key: cord-0729297-ovh2x7qo authors: Freimanis, Graham L.; Oade, Michael S. title: Whole-Genome Sequencing Protocols for IBV and Other Coronaviruses Using High-Throughput Sequencing date: 2020-05-11 journal: Coronaviruses DOI: 10.1007/978-1-0716-0900-2_5 sha: 8e2e30bb79a9d672889e29061e418db8aa7fe469 doc_id: 729297 cord_uid: ovh2x7qo This chapter reports the high-throughput sequencing protocol for sequencing Coronaviruses and other positive strand viruses to produce a dataset of significant depth of coverage. The protocol describes sequencing of infectious bronchitis virus propagated in embryonated eggs and harvested in the allantoic fluid. The protocol is composed of three main steps—enrichment of the allantoic fluid using ultracentrifugation, extraction of total RNA from allantoic fluid, and library preparation from total RNA to DNA sequencing libraries. The workflow will be suitable for all coronaviruses using high-throughput sequencing platforms. High-throughput sequencing (HTS) technologies provide the opportunity to rapidly obtain full genome sequence data of pathogenic organisms. In the recent COVID-19 pandemic, HTS was been used to quickly identify the causative agent as a coronavirus, which has subsequently been named SARS-COV-2 [1] . The subsequent dissemination of sequence information online has allowed laboratories worldwide to study the virus and begin the development of vaccines eliminating the requirement of access to live virus samples. HTS and whole-genome sequencing (WGS) are therefore powerful, versatile tools for the development of novel treatments and vaccines, for studying virus evolution and genetic associations to disease or for tracking outbreaks with notable recent publications investigating viral outbreaks in health-care settings [2, 3] . The depth of data and high sequence quality obtained by such HTS also affords the possibility to dissect and study virus populations leading to the identification of low frequency mutations that would otherwise be undetected by conventional Sanger-based sequencing. This is particularly relevant when working with RNA viruses, like coronaviruses, which have multiple variant viruses within a viral swarm due to factors including rapid replication rates, an error prone RNA-dependent RNA polymerase and large population sizes [4] . Despite the relatively small size of virus genomes, their sequencing is complicated by the atypical low qualities of viral nucleic acid template extracted from samples. Sample preparation therefore is the key to deep sequencing, in that it is important to enrich for viral RNA whist maintaining the integrity of input material, resulting in a DNA-free high-quality preparation. The protocol described here can produce high coverage depth datasets for Coronavirus genomes, such as Infectious Bronchitis Virus (IBV) grown in ovo from around 50 ng of total RNA. The growth of IBV in embryonated eggs has been described previously and is not discussed in this protocol [5] . Allantoic fluid must been free of membrane or other solid masses and ideally be free from blood. If the IBV causes hemorrhaging of the blood vessels, it is important to harvest the allantoic fluid before this happens. Due to the low sample matrix content in allantoic fluid and the polyA enrichment component of this protocol, some host mRNAs are also captured during sequencing, however this is dependent of the purity of the original sample. This protocol is suitable for the capture of both viral gRNA and mRNA (combined) from allantoic fluid, is robust in terms of coverage depth, producing >99% genome recovery. Perform all activities prior to library preparation in a clean RNase free environment with dedicated RNA pipettes, RNase-free wipes, and other consumables. Always clean workspace with RNase cleaning agents prior to starting protocol. A schematic of the whole protocol is shown in Fig. 1 . 1. Place 3-10 ml of IBV infected allantoic fluid into a 15 ml tube and centrifuge for 10 min, 1150  g at 4 C in a benchtop centrifuge in order to remove large solid matter from the IBV infected allantoic fluid. 2. Take the supernatant and add it to a 5 ml ultracentrifuge tube. A cushion of 0.5 ml 30% Sucrose is placed at the base of the tube under the allantoic fluid using a canula being careful not to disturb the allantoic fluid. Tubes must be balanced carefully. 