TITLE: Viral Sequencing, from Gunk to Graph (Two-step, strand-switching)
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"><span>An alternative protocol using both forward and reverse primers can be found </span><a href="https://dx.doi.org/10.17504/protocols.io.bd5ni85e" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span style = ":;">.</span></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]
Transfer disc to a new  PCR tube using a pipette tip
200 µl
?. [cDNA Synthesis]
Add the following additional components into the  PCR tube (see the Nanopore protocol for Sequence-specific cDNA-PCR Sequencing (SQK-PCS109)) in a  reaction: x  x Reverse 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']A potential primer pool are the reverse/right ARCTIC primers (and amplification control) with barcodes and ONT anchor sequences from here.An alternative protocol using both forward and reverse primers can be found here.
200 µl
11 µl
1 µl
[reverse primers]
1 µl
[dNTPs]
[RNAse-free water]
?. [cDNA Synthesis]
Mix gently by flicking the tube and spin down
?. [cDNA Synthesis]
Denature RNA and anneal primers at  for  and then snap cool on a pre-chilled freezer block for
65 °C
?. [cDNA Synthesis]
In a separate tube, mix together the following in an  reaction: 5X RT Buffer RNAseOUT Nuclease-free water x  ONT Strand-switching primer (SSP)
8 µl
4 µl
1 µl
1 µl
2 µl
?. [cDNA Synthesis]
Mix gently by flicking the tube and spin down
?. [cDNA Synthesis]
Add the strand-switching buffer to the snap-cooled, annealed RNA, mix by flicking the tube and spin down
?. [cDNA Synthesis]
Incubate at  for
42 °C
?. [cDNA Synthesis]
Add  of Maxima H Minus Reverse Transcriptase, to a total volume of
1 µl
20 µl
?. [cDNA Synthesis]
Mix gently by flicking the tube and spin down
?. [cDNA Synthesis]
Incubate using the following protocol:  ABCD1Cycle stepTemperatureTimeNo. of cycles2Reverse transcription and strand-switching42° C90 mins13Heat inactivation85° C5 mins14Hold4° C∞
ABCD1Cycle stepTemperatureTimeNo. of cycles2Reverse transcription and strand-switching42° C90 mins13Heat inactivation85° C5 mins14Hold4° C∞
?. [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
?. [PCR amplification]
Amplify using the following cycling conditions:  ABCD1Cycle stepTemperatureTimeNo. of cycles2Initial denaturation95 °C30 secs13Denaturation95 °C15 secs10-40*4Annealing62 °C15 secs10-40*5Extension65 °C50 secs per kb10-40*6Final extension65 °C6 mins17Hold4 °C∞* Starting from viral RNA, the recommended starting point is 20 cycles - adjust this depending on experimental needs.
ABCD1Cycle stepTemperatureTimeNo. of cycles2Initial denaturation95 °C30 secs13Denaturation95 °C15 secs10-40*4Annealing62 °C15 secs10-40*5Extension65 °C50 secs per kb10-40*6Final extension65 °C6 mins17Hold4 °C∞
?. [PCR amplification]
Add  of NEB Exonuclease 1 (20 units) directly to each PCR tube to remove unextended primers. Mix by pipetting.
1 µl
?. [PCR amplification]
Incubate the reaction at  for , followed by  for
37 °C
80 °C
?. [PCR amplification]
Run 1μl of amplified product on a gel (or similar length-based QC device) to verify that amplified products exist at the expected length. Because this is a strand-switch protocol, there may be a smear of template DNA rather than specific bands.
?. [Bead Cleanup]
Add 160 µl of resuspended AMPure XP beads to the  tube and mix by pipetting
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.
200 µl
[ethanol]
?. [Bead Cleanup]
Repeat the previous step: wash with  , and discard the ethanol / wash liquid.
200 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 into a clean Eppendorf DNA LoBind tube
12 µl
1.5 ml
?. [Bead Cleanup]
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)
?. [Swab Lysis]
Break swab and place into the prepared  tube with lysis buffer to remove the outer viral shell and release RNA.
1.5 ml
?. [Swab Lysis]
Vortex tube for  , then incubate tube at  for   to inactivate viral proteins.
60 °C
[[10-30 mins]]