TITLE: Rabies virus MinION sequencing protocol
AUTHORS: Kirstyn Brunker, Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a sample-to-sequence protocol for sequencing rabies virus genomes using the MinION platform ( may also be used with Illumina). This pipeline is feasible in low resource settings using a lab-in-a-suitcase approach and has been applied successfully under a range of conditions (from in the field to diagnostic labs) across East Africa, the Philippines and Peru. </div><div class = "text-block"><span>The protocol was developed based on resources from the </span><a href="https://artic.network/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">ARTIC network</span></a><span> and is published and described in </span><a href="https://wellcomeopenresearch.org/articles/5-3" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;">Rapid in-country sequencing of whole virus genomes to inform rabies elimination programmes</span></a><span style = "font-weight:bold;"> (Brunker et al, 2019, Wellcome Open Research). </span></div></div>

[STEPS]
?. [Primer pool preparation]
If required resuspend lyophilised primers at a concentration of  each
RABV primers for this protocol were designed using Primal Scheme and generate overlapping 400nt amplicons. There are currently primer schemes available for East Africa, Philippines and Peru, primer sequences are listed in attached documents here:
?. [Primer pool preparation]
Generate primer pool stocks by adding  of each primer pair to a  Eppendorf labelled with the primer scheme name and either “Pool 1 (100µM)” or “Pool 2 (100µM)”. These are your  stocks of each primer pool.
5 µl
1.5 ml
Primers should be prepped and aliquoted in a sterile PCR cabinet. At no stage should primers or PCR reagents be anywhere near the template until cDNA addition.
?. [Primer pool preparation]
Dilute this primer pool 1:10 in molecular grade water, to generate  primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination.
Primers need to be used at a final concentration of 0.015µM per primer. For example, for the rabvSEasia scheme, Pool 1 has 42 primers in it so the requirement is 1.575 µL of Pool 1 (10µM) primers per 25µL reaction (i.e. [number of primers X reaction volume (25) x 0.015]/ concentration of primer stock). Pool 2 has 40 primers the requirement is 1.5 µL of Pool 2 (10µM) primers per 25µL reaction. For other schemes, adjust the volume added appropriately.
?. [Multiplex PCR]
In the mastermix hood prepare a mastermix of the components below according to the number of samples (plus 1 extra) and aliquot  for each reaction into labelled 0.2mL 8-strip PCR tubes:Important: Adjust primer volume below according to the scheme used and make up to 22.5ul with water, example shown is for scheme rabvSEasiaComponent                                                                     Pool 1                Pool 2 NEB Q5® Hot Start Polymerase 2X MasterMix    *Primer Pool 1 or 2 (10µM)                                       Nuclease-free water                                                  Total
22.5 µl
12.5 µl
12.5 µl
1.58 µl
1.5 µl
8.42 µl
8.5 µl
22.5 µl
22.5 µl
The mastermix hood should be prepared by sterilising with UV and treated with MediPal wipes/10% bleach solution, DNAway and RNAseZap reagents. Wipe down the hood with each sequentially, allowing 5 minutes for drying between each. Pipettes should also be treated in the same way, and UV treated for 30 mins between library preparations.
?. [Multiplex PCR]
In the template hood add  cDNA to each tube and mix well by pipetting. Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
2.5 µl
The template hood should prepared by sterilising with UV and treated with MediPal wipes, DNAway and RNAseZap reagents. Wipe down the hood with each sequentially, allowing 5 minutes for drying between each. Pipettes should also be treated in the same way, and UV treated for 30 mins between library preparations.
?. [Multiplex PCR]
Set-up the following program on the thermal cycler:  Step                        Temperature     Time                    CyclesHeat Activation         1Denaturation              25-35Annealing                   25-35Hold                                   Indefinite              1
98 °C
98 °C
65 °C
4 °C
Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35. If you don't have realtime PCR results use 30 cycles!Samples do not need to be refrigurated immediately after PCR - in fact the DNA will be stable for a few days at room temperature without suffering from degradation (e.g. see here). Therefore a holding step at   is not entirely necessary e.g. this temperature may unavailable (e.g. using a MiniPCR machine).
?. [PCR clean-up]
From this point on samples should be prepared in a designated post-PCR area (can be conducted on the bench, if this is separate to mastermix and template areas). Keep the contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample separate.
In future, when you are happy that amplification is relatively equal in each pool, you may want to combine the contents of these tubes for each biological sample prior to clean-up.
?. [PCR clean-up]
Clean-up each pool of amplicons using the following protocol:
Money saving tips:Aline PCRCLEAN DX beads can be used as a direct replacement for Ampure XT beads in this protocol - they cost a lot less!In addition, 3-D printed versions of magnetic racks (e.g. tried-and-tested Sam and Tom Industrys Separatron https://samandtomindustrys.science/borchure.html) offer a cheaper alternative to big company versions (just make sure your tubes fit!)
?. [PCR clean-up]
Quantify the amplicon pools using one of the methods below:
?. [Native barcoding]
Quantify the final library using the Quantus Fluorometer using the ONE dsDNA assay/ Qubit Fluorometer with the high sensitivity dsDNA assay
For best results proceed directly to MinION sequencing but the final library can be now be stored in EB at 4°C for up to a week if needed.
?. [MinION sequencing]
Prime the flowcell and load  sequencing library onto the flowcell.
20 ng
From experience we know 20 ng is optimum loading input for short amplicons.
