workflow. <a href="https://www.jove.com/files/ftp_upload/65414/Table 6.xlsx" target="_blank">Please click here to download this Table.</a>
Table 7: Major and minor clade assignments from RABV-GLUE and lineage assignments from MADDOG for sequences generated using the workflow. <a href="https://www.jove.com/files/ftp_upload/65414/Table 7.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Primer scheme design and optimization, and amplicon read depth analysis. <a href="https://www.jove.com/files/ftp_upload/65414/Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Computational setup <a href="https://www.jove.com/files/ftp_upload/65414/Supplementary File 2.docx" target="_blank">Please click here to download this File.</a>
Supplementary File 3: RABV WGS protocol worksheet <a href="https://www.jove.com/files/ftp_upload/65414/Supplementary File 3- Worksheet.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about A Cost-Effective Genomic Workflow for Advancing Rabies Control in Resource-Limited Settings at JoVE.com

Author Spotlight: A Cost-Effective Genomic Workflow for Advancing Rabies Control in Resource-Limited Settings 

Here, we present a rapid and cost-effective workflow for characterizing rabies virus (RABV) genomes using nanopore technology. The workflow is intended to support genomics-informed surveillance at a local level, providing information on circulating RABV lineages and their placement within regional phylogenies to guide rabies control measures.

Whole Genome Sequencing for Rapid Characterization of Rabies Virus Using Nanopore Technology

Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic ...

whole-genome-sequencing-for-rapid-characterization-rabies-virus-using

Research

JoVE Journal

Biology

5.0K Views.  University of Glasgow. Ons onderzoek maakt gebruik van geavanceerde genomica om de surveillance van ziekteverwekkers in lage- en middeninkomenslanden te verbeteren. En dit helpt ons om lokale onderzoekers in staat te stellen vijf grote vragen te beantwoorden: wie, wat, waar, waarom en wanneer. En dit kan praktische oplossingen op het terrein opleveren.We gebruiken nanoporietechnologie om sequencing te decentraliseren. Het gebruiksgemak, de draagbaarheid en de lagere kosten in vergelijking met andere platform...

Video: Author Spotlight: een kosteneffectieve genomische workflow voor het bevorderen van rabiëscontrole in omgevingen met beperkte middelen 

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Genetics

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R10 flow cell and Illumina sequencing as an example.

We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell.

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 2

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Quantitation of Rabies Virus in Various Bovine Brain Structures

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon&#8217;s horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major ...

Whole Genome Sequencing for Rapid Characterization of Rabies Virus Using Nanopore Technology

Samenvatting

Meer video's verkennen