TITLE: Viral Sequencing, from Gunk to Graph (One-Step four-primer PCR alternative)
AUTHORS: David Eccles

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a fast "gunk to graph" protocol for analysing viral RNA from nasopharyngeal swabs. The approach involves swab lysis and inactivation at the point of sampling, uses a cellulose binding / wash protocol to reduce extraction cost, incorporates sample-specific barcodes during first-strand synthesis, nanopore rapid-attachment primers during PCR amplification, and nanopore sequencing with parallel RAMPART analysis for fast assembly and phylogenetics.</div><div class = "text-block">Note: this is a one-step RT-PCR reaction protocol. For an alternative method that allows more multiplexing flexibility, see the protocol that this has been forked from:</div><div class = "text-block"><a href="https://dx.doi.org/10.17504/protocols.io.bd3yi8pw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://dx.doi.org/10.17504/protocols.io.bd3yi8pw</span></a></div></div>

[STEPS]
?. [Swab Lysis]
Prepare a centrifuge tube with heated lysis buffer and a cellulose disc
1.5 ml
?. [Swab Lysis]
Add  lysis / RNAse inactivation buffer (Twitter reference) to 1.5ml centrifuge tube:OR  extraction buffer #2 (see paper):
500 µl
[Tris]
[EDTA]
[SDS]
[NaCl]
500 µl
[guanidine hydrochloride]
[Tris [pH 8]]
[Triton X100]
[Tween-20]
?. [Swab Lysis]
Add a  punched disc from Whatman #1 filter paper (see paper)
?. [Swab Lysis]
Preheat  tube to
1.5 ml
60 °C
?. [Swab Lysis]
Collect sample using a sterile polystyrene swab with a 30mm breakpoint (e.g. Puritan 25-3606-U; PurFlock Ultra 6" Sterile Elongated Flock Swab w/Polystryene Handle, 30mm Breakpoint).
?. [RNA Wash]
Transfer disc to a new  tube containing wash buffer using a pipette tip to remove contaminants:
1.5 ml
200 µl
[Tris [pH 8.0]]
[Tween-20]
?. [RNA Wash]
Incubate tube at  for
60 Room temperature
?. [cDNA Synthesis setup]
Add the following additional components into the  PCR tube (see the TaqMan Fast Virus 1-Step Master Mix Product Sheet and Nanopore protocol for Sequence-specific cDNA-PCR Sequencing (SQK-PCS109)) in a  reaction: x  x  cDNA primer (cPRM) TaqMan Fast Virus 1-StepMaster MixReverse primers should be prefixed with sample-specific barcode sequences (if used) and the ONT reverse anchor sequence, i.e. [5' - ACTTGCCTGTCGCTCTATCTTC - [barcode] - [sequence-specific] - 3']Forward primers should be prefixed with sample-specific barcode sequences (if used) and the ONT forward anchor sequence, i.e. [5' - TTTCTGTTGGTGCTGATATTGC - [barcode] - [sequence-specific] - 3']
200 µl
50 µl
1 µl
[reverse primers]
1 µl
[forward primers]
1.5 µl
12.5 µl
[RNAse-free water]
?. [cDNA Synthesis setup]
Mix gently by flicking the tube and spin down
?. [cDNA Synthesis setup]
Denature RNA and anneal reverse primers at  for  and then snap cool on a pre-chilled freezer block for [Note: this step may not be needed]
65 °C
?. [cDNA Synthesis setup]
Mix gently by flicking the tube and spin down
?. [RT and PCR amplification]
In four new  PCR tubes, prepare the following reaction at   in a  reaction: 2X Q5 Hot Start High-Fidelity Master Mix cDNA primer (cPRM) Nuclease-free water Reverse-transcribed cDNA from the previous step (pool, or single sample)
200 µl
42 Room temperature
50 µl
25 µl
1.5 µl
18.5 µl
5 µl
?. [RT and PCR amplification]
Amplify using the following cycling conditions:  ABCD1Cycle stepTemperatureTimeNo. of cycles2Reverse transcription50° C5 mins13RT inactivation /initial denaturation95° C30 secs14Denaturation95 °C15 secs10-40*5Anneal / Extend62 °C60 secs10-40*6Final extension65 °C6 mins17Hold4 °C∞* The recommended starting point is 20 cycles (i.e. 14 cycles + 6 for direct RNA amplification penalty, see here) - adjust this depending on experimental needs.