?. [MinION sequencing]
Start the sequencing run using MinKNOW.
?. [cDNA preparation]
In the mastermix hood: prepare a mastermix of the reagents below according to the number of samples and controls to be prepared (plus 1 extra to ensure some excess reagent). Important: A negative control must be included here and taken right through to sequencing stage.Component                                        Volume LunaScript RT SuperMix (5X)         Nuclease-free water                         Aliqout 5µL of mastermix for each reaction into labelled 0.2ml PCR strip tubes.
2 µl
3 µl
We use half the reaction volume stated in the manufacturer's protocol, using only   of Lunascript reagent per   reaction and have found this is sufficient to generate enough cDNA.The Lunascript reagent is quite viscous so take care pipetting, pipette slowly and pause before final purge to ensure reagent is in the tip before dispensing.
?. [cDNA preparation]
Take the prepared tubes to the template hood. Add  of RNA to each tube. Mix gently by pipetting or gently flicking tube and pulse centrifuge to collect liquid at the bottom of the tube. Incubate the reaction as follows on a thermocycler:Hold at
5 µl
25 °C
55 °C
95 °C
4 °C
?. [Quantification and normalisation]
?. [Quantification and normalisation]
?. [Quantification and normalisation]
?. [cDNA preparation]
?. [Quantification and normalisation]
?. [Quantification and normalisation]
?. [Quantification and normalisation]
?. [Quantification and normalisation]
?. [Quantification and normalisation]
?. [Quantification and normalisation]
?. [Quantification and normalisation]
?. [Quantification and normalisation]
?. [Normalisation]
Amplicon pools for each sample need to be combined in equimolar amounts to a total input of  prior to  barcode ligation in the next step. To do this first dilute each amplicon pool to   and then combine  from each pool in a new tube with  of nuclease-free water. A worked example and a normalisation template are provided below.   ABCDEFGH11. dilute each pool to 1ng/ul in a new tube (keep pools separate)2. Combine normalised pools in a new tube2Sample idPoolConcentration (ng/uL)Volume of sample (ul)Volume of water (ul)Volume of dilution required
  (ul)Volume of water (ul)3Sample 1152.05.0255.02.507.54236.05.0175.02.505
5 ng
2.5 µl
7.5 µl
ABCDEFGH11. dilute each pool to 1ng/ul in a new tube (keep pools separate)2. Combine normalised pools in a new tube2Sample idPoolConcentration (ng/uL)Volume of sample (ul)Volume of water (ul)Volume of dilution required
  (ul)Volume of water (ul)3Sample 1152.05.0255.02.507.54236.05.0175.02.505
?. [Native barcoding]
We use the ‘one-pot ligation’ protocol by Josh Quick for native barcoded ligation libraries. Methods to improve barcoding efficiency are ongoing, therefore this part of the protocol may be subject to change (take note of any comments left on the protocol for new tweaks!).
{"blocks":[{"key":"qnlk","text":"This is a \u2018one-pot ligation\u2019 protocol for Oxford Nanopore native barcoded ligation libraries using shearing. ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"28bgi","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"635hs","text":" ","type":"atomic","depth":0,"inlineStyleRanges":[],"entityRanges":[{"key":0,"length":1,"offset":0}],"data":[]},{"key":"6vokj","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[{"type":"notes","data":{"blocks":[{"data":[],"depth":0,"entityRanges":[],"inlineStyleRanges":[{"length":4,"offset":111,"style":"italic"}],"key":"dijah","text":"This protocol has been slightly modified from Josh Quick's original to use the Ultra II ligation for barcoding and adaptor ligation","type":"unstyled"}],"entityMap":[],"guid":"8FE2F3D05F9611EA81A6D5CE3A9520A3"}}]}
We use the Ultra II® ligation module for barcoding and adaptor ligation steps but adaptor ligation may be performed with the NEBNext Quick Ligation module as per Josh Quick's original protocol https://www.protocols.io/view/one-pot-native-barcoding-of-amplicons-sg2ebye
?. [PCR clean-up]
Quantus Fluorometer with the ONE dsDNA assay
?. [PCR clean-up]
Qubit Fluorometer with the high sensitivity dsDNA assay
{"blocks":[{"key":"fi1ru","text":"Instructions to quantify double stranded DNA on a Qubit Fluorometer using the high sensitivity dsDNA assay kit","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [cDNA preparation]
?. [Sample preparation and RNA extraction]
Extract RNA from brain tissue sample in a dedicated sample extraction area (biosafety cabinet/portable glove box)
{"blocks":[{"key":"a4945","text":"This protocol details the steps involved to perform RNA extraction on rabies virus brain tissue samples collected by the WHO recommended \"straw method\" in the field (Meslin F-X, Kaplan MM, Koprowski H. Laboratory techniques in rabies. World Health Organization; 1996.[10]). The protocol is field-friendly and can be performed using portable, battery powered equipment.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [cDNA preparation]
OPTIONAL PAUSE POINT: cDNA can be stored at for up to a month if necessary but it is better to continue to PCR setup if possible.
-20 °C
?. [Multiplex PCR]
OPTIONAL PAUSE POINT: PCR products can be stored in the fridge for up to a month or longer term at.
-20 °C
?. [PCR clean-up]
OPTIONAL PAUSE POINT: PCR products can be stored in the fridge for up to a month or longer term at.
-20 °C