TITLE: Whole-genome sequencing of two human rhinovirus A types - A15 and A101
AUTHORS: Martha   Luka, Everlyn  Kamau, Zaydah  R. de Laurent, John  Mwita Morobe, Leonard  K. Alii, Charles Agoti, D.  James Nokes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Genome sequencing of pathogenic viruses has become an established tool for studying transmission, evolution and drug/vaccine design. Viral genome sequencing can either take up a targeted/enrichment approach or an agnostic/metagenomics approach. A standardised enrichment protocol for the whole-genome sequencing (WGS) of human rhinovirus (HRV) remains a challenge due to high viral genetic diversity. We present a type-specific WGS approach for two types - A15 and A101 using the Illumina Miseq. </div></div><div class = "text-block">Our protocol comprises of:-</div><div class = "text-block">    Primer design</div><div class = "text-block">    Polymerase Chain Reaction</div><div class = "text-block">    Library preparation/ sequencing</div></div>

[STEPS]
?. [RNA extraction]
Extract viral RNA from 140 μl of sample using the QIAamp viral RNA mini kit according to the manufacturer's recommendations, eluting in 60 μl of EB buffer
?. [Reverse transcription]
Combine the following in a 1.5 ml tube:ComponentVolumeRNA5 μlDEPC-treated water3 μlrandom primer (10 μM)1 μl10 mM dNTP mix1 μlTotal Volume10 μlMix thoroughly by pipetting up and down.
ComponentVolumeRNA5 μlDEPC-treated water3 μlrandom primer (10 μM)1 μl10 mM dNTP mix1 μlTotal Volume10 μl
Note: One can also use the reverse HRV primers in place of random hexamers to generate consistently longer fragments…. see Troubleshooting table in Guidelines
?. [Primer design]
1. To retrieve publicly available (near) whole genomes, search GenBank using the search term: "Rhinovirus" AND {HRV type} AND 6000:8000 [SLEN].
?. [Primer design]
Design suitable primers using a suitable primer design tool. We used Geneious Prime 2019.2.1 (https://www.geneious.com), ensuring:a) A moderate GC content (40-60%)b) Primer length of 18-27 bp, an annealing temperature between 55 °C and 65°Cc) Less than degenerate bases to minimize non-specific annealing. d) No secondary structures (hairpins, self-dimers and primer dimers) or if present, kept below standard room temperature (18°C)e) Primer pairs selected did not have a melting temperature (Tm) difference of more than 5 °Cf) Where possible, a GC clamp to improve PCR primer specificity.
?. [Primer design]
Filter out any unsuitable genomes e.g. non-human.
?. [Reverse transcription]
Place the sample in a thermal cycler and incubate the sample at 65°C for 5 minutes.
?. [Reverse transcription]
Immediately transfer the tube to ice for at least 1 minute.
?. [Reverse transcription]
Assemble the first strand synthesis reaction on ice by adding the following components to the fragmented and primed RNA from step aboveComponentVolumeFragmented and primed RNA (Step 4)10 μl10X RT buffer2 μl25 mM MgCl24 μl0.1 M DTT2 μlRNaseOUT™ (40 U/μL)1 μlSuperScript® III RT (200 U/μL)1 μlTotal volume20 μlMix thoroughly by pipetting up and down several times.
ComponentVolumeFragmented and primed RNA (Step 4)10 μl10X RT buffer2 μl25 mM MgCl24 μl0.1 M DTT2 μlRNaseOUT™ (40 U/μL)1 μlSuperScript® III RT (200 U/μL)1 μlTotal volume20 μl
?. [Reverse transcription]
Incubate the sample in a preheated thermal cycler with the heated lid set at ≥ 80°C as follows:Step 1: 10 minutes at 25°CStep 2: 50 minutes at 50°CStep 3: 5 minutes at 85°C
?. [Reverse transcription]
(Optional) Briefly spin the plate. Add 1 μl of RNase H to each reaction. Incubate for 20 minutes at 37°C.
Safe stop: Proceed directly to PCR or store at 4°C overnight
?. [Preparing primer pools]
Create a working concentration of 10 μM of each primer from the stock solution (100 μM) using nuclease free water. Alternatively, pool primers from the 100 μM solutions and dilute to 10 μM working solutions.
Note: To create the stock primer solution (100 μM), add the volume specified (of nuclease-free water) in the oligonucleotide QC report to the lyophilized primers.
