TITLE: DNA barcoding on Oxford Nanopore:  multiplexing up to 24 x 96-well plates
AUTHORS: Robin Floyd, Sean Prosser, Saeideh Jafarpour

[DESCRIPTION]
This protocol describes laboratory methods for sequencing a standard COI marker (i.e. DNA barcoding), multiplexing up to 2,280 specimens (24 x 96 well plates, with one negative control well per plate), to run on an Oxford Nanopore Technologies Flongle 10.4.1 Flow Cell on a MinION sequencer. All indexing is accomplished by PCR using tagged primers, meaning that library prep is only carried out in a single tube with all 2,280 PCRs pooled. This is accomplished by asymmetrical indexing, where forward primers carrying 96 Unique Molecular Identifiers (UMIs) provide mapping to a well of a 96-well plate, and reverse primers with 24 UMIs provide mapping to the plate.

The protocol details all primer sequences (attached as a text table) and gives instructions for manufacturing batches of pre-made indexed plates, carrying out PCR, pooling and library prep for the ONT platform, and loading onto the sequencing device.

[BEFORE_START]
DNA extracts or lysates from specimens of interest should be arrayed in 96-well plates. 
Forward indexing primers should be ordered in 96-well plate format. 


[GUIDELINES]
This protocol describes laboratory protocols for sequencing a standard COI marker (i.e. DNA barcoding), multiplexing up to 2,280 specimens (24 x 96 well plates, with one negative control well per plate), to run on an Oxford Nanopore Technologies Flongle 10.4.1 Flow Cell on a MinION sequencer. All indexing is accomplished by PCR using tagged primers, meaning that library prep is only carried out in a single tube with all 2,280 PCRs pooled. This is accomplished by asymmetrical indexing, where forward primers carrying 96 Unique Molecular Identifiers (UMIs) provide mapping to a well of a 96-well plate, and reverse primers with 24 UMIs provide mapping to the plate.

The protocol details all primer sequences (attached as a text table) and gives instructions for manufacturing batches of pre-made indexed plates, carrying out PCR, pooling and library prep for the ONT platform, and loading onto the sequencing device.

It is recommended that:
-reagent prep
-DNA extraction
-all post-PCR steps including Library prep 
should be carried out in three different rooms or otherwise physically separated spaces to avoid cross-contamination.

[STEPS]
SECTION: 1. Prepare Indexed PCR Plates
1. To prepare the partial mastermix (no primers), combine the following components in a sterile bottle of at least 250 mL volume: 
 

This volume is calculated to make a slight excess of partial PCR mix, more than sufficient for 8 reactions x 96 wells x 24 plates.
SECTION: 1. Prepare Indexed PCR Plates
2. Dilute forward UMI primer plate to 2 uM (e.g. 20 uL of stock primer at 10 uM, added to 80 uL molecular-grade water or Tris buffer pH 8), e.g. using Liquidator™ to transfer the contents from all wells to a new 96-well plate in the same orientation.
2 micromolar (µM) 


SECTION: 2. COI PCR
8. Thaw the number of PCR plates required. Add 2 uL of extracted DNA or specimen lysate to each 10.5 uL PCR reaction. Mix by pipetting up and down several times and spin down plate in centrifuge. 
2 µL 
Label all plates carefully, indicating both plate ID/name and UMI index (i.e. reverse primer).
SECTION: 2. COI PCR
9. Place in thermocycler and carry out the following program:

 

SECTION: 3. Pool and purify PCRs
11. Using a multichannel pipette or liquid handling robot, pool each PCR plate into a single 1.5ml Eppendorf tube. Mix well by pipetting and label all tubes carefully with both plate ID/name and UMI index (i.e. reverse primer).
SECTION: 1. Prepare Indexed PCR Plates
3. Dispense 9,552 uL of partial mastermix (from Step 1) into each of 24 sterile falcon tubes.
9552 µL 

