Oct 07, 2024

Public workspaceSequencing the Virome of Wild Eastern Cottontail Rabbits using Oxford Nanopore Technologies (ONT) V.1

DOI
dx.doi.org/10.17504/protocols.io.5jyl82ywrl2w/v1
  • Paula Glover1
  • 1North Carolina State University
  • Paula Glover: Created for BIT 595 Portable Genome Sequencing Course Fall 2024
Paula Glover
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DOI: dx.doi.org/10.17504/protocols.io.5jyl82ywrl2w/v1
Protocol Citation: Paula Glover 2024. Sequencing the Virome of Wild Eastern Cottontail Rabbits using Oxford Nanopore Technologies (ONT). protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82ywrl2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 04, 2024
Last Modified: October 07, 2024
Protocol Integer ID: 109176
Keywords: Oxford Nanopores Technologies, ONT, Nanopore, Nanopore sequencing
Disclaimer
Protocols are linked.
Abstract
This protocol can be used to perform multiplex sequencing of a virome using Oxford Nanopore Technologies (ONT).

Forty RNA samples, including blood (n = 10), fecal (n = 10), epithelial (n = 10) and mucosal (n = 10) collected using cotton swabs from 10 wild Eastern Cottontail rabbits and stored at -80 °C. The 40 samples were combined into four pools each composed of ten samples of the same type. Sequencing was performed using the cDNA-PCR kit from
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Created with BioRender.
Materials
RNA Extraction Using the TRIzol™ Protocol by Invitrogen
  • 100–200 µl of pre-processed sample (blood, fecal, epithelial, and mucosal separately)
  • TRIzol (Invitrogen)
  • Phase Lock GelTM tubes (VWR) - optional
  • RNase-free glycogen or GlycoBlue‱ Coprecipitant (ThermoFisher)- optional
  • 75% freshly-prepared ethanol
  • Isopropanol
  • Nuclease-free water or TE buffer
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 1.5 ml Eppendorf tubes

Polyadenylation of Native RNA
  • E. coli Poly(A) Polymerase (NEB: M0276L)
  • 50 mM RNase-free EDTA (Invitrogen: AM9260G)
  • Nuclease-free or DEPC-treated water, or 10 mM Tris-HCL, pH7.5
  • Freshly-prepared 70% ethanol
  • Agencourt RNAClean XP beads (Beckman Coulter: A63987) 
  • Nuclease-free 1.5 ml microcentrifuge tubes
  • Heat block set to 37°C
  • Centrifuge fitted for microcentrifuge tubes
  • Vortex mixer
  • Magnetic rack
  • RNA HS Qubit kit (Invitrogen: 10320093)
  • Qubit fluorometer (Invitrogen: 16223001)
  • Hula mixer

Library Preparation with Oxford Nanopores Technologies (ONT)
  • 10 ng enriched RNA (Poly(A)+ RNA or ribodepleted) or 500 ng total RNA
  • cDNA-PCR Sequencing Kit V14 (SQK-PCS114) 
  • R10.4.1 flow cells (FLO-PRO114M)
  • Flow Cell Wash Kit (EXP-WSH004)
  • RNA Control Expansion (EXP-RCS001)
  • Rapid Adapter Auxiliary V14 (EXP-RAA114)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)
  • Flow Cell Priming Kit V14 (EXP-FLP004)
  • GridION GridION Mk1 IT Requirements
Pre-processing of Samples: Blood, Fecal, Epithelial, and Mucosal (Salival)
Pre-processing of Samples: Blood, Fecal, Epithelial, and Mucosal (Salival)
10s
10s
Blood, Fecal, and Epithelial Sample Pre-processing
Place each swab in a 1.5-mL centrifuge tube with Amount500 µL of phosphate buffered saline (PBS). PBS is an isotonic solution that prevents the rupture of cells or shriveling of cells due to osmosis and is a non-toxic formulation for the cells.

Vortex the samples for Duration00:05:00 at room temperature, then centrifuge for Duration00:05:00 atCentrifigation10000 rpm . Collect Amount400 µL in a fresh 1.5 ml Eppendorf DNA LoBind tube.

10m
Mucosal (Salival) Sample Pre-Processing (Dilution)
Dilute Amount100 µL of the sample in Amount300 µL of Saline Solution Concentration0.8 Mass Percent in a fresh 1.5 ml Eppendorf DNA LoBind tube. Normal saline solution can serve as RNA preservative, eliminating the need for snap-freezing the sample.

Vortex mix Duration00:00:10 s .

10s
RNA Extraction Using the TRIzol™ Protocol by Invitrogen
RNA Extraction Using the TRIzol™ Protocol by Invitrogen
Critical Step: Ensure you start with at least 3X more TRIzol‱ than the volume of your starting material. Extract Amount250 µL of each sample in a 1.5 ml Eppendorf DNA LoBind tube.

Critical
Follow the standard TRIzol™ protocol, using a Phase Lock GelTM tube to trap the organic phase. Add Amount1 µg RNase-free glycogen to the first isopropanol precipitation. Then use standard 1.5 ml tubes for the pelleting steps.