?. [Preparing primer pools]
Label four 1.5 ml Eppendorf tubes (pool 1- pool 4). Add equal volumes of 10 μM paired forward and reverse primers to either of the pools, e.g. Primer pool 1 contains HRV-A101_amp1F, HRV-A101_amp1R, HRV-A101_amp3F and HRV-A101_amp3R.Primer pool 2 contains HRV-A101_amp2F, HRV-A101_amp2R, HRV-A101_amp4F and HRV-A101_amp4R
Note: Subsequent (overlapping) primer pairs should not be pooled into one reaction to avoid preferentially amplifying the shorter overlapping region. Non-overlapping primer pairs with same annealing conditions can be pooled, with two primer pairs per reaction. Pooling more primer pairs into one reaction reduced PCR success.
?. [Duplex PCR]
Assemble the reaction on ice by adding the following to a reaction wellComponent20 µl ReactionFinal ConcentrationQ5 High-Fidelity 2X Master Mix10 µl1XPrimer pool 10 μM (1-4) 2 µl0.25 µM per primerTemplate DNAVariableNuclease free waterUp to 20 µl
Component20 µl ReactionFinal ConcentrationQ5 High-Fidelity 2X Master Mix10 µl1XPrimer pool 10 μM (1-4) 2 µl0.25 µM per primerTemplate DNAVariableNuclease free waterUp to 20 µl
Note: We recommend a primer concentration of below 0.5 µM per primer, though this can vary 0.1 – 1 µM per primer.
?. [Duplex PCR]
Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
?. [Duplex PCR]
Transfer PCR tubes to a PCR machine and begin thermocycling. Thermocycling conditions:STEPTEMPTIMEInitial Denaturation98°C30 seconds35 Cycles98°C10 secondsPrimer annealing temp.30 seconds72°C2 minutesFinal Extension72°C2 minutesHold4°C
Note: The most suitable annealing temperature for individual primer pairs was sought by setting up a gradient PCR of annealing temperatures +3°C from the estimated enzyme-specific annealing temperature.
STEPTEMPTIMEInitial Denaturation98°C30 seconds35 Cycles98°C10 secondsPrimer annealing temp.30 seconds72°C2 minutesFinal Extension72°C2 minutesHold4°C
Safe stop: You can store the PCR product at 4°C for up to one week or at -20°C for two months.
?. [Gel electrophoresis]
Preparing the gel
?. [Gel electrophoresis]
Load samples
?. [Gel electrophoresis]
Switch on the power pack set 90 Volts, 400 mA and 50- 60 minutes start the run.
Note: Check that the terminals of the electrophoresis tank are connected correctly to avoid the products out into the buffer due to the wrong connections.
?. [Gel electrophoresis]
Using agarose powder and 0.5 % Tris Borate EDTA, prepare 1% Agarose Gel: It can effectively resolve small and large fragments.
?. [Gel electrophoresis]
Prepare the casting block; put in the comb(s) depending on the number of samples to be run.
?. [Gel electrophoresis]
Pour the gel into the casting plate and wait for 10-15 minutes for the gel to set and harden. When it hardens, remove the combs and put the gel into the electrophoresis tank containing 0.5 % TBE buffer. Ensure the gel is submerged in the buffer.
?. [Gel electrophoresis]
On a parafilm dispense 2µl of loading dye depending on the number of samples plus an extra for the ladder.
?. [Gel electrophoresis]
Pipette 4 µl of the ladder mix with the dye and load into the first well. Pipette 6 µl of each sample mix with the loading dye and load onto the well, do the same for all PCR products
?. [Gel electrophoresis]
Remove the gel after time lapses put in the gel doc machine; adjust the focus of the gel until a sharp focus is achieved. Capture the image
Quality ControlCheck that the ladder has separated. If the ladder did not separate then the results are invalid.
?. [Amplicon clean-up]
Purify PCR product using 1.0X Ampure beads according to manufacturer's instructions
?. [Primer design]
Align the retrieved genomes.
?. [DNA quantification and pooling per sample]
Run Qubit according to manufacturer's recommendations.
?. [DNA quantification and pooling per sample]
Pool the amplicons per sample (samples with a dsDNA quantity of approximately 0.2ng/μl).
Safe stop: You can store the PCR product at 4°C for up to one week or at -20°C for a couple of months.
?. [Library preparation and sequencing]
The PCR products were tagmented, cleaned up and amplified. The libraries were then cleaned up, normalized and pooled as per the Nextera DNA Library Prep guidelines.
?. [Library preparation and sequencing]
Sequencing was performed using the the Miseq Reagent kit v3 600 cycles with an output of 2 x 200 bp paired-end reads.