SECTION: 1. Prepare Indexed PCR Plates
4. Add 12 uL of each 100 uM reverse primer to a tube of mastermix, carefully labelling which tube contains which reverse primer. Mix well.
12 µL 
Tubes can be stored in fridge for use later the same day, or at -20°C for later use.
SECTION: 1. Prepare Indexed PCR Plates
5. Transfer mastermix (containing reverse primer) to a reservoir and dispense 87 uL to each well of a 96-well plate using a multichannel pipette.
87 µL 

SECTION: 1. Prepare Indexed PCR Plates
6. Using a Liquidator™ or multichannel pipette, add 5.5 uL of primer from the diluted (2uM) forward primer plate to each well of the 96-well plate, in the same orientation. 
5.5 µL 

SECTION: 1. Prepare Indexed PCR Plates
7. Using a Liquidator™ or multichannel pipette, mix all wells by pipetting up and down several times, then dispense 10.5 uL of complete mastermix (containing forward and reverse primers) into eight (8) 96-well PCR plates. Seal each plate and store at -20C for up to 6 months.
10.5 µL 
Ensure all plates are clearly labelled to indicate primers and plate UMI used.
SECTION: 2. COI PCR
10. Optional: run 4 uL of each PCR product on an E-gel™ or other gel electrophoresis device to confirm amplification.
4 µL 

SECTION: 4. Quantification and End Prep
14. Quantify 1 uL of the eluted DNA using Qubit HS kit (following manufacturer's instructions).
SECTION: 4. Quantification and End Prep
15. Set up End Prep reaction:
SECTION: 4. Quantification and End Prep
15.1. From the Qubit result in step 14, calculate the volume of DNA needed for ~100ng (this is a guide, not a critical amount). Transfer this volume to a fresh 0.2ml PCR tube. Unused purified pool may be stored at -20°C for future use.
100 ng 

SECTION: 4. Quantification and End Prep
15.2. Add molecular grade water to make up a total volume of DNA+water to 12.5 uL.
SECTION: 4. Quantification and End Prep
15.3. Combine the following components in the tube:


 

SECTION: 4. Quantification and End Prep
15.4. Carry out the following program in thermocycler 20°C for 5 minutes, 65°C for 5 minutes, hold at 4°C. 
20 °C 5 min 
65 °C 5 min 

SECTION: 4. Quantification and End Prep
16. End Prep cleanup:
SECTION: 4. Quantification and End Prep
16.1. Transfer all 15 uL End Prep reaction to a new 1.5 mL LoBind tube and mix with 15 uL Ampure XP beads. Mix well by pipetting.
SECTION: 4. Quantification and End Prep
16.2. Incubate 5 minutes at room temperature, mixing occasionally by gently flicking the tube.
SECTION: 4. Quantification and End Prep
16.3. Place tube on magnetic rack. Wait 1-2 minutes until supernatant is clear, then remove and discard supernatant. 
SECTION: 4. Quantification and End Prep
16.4. Leaving tube on magnetic rack, wash with 200 uL of 70% ethanol (prepared on same day).
200 µL 

SECTION: 4. Quantification and End Prep
16.5. Remove and discard ethanol. 
SECTION: 4. Quantification and End Prep
16.6. Repeat steps 16.4 and 16.5 for a total of two washes.
SECTION: 4. Quantification and End Prep
16.7. After the second ethanol removal, place tube in mini centrifuge and spin for a few seconds to collect all remaining ethanol at the bottom of the tube. Return tube to magnetic rack and use a P20 pipette to remove the last ethanol.
SECTION: 4. Quantification and End Prep
16.8. Leave tube open at room temperature for 1-2 minutes to allow residual ethanol to dry (beads are sufficiently dry when they take on a dull appearance; do not over-dry).
SECTION: 5. Adapter Ligation and Final Cleanup
17. Combine the following components in the 1.5ml tube: 

 
* From ONT Native Barcoding Kit (SQK-LSK114).

 

Mix by pipetting and incubate at room temperature (~25°C) for 10 minutes.