3. Ultracentrifuge for 60 min, 236,880  g at 4 C in an ultracentrifuge. 4. Remove supernatant in layers, taking care not to disturb the pellet (see Note 1). Resuspend the pellet in 350 μl of RLT lysis buffer or TRIzol. 6. Either store at À20 C or progress directly onto total RNA extraction. In our experience although TRIzol produced higher-quality RNA, RNeasy extractions by comparison yielded more RNA. A greater RNA yield and the easier scalability of RNeasy versus TRIzol resulted in RNeasy being our preferred method of RNA extraction for this protocol. Other RNA extraction kits and methods may also be used. 1. Extract total RNA from the 350 μl of nucleic acid dissolved in lysis buffer (RLT) using the Qiagen RNeasy RNA extract kit (or equivalent) according to the manufacturer's protocol, following the optional additional spin to remove excess RPE. Elute in a total of 30 μl of elution buffer. Sometimes running elution buffer, prewarmed at 37 C, through the column twice can improve the yield of the kit. 1. Quantify total RNA content using Qubit and the Qubit High sensitivity RNA kit (or equivalent) following the manufacturer's instructions with each sample measured in duplicate. Fig. 1 Schematic for the overall IBV library preparation pipeline 2. Identify size distribution of RNA using the Bioanalyzer 2100 and Bioanalyzer RNA nano chip (or equivalent) following the manufacturer's protocol (see Note 2). Preparation (1-2 Days Dependent Upon Kit) 1. Calculate a minimum input of 50-200 ng of total RNA and dilute into a total volume of 50 μl to satisfy input requirements for the NEBNEXT Ultra II stranded mRNA RNA-Seq kit (or equivalent) (see Note 3). 2. The remainder of the protocol is carried out following the manufacturer's instructions, fragmenting for 200-300 bp fragments in terms of fragmentation and size selection protocols. However, if the amount of input material is low (<50 ng) then 200 bp is recommended. 3. Elute the final libraries in 20 μl of 10 mM Tris-HCl, pH 8. Store at À20 C for a maximum of 4 weeks (see Note 4). 1. Run generated libraries on a Bioanalyzer 2100 using Bioanalyzer DNA 1000 kit (or equivalent) following the manufacturer's instructions (see Note 5) . A typical library profile is as below (Fig. 2) . Record the average library size for each library using smear analysis to increase accuracy of average size. 2. Manually quantitate each sample using the Qubit high sensitivity kit measuring each sample in duplicate to get an accurate reading. 3. Convert the average Qubit reading from ng/μl to nM using the average library size from the Bioanalyzer results using the formula below; concentration in ng=μl 660 g=mol  average library size in bp ð Þ Â 10 6 ¼ concentration in nM 4. For longer term storage, dilute in 10 mM Tris-HCl, pH 8.5. Do not pipette volumes <2 μl (see Note 6). Otherwise, proceed to step 5. Dilute libraries to a concentration of 5 nM using the formula below. A minimum of 4 nM is required before addition to the pool. V 2 ¼ volume of the initiation solution that will be diluted. C 2 ¼ concentration of final solution. V 2 ¼ desired volume of final solution. 6. Take 5 μl from all samples and pool in a single 1.5 ml LoBind tube and store at À20 C. If you do not intend to progress to the next step immediately then ensure sequencing is performed within 2-3 weeks for optimal cluster densities where possible. Fig. 3. 3. Using the poly-A enrichment module will directly enrich for both genomic vRNA and viral mRNAs due to the presence of a poly-A tail at the 3 0 end of the IBV genome. 4. Libraries should not be left for longer than 4 weeks at À20 C or this can result in a reduction in clustering and sequencing quality. 5. Performing library QC is important for indicating inefficient library preparation/low library yield. 6. Where possible, do not dilute values less than 2 μl. Pipetting smaller volumes is highly inaccurate and will unduly bias the proportion of this library on the flow cell. 7. Loading concentrations may need to be optimized for individual machines. Use of whole-genome sequencing in the investigation of a nosocomial influenza virus outbreak Viral quasispecies Isolation and propagation of coronaviruses in embryonated eggs