Introduction

• In the 16S metabarcoding library preparation 16S primers have index sequences and a sequencing adapter. After pooling the motor protein is attached to the sequencing adapters.

Materials checklist

Reagents

• 16S Barcoding kit (SQK-16S114.24)

• DNA template (Suggested input 30ng)

• LongAmp Taq Polymerase

• Freshly prepared 80% ethanol in nuclease-free water (500 µL per PCR reaction to be cleaned up)

• Qubit (™) reagents

• MinION Mk1B

• Flow cell (R10.4.1), Flongle (R10.4.1) and Flongle adapter

• Flongle Expansion Kit (EXP-FLP002)

Shared Supplies and Equipment

• Container with cracked or crushed ice

• Micropipettes and tips (2–100 µL)

• Microcentrifuge

• Microcentrifuge tube rack

• PCR tubes

• 1.5ml Microcentrifuge tubes

• Qubit (™) tubes

• Qubit™ Fluorometer (Invitrogen™), Q32856), Nanodrop™, QIAxpert® or other for DNA quantification

16S PCR

1. In a PCR tube mix:

Reagent	Volume Per Sample
DNA (~10ng)	15ul
LongAmp Hot Start Taq 2X Master Mix	25ul
16S Primers (SQK-16S114.24)	10ul
Total	50ul

2. Place tubes in thermocycler and run the following program:

Reagent	Temperature	Time	Cycles
Initial Denaturation	95 °C	1 min	1
Denature	95 °C	20 sec	3
Anneal	50 °C	30 sec	3
Extend	65 °C	2 min	3
Denaturation	95 °C	20 sec	38
Annealing	55 °C	30 sec	38
Extension	65 °C	2 min	38
Final Extension	65 °C	5 min	1
Hold	4 °C	Infinite

3 Add 4ul of EDTA to each tube to stop the reaction.
4. Check PCR products on a 2% Agarose gel

Clean up

1. Pool PCR products into a 1.5ml tube
2. Resuspend Ampure beads by vortexing or pipetting up and down.
3. Add .6X ul the total volume of combined PCRs of Ampure beads to the tube with PCR products.
4. Incubate for 5 minutes at room temperature, flicking occasionally.
5. Spin down 15 seconds.
6. Place a sample on a magnetic rack and allow magnetic beads to pellet.
7. Pipette off and discard supernatant.
8. Add 1ml of 80% ethanol then pipette off supernatant without disturbing pellet
9. Repeat previous step
10. Briefly spin down, remove any residual ethanol. Allow to dry for 30 seconds.
11. Add 15ul of elution buffer (EB) and remove from the magnetic rack.
12. Incubate for 5 minutes at room temperature.
13. Add tube to magnetic rack and allow beads to pellet.
14. Transfer 15ul to a new 1.5ml tube.
15. Quantify DNA concentration by Nanodrop of Qubit.
16. Transfer 50 fmol to a new tube. Use the elution buffer (EB) to bring the volume up to 11ul.
*For 16S amplicons ~1500bp, 50-100fmol equates to 50-100ng.

Library Preparation

1. In a 1.5ml tube dilute Rapid Adapter (RA) as follows:

Reagent	Volume
Rapid Adapter (RA)	1.5ul
Adapter Buffer (ADB)	3.5ul
Total	5ul

2. Add 1ul of diluted Rapid Adapter to indexed DNA.
3. Incubate for 5 min at room temperature.

Flow cell Priming for Flongle (Instructor)

1. Mix 117ul FCF (from the Flongle expansion kit) and 3ul of FCT (from the Rapid Barcoding kit).

2. Peel back the seal from the Flongle flow cell

3. Slowly expel liquid from the pipette by turning the dial clockwise until a bead of liquid forms at the tip.

4. Place the pipette tip in the sample port, keeping the pipette vertical.

5. Turn the dial clockwise to load the flush buffer and primer into the flow cell, stopping right before all liquid is entirely expelled to avoid pipetting air into the flow cell.

Flow cell Loading (Instructor)

1. Make sequencing mix:

Reagents	Volume
Sequencing Buffer (SB)	15ul
Loading Beads (LIB)	10ul
DNA Library	5ul
Total	30ul

2. Pipette up sequencing mix, slowly expel liquid from the pipette by turning the dial clockwise until a bead of liquid forms at the tip.

3. Place the pipette tip in the sample port, keeping the pipette vertical.

4. Turn the dial clockwise to load to the flush buffer and primer the flow cell port slowly. Stop rotation right before all liquid is entirely expelled to avoid pipetting air into the flow cell.

5. Seal flow cell port with sticker and run sequencing.