SECTION: 5. Adapter Ligation and Final Cleanup
18. Cleanup final library using Ampure XP beads: 
SECTION: 5. Adapter Ligation and Final Cleanup
18.1. Add 12.5 uL (0.5x) volume of Ampure XP beads and mix well by pipetting.
12.5 µL 

SECTION: 5. Adapter Ligation and Final Cleanup
18.2. Incubate at room temperature for 10 minutes.
SECTION: 5. Adapter Ligation and Final Cleanup
18.3. Place tube on magnetic rack. Wait 1-2 minutes until supernatant is clear, then remove and discard supernatant.
SECTION: 5. Adapter Ligation and Final Cleanup
18.4. Remove tube from rack and add 100 uL Short Fragment Buffer (SFB - from Ligation Sequencing Kit SQK-LSK114). Pipette up and down or flick tube to resuspend beads. 
100 µL 
SECTION: 5. Adapter Ligation and Final Cleanup
18.5. Return tube to magnetic rack. Wait 1-2 minutes until supernatant is clear, then remove and discard supernatant.
SECTION: 5. Adapter Ligation and Final Cleanup
18.6. Repeat steps 18.4 and 18.5 for a total of two washes in SFB.
SECTION: 5. Adapter Ligation and Final Cleanup
18.7. After the second wash, place tube in mini centrifuge and spin for a few seconds to collect all remaining SFB at the bottom of the tube. Return tube to magnetic rack and use a P20 pipette to remove the last SFB.
SECTION: 5. Adapter Ligation and Final Cleanup
18.8. Air dry pellet for no more than 30 seconds, then add 11 uL Elution Buffer (EB - from Ligation Sequencing Kit SQK-LSK114) and resuspend pellet by pipetting up and down.
11 µL 

SECTION: 5. Adapter Ligation and Final Cleanup
18.9. Incubate for 10 minutes at room temperature or optionally, for increased recovery, place on thermocycler block set to 37C. 
SECTION: 5. Adapter Ligation and Final Cleanup
18.10. Return tube to magnetic rack. Wait ~ 1 minute until supernatant is clear then collect 10 uL supernatant to fresh 1.5ml tube.
10 µL 

19. Quantify 1ul of library using Qubit HS kit (following manufacturer's instructions).


SECTION: 6. Flow Cell Loading
20. Prepare Flongle and carry out Flow Cell Check:
SECTION: 6. Flow Cell Loading
20.1. Remove one Flongle Flow Cell from the fridge and insert into MinION, ensuring the Flongle Adapter is in place. 
  

SECTION: 6. Flow Cell Loading
20.2. In MinKNOW software, navigate to “Flow Cell Check”. Enter the Flongle ID (3 letter+3 number code written on the Flongle) and select “FLO-FLG-114” if using 10.4.1 cell.

The check takes ~5 minutes. When complete, record the pore count. If below 50, it is considered to fail warranty and is eligible for a free replacement if within 28 days of receipt; record details and report to ONT.
SECTION: 6. Flow Cell Loading
21. While Flow Cell Check is running, prepare flow cell priming mix and sequencing mix:
SECTION: 6. Flow Cell Loading
21.1. In 1.5ml Eppendorf tube, mix 117 uL Flow Cell Flush (FCF) with 3 uL Flush Tether (FT) – both from Flongle Expansion Kit. 
117 µL FCF
3 µL FCT

SECTION: 6. Flow Cell Loading
21.2. Pull back the plastic film covering the Flongle loading port, holding in place by pressing the dots (adhesive) against the lid of the MinION device.
      

SECTION: 6. Flow Cell Loading
21.3. Pipette the priming mix (FCF/FT) into the loading port using a P200 pipette. It is important to dispense slowly and not to introduce air bubbles; draw all 120 uL into the pipette, ensure there is no air gap at the tip and dispense by turning the pipette plunger rather than pushing down. Leave the last 1-2 uL in the tip and discard to ensure no air is introduced.
120 µL 
 
SECTION: 6. Flow Cell Loading
21.4. From Qubit reading in Step 19, calculate volume of library needed for 5ng DNA in a final volume of 5 uL. If dilution is necessary, dilute using EB. 
5 ng 
 

When ready to load, combine the following components in a 1.5ml Eppendorf tube: 

 
* Library beads must be thoroughly mixed by vortexing immediately before use and pipetting up and down several times before taking from vial.

