TITLE: Ultra-Rapid Sequencing (LAMP)
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 less than 30 minutes. The protocol outlines DNA extraction, LAMP, library preparation for Oxford Nanopore Sequencing, and sequencer preparation and loading.</div></div>

[STEPS]
?. [Preparation]
Prepare the LAMP reaction tube by mixing the following components in a PCR tube  LAMP primer mix
0.2 mL
12.5 µl
2.5 µl
9 µl
?. [Preparation]
Prepare the DNA extraction tube by mixing the following components in a  PCR tube  of
0.2 mL
100 µl
?. [DNA Extraction]
When acquired, place  tumor tissue into DNA extraction tube
20 µg
?. [DNA Extraction]
Vortex on high for
?. [DNA Extraction]
Place in pre-heated thermocycler and unpause combined program
?. [DNA Extraction]
Let thermocycler run through the DNA extraction protocol
?. [DNA Extraction]
Either let thermocycler draw DNA down to room temperature or quench in ice for . Pause thermocycler program to pre-heat for LAMP. Briefly vortex on high and spin down.
?. [Target Amplification]
Add  extracted DNA to LAMP reaction tube, being careful to avoid leftover tissue or other debris in tube.
1 µl
?. [Target Amplification]
Place in thermocycler and unpause combined protocol
?. During LAMP, prepare ONT tagmentation mix by mixing the following in a PCR tube
0.2 mL
2.5 µl
5.6 µl
?. [Target Amplification]
Let thermocycler run through the LAMP protocol
?. [Target Amplification]
Either let thermocycler draw DNA down to room temperature or quench in ice water for . Pause thermocycler program to pre-heat for ONT rapid library preparation tagmentation.
?. [Library Preparation]
Add  LAMP product to tagmentation mix, flick to mix, and spin down
1.9 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 water for
?. [Library Preparation]
Add   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  mix
200 µl
?. [Sequencing]
Add the entire 11ul library to the sequencing mix tube
?. [Sequencing]
Pipette entire  sequencing mix into the MinION Spot On port, by slowly squeezing droplets onto the ramp/port.
75 µl
?. [Sequencing]
Unpause sequencing run in MinKNOW software
?. [Library Preparation]
Let thermocycler run through the tagmentation protocol
?. [Preparation]
Prepare ONT sequencing buffer SQB
34 µl
25.5 µl
4.5 µl
?. [Preparation]
Prepare ONT loading bufferAdd into tube of
30 µl
?. [Preparation]
Pre-heat thermocycler initiate, then pause, combined Ultra-Rapid ProgramDNA Extraction  for  for     hold at LAMP for Fragmentation for  for     hold at
65 °C
98 °C
20 °C
65 °C
30 °C
80 °C
20 °C
?. [Preparation]
Prepare ONT fragmentation mix
2.5 µl
5.6 µl
?. [Preparation]
Prime flow cell with  of loading buffer and let incubate for
800 µl
?. [Preparation]
Start sequencing run in MinKNOW software and pause run immediately after flow cell QC check