ABCD1Cycle stepTemperatureTimeNo. of cycles2Reverse transcription50° C5 mins13RT inactivation /initial denaturation95° C30 secs14Denaturation95 °C15 secs10-40*5Anneal / Extend62 °C60 secs10-40*6Final extension65 °C6 mins17Hold4 °C∞
?. [RT and PCR amplification]
Add  of NEB Exonuclease 1 (20 units) directly to each PCR tube to remove unextended primers. Mix by pipetting.
1 µl
?. [RT and PCR amplification]
Incubate the reaction at  for , followed by  for  to
37 °C
80 °C
?. [Bead Cleanup]
Add 2X resuspended AMPure XP beads (e.g.  XP beads to pooled sample) to the  tube and mix by pipetting
1000 µl
500 µl
1.5 ml
?. [Bead Cleanup]
Incubate on a gentle agitator (e.g. hula mixer or rotator mixer) for  at
80 Room temperature
?. [Bead Cleanup]
Spin down   the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
?. [Bead Cleanup]
Keep the tube on the magnet and wash the beads with  of  freshly-prepared  without disturbing the pellet. Remove the ethanol using a pipette and discard.
500 µl
[ethanol]
?. [Bead Cleanup]
Repeat the previous step: wash with  , and discard the ethanol / wash liquid.
500 µl
[ethanol]
?. [Bead Cleanup]
Spin down  and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for  but do not dry the pellet to the point of cracking (the magnetic beads should just start to lose their shiny sheen).
[[at most]]
?. [Bead Cleanup]
Remove the tube from the magnetic rack and resuspend pellet in  of Elution Buffer (EB).
12 µl
?. [Bead Cleanup]
Incubate at  for
80 Room temperature
?. [Bead Cleanup]
Pellet beads on magnet  until the eluate is clear and colourless
?. [Bead Cleanup]
While still on the magnet, carefully remove and retain  of eluate from each pooled sample into a clean Eppendorf DNA LoBind tube
11 µl
1.5 ml
?. [Bead Cleanup]
While still on the magnet, quantify 1 µl of the amplified cDNA library using the Quantus Fluorometer using the ONE dsDNA assay (see ncov 2019 sequencing protocol, step 16)
?. [Adapter Addition]
Add  of Rapid Adapter (RAP) to the amplified cDNA library
1 µl
?. [Adapter Addition]
Mix by pipetting and spin down
?. [Adapter Addition]
Incubate the reaction for  at
80 Room temperature
?. [Adapter Addition]
Store the prepared library  until ready to load onto a flow cell.
on ice
?. [Nanopore Sequencing]
Load  sequencing library onto a MinION flow cell (see ncov 2019 sequencing protocol, step 21)
20 ng
?. [Nanopore Sequencing]
Start the sequencing run using MinKNOW, using SQK-PCS109 as the sample preparation protocol (see  ncov 2019 sequencing protocol, step 22)
?. [RAMPART Analysis]
Analyse the run results using RAMPART (see https://artic.network/ncov-2019/ncov2019-using-rampart.html)
?. [cDNA Synthesis setup]
Transfer disc to a new  PCR tube using a pipette tip
200 µl
?. [Bead Cleanup]
Pool liquid from amplified samples into  Eppendorf DNA LoBind tubes, with no more than  in each tube
1.5 µl
500 µl
?. [Adapter Addition]
Transfer  from the total pool into a clean Eppendorf DNA LoBind tube
11 µl
1.5 ml