TITLE: Ultra-Rapid Sequencing (PCR)
AUTHORS: Jack Wadden

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol accompanies the paper "Ultra-Rapid Somatic Variant Detection via Real-Time Threshold Sequencing." This protocol was followed to initiate a sequencing run that resulted in a somatic variant call from known tumor tissue in ~52 minutes. The protocol outlines DNA extraction, PCR, library preparation for Oxford Nanopore Sequencing, and sequencer preparation and loading as executed for the 52 minute demonstration.</div></div>

[STEPS]
?. [DNA Extraction]
When acquired, place  tumor tissue into  DNA extraction tube
20 µg
2 mL
?. [Target Amplification]
Add  extracted DNA to PCR reaction tube
1 µl
?. [Library Preparation]
Add  PCR product to tagmentation mix tube, flick to mix, and spin down
7.5 µl
?. [Preparation]
Prepare the PCR reaction tube by mixing the following components in a PCR tube  forward primer reverse primer
0.2 mL
12.5 µl
1.25 µl
1.25 µl
9 µl
?. [Sequencing]
Add the entire  library to the sequencing mix tube
11 µl
?. [Preparation]
Prepare the DNA extraction tube by mixing the following components in a  Eppendorf tube  of
2 mL
500 µl
?. [Preparation]
Pre-heat thermocycler and pause for combined PCR and fragmentation protocolPre-heat two heat blocks (  and  )DNA Extraction (in heat blocks)  for  for PCR (in thermocycler) for     28 cycles of for  for  for  for     hold at Fragmentation (in thermocycler) for  for     hold at
65 °C
98 °C
65 °C
98 °C
98 °C
98 °C
64 °C
72 °C
72 °C
20 °C
30 °C
80 °C
20 °C
?. [DNA Extraction]
Vortex on high for
?. [DNA Extraction]
Place in pre-heated heat block at
65 °C
?. [DNA Extraction]
Remove tube, vortex briefly, spin down
?. [DNA Extraction]
Place in pre-heated heat block at
98 °C
?. [Target Amplification]
Place in thermocycler and unpause combined protocol
?. [Target Amplification]
Let thermocycler run through the PCR protocol
?. [Target Amplification]
Either let thermocycler draw DNA down to room temperature or quench in ice for . Pause thermocycler program to pre-heat for ONT rapid library preparation tagmentation.
?. [Target Amplification]
During PCR, prepare ONT tagmentation mix by mixing the following in a  PCR tube
0.2 mL
2.5 µl
?. [Target Amplification]
During PCR, prepare ONT sequencing buffer SQB
34 µl
25.5 µl
4.5 µl
?. [Target Amplification]
During PCR, prepare ONT flush bufferAdd into tube of Mix and spin down
30 µl
?. [Target Amplification]
Prime flow cell with  of mixed  and let incubate for
800 µl
?. [Library Preparation]
Place in thermocycler and unpause combined protocol
?. [Library Preparation]
Either let thermocycler draw DNA down to room temperature or quench in ice for
?. [Library Preparation]
Add ONT Rapid Adapter to tagmentation tube, flick to mix, and spin down
1 µl
?. [Library Preparation]
Incubate sequencing library for  at room temperature, flicking to mix occasionally and spinning down
?. [Library Preparation]
During the final 1m of incubation, re-prime the ONT flow-cell with SpotOn port open, using  of  loading buffer
200 µl
?. [Sequencing]
Pipette entire  sequencing mix into the SpotOn port, by slowly squeezing droplets onto the ramp/port
75 µl
?. [Sequencing]
Unpause sequencing run in MinKNOW software
?. [Target Amplification]
Start sequencing run in MinKNOW software and pause run immediately after flow cell QC check
?. [Library Preparation]
Let thermocycler run through fragmentation protocol
?. [DNA Extraction]
Incubate on ice for