SECTION: 6. Flow Cell Loading
21.5. Pipette library mix up and down several times in the tube to resuspend Library Beads, then pipette into the loading port. NOTE: as in Step 22.4, dispense slowly to avoid introducing air bubbles and leave the last 1-2 uL in the tip. 
30 µL 
 
SECTION: 6. Flow Cell Loading
21.6. Close the seal tab over the loading port, pressing down on the adhesive spots to stick down. Close the lid of the MinION. 
    

SECTION: 6. Flow Cell Loading
21.7. Navigate to “Start Sequencing” in MinKNOW software and begin run. 
SECTION: 3. Pool and purify PCRs
13. Carry out magnetic bead cleanup:
SECTION: 3. Pool and purify PCRs
13.4. Leaving tube on magnetic rack, add 500 ul of freshly-prepared 70% ethanol.
500 µL 

SECTION: 3. Pool and purify PCRs
13.5. Remove and discard ethanol.
SECTION: 3. Pool and purify PCRs
13.6. Repeat steps 13.4 and 13.5 for a total of two ethanol washes. 
SECTION: 3. Pool and purify PCRs
13.7. After the second ethanol removal, place tube in mini centrifuge and spin for a few seconds to collect all remaining ethanol at the bottom of the tube. Return tube to magnetic rack and use a P20 pipette to remove the last ethanol.
SECTION: 3. Pool and purify PCRs
13.8. Leave tube open at room temperature for 1-2 minutes to allow residual ethanol to dry (beads are sufficiently dry when they take on a matte appearance; do not over-dry).

SECTION: 3. Pool and purify PCRs
13.9. To elute DNA, remove tube from magnetic rack and add 40 ul of molecular-grade water. Resuspend beads by pipetting up and down or by flicking tube.
40 µL 

SECTION: 3. Pool and purify PCRs
13.10. Incubate 10 minutes at room temperature, mixing occasionally by gently flicking the tube. 
SECTION: 3. Pool and purify PCRs
13.11. Return tube to magnetic rack. Wait 1 minute until supernatant is clear, then transfer 38 uL  supernatant to fresh 1.5 ml eppendorf tube. 
38 µL 

SECTION: 3. Pool and purify PCRs
12. Combine equal volumes of each plate pool (maximum 24), such that the final volume will be at least 200 uL (e.g. 50 uL from each tube). Mix well and take 200 uL of the single pool into a new 1.5ml eppendorf tube (exact amount is not critical, 200 uL provides an excess of final DNA in most cases). Store excess pools at -20°C in case repeat runs are needed.
SECTION: 3. Pool and purify PCRs
13.1. Add 0.8x volume of Ampure XP beads (e.g. for 200 uL pooled PCRs, add 160 uL beads). Mix well by pipetting.
160 µL 

SECTION: 3. Pool and purify PCRs
13.2. Incubate ~10 minutes at room temperature, mixing occasionally by gently flicking the tube.
During this incubation, prepare 70% ethanol (e.g. mix 1.4 mL 100% ethanol + 0.6 mL molecular-grade water for 2 ml, sufficient for this wash and the later washes in Step 16).
SECTION: 3. Pool and purify PCRs
13.3. Place bead-pool tube on magnetic rack. Wait 1-2 minutes until supernatant is clear, then remove and discard supernatant. 
SECTION: 4. Quantification and End Prep
16.9. To elute DNA, Remove tube from magnetic rack and add 16 uL of molecular-grade water. Resuspend beads by pipetting up and down or by flicking tube.
16 µL 

SECTION: 4. Quantification and End Prep
16.10. Incubate 5 minutes at room temperature, mixing occasionally by gently flicking the tube. 
SECTION: 4. Quantification and End Prep
16.11. Return tube to magnetic rack. Wait ~1 minute until supernatant is clear, then transfer 15 uL supernatant to fresh 1.5 ml LoBind tube.
15 µL