Optional Step: GlycoBlue Coprecipitant can be used to make the pellet visible.
Optional
Elute the RNA in Amount100 µL nuclease-free water or TE buffer. (Yields 10-20 ng of RNA)

Assessing input RNA
Assessing input RNA
Oxford Nanopore Technologies (ONT) protocols recommend an input quantity in mass. The Direct RNA Sequencing Kit (SQK-RNA004) from ONT is used to prepare RNA for nanopore sequencing from an input of as low as Amount300 ng poly(A)+ RNA or Amount1 µg of total RNA (additional optimization may be required for total RNA). Therefore, it is important to assess the input RNA to determine if it is of high enough quality and amount to be used in direct RNA sequencing. The following protocol is described by ONT for assessing input RNA:


Polyadenylating with E. coli poly(A) polymerase
Polyadenylating with E. coli poly(A) polymerase
In a 1.5 ml microcentrifuge tube, set up the 3’ polyadenylation reaction as follows:



Incubate the reaction mixture atTemperature37 °C for Duration00:01:00 .

1m
Stop the reaction by adding Amount5 µL of 50 mM EDTA (to a final concentration of 10 mM EDTA). The final volume will be Amount25 µL
Note: An incubation time of 30–90 seconds to add a 3’ adenosine homopolymer (poly(A) tail) of approximately 50-100 nucleotides in length. An increase in incubation time will result in longer poly(A) tails. The maximum incubation time is 5 minutes and longer timings may result in a lower total sequencing yield.
Optional
Optional Step: It is recommended that EDTA be removed before starting any sequencing kit protocols.
Add Amount45 µL of RNase-free SPRI beads to the reaction.

Incubate on a Hula Mixer for Duration00:05:00 at room temperature.

5m
Spin down the sample and pellet on a magnet.
. Keep the tube on the magnet and pipette off and discard the supernatant.
Keep the tube on the magnet and wash the beads with 200 µl of freshly-prepared 70% ethanol. Carefully turn the tube 180° twice in the rack to wash pelleted beads. Pippette off and discard the supernatant.
Repeat the previous step for a total of two washes.
Briefly spin down and place the tube back on the magnet.
Pipette off any residual ethanol. Allow to dry for Duration00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend the pellet in Amount12 µL of nuclease-free water. Incubate on ice for Duration00:05:00 .
5m
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain Amount12 µL of eluate containing the 3’-polyadenylated RNA in a clean 1.5 ml microcentrifuge tube.
Remove Amount1 µL of final eluate and quantify the final concentration using HS RNA Qubit kit.

Optional step: Dilute the final eluate to 100 ng/µl and quantify Amount1 µL using Agilent 6000 Nano RNA kit, using total RNA or mRNA setting depending on the sample.

Store the final product at Temperature-80 °C or proceed immediately with the library preparation, keeping your sample on ice.

Library Preparation with the Direct RNA Sequencing Kit (SQK-RNA004)
Library Preparation with the Direct RNA Sequencing Kit (SQK-RNA004)
Use the Direct RNA Sequencing (SQK-RNA004)Protocol from Oxford Nanopore Technologies (ONT). The table below is an overview of the steps required in the library preparation, including timings and stopping points.





Protocol references
Caro-Quintero, Alejandro & Ruiz-Avila, Camilo & Navarrete, Andrea & Bernal Morales, Johan & Daza, Giuliana & Yockteng, Roxana. (2020). Viral RNA extraction low-cost protocol optimized for SARS-Cov2 at AGROSAVIA v1. 10.17504/protocols.io.bggvjtw6.

Keller, M.W. et al., Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome. Nature Scientific Reports, 8, 14408. Adapted from Oxford Nanopores Technologies.

Viehweger A, Krautwurst S, Lamkiewicz K, Madhugiri R, Ziebuhr J, Hölzer M, Marz M. Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis. Genome Res. 2019 Sep;29(9):1545-1554. doi: 10.1101/gr.247064.118. Epub 2019 Aug 22. PMID: 31439691; PMCID: PMC6724671.

Vincek, V., Nassiri, M., Knowles, J. et al. Preservation of Tissue RNA in Normal Saline. Lab Invest 83, 137–138 (2003). https://doi.org/10.1097/01.LAB.0000047490.26282.CF.

Xiao Y, Wang H, Feng L, Pan J, Chen Z, Wang H, Yang S, Shen Q, Wang X, Shan T, Zhang W. Fecal, oral, blood and skin virome of laboratory rabbits. Arch Virol. 2020 Dec;165(12):2847-2856. doi: 10.1007/s00705-020-04808-y. Epub 2020 Oct 9. PMID: 33034764; PMCID: PMC7546134.

Oxford Nanopore Technologies. 3’ polyadenylation of RNA transcripts using E. coli poly(A) polymerase (PAP). https://nanoporetech.com/document/extraction-method/3-poly-rna-ecoli-pap

Oxford Nanopore Technologies. Assessing input RNA. https://nanoporetech.com/document/input-dna-